Peptidic antifungal and antibacterial coating compositions

ANTIFUNGAL AND ANTIBACTERIAL COATING COMPOSITIONS

C. STEVEN MCDANIEL

C: 75504

BACKGROUND OF THE INVENTION

Field of the Invention

[00031 The present Invention generally relates to antifungal and antibacterial compositions and methods employing such compositions to deter or prevent fungal growth in stored coatings and on susceptible surfaces. More particularly, the present invention relates to such compositions containing antifungal and antibacterial peptides, polypeptides or ._kproteins and to methods of making and using such compositions.

Description of the Related Art

[0004] Fungal growth on indoor and outdoor surfaces is a major environmental concern today affecting home, work and recreational environments. Not only can fungus (e.g., mold, mildew) be unsightly on exposed surfaces, it can destroy wood, fiber and other materials if left untreated, causing severe damage to buildings and other structures and equipment. Over the past few years it has become increasingly apparent that exposure to certain fungi or their spores can seriously Impact the health of humans, pets and other animals. Although fungi are certainly not the only factors that detrimentally affect indoor _;air quality, in many Instances they have been identified as a primary contributor to indoor air quality problems. In fact, the term sick building syndrome was recently coined to describe buildings In which various physical, chemical and biological factors, including growing fungi and/or their spores, have severely compromised the air quality leading to discomfort or illness of the occupants. Concerns such as allergies, asthma, Infections, and the long-term repercussions of mold toxins are just a few of the many real health effects associated with mold contamination of indoor and outdoor environments.

[0005] Fungi (including true fungi, molds and mildews) are eukaryotic organisms that have cell walls, similar to plants, but do not contain chlorophyll. There are between 100,000 1

200,000 species of fungi, mold and mildew, depending on which classification methods are used. Of particular concern are the pathogenic fungi, which can cause significant harm to individuals who are exposed to them. About 300 species are presently known to be pathogenic for man, but It Is thought that there are many other as yet unrecognized fungal pathogens. The field of medical mycology has emerged as a result of the growing number of fungal-related illnesses and deaths.

[0006] Fungi grow as saprophytes, /.6., in a suitable moist environment they are able to decompose organic matter to obtain the nourishment needed for growth. Building and decorative materials such as wood, paper-coated wallboard, wallpaper, fabrics, carpet and leather can provide the necessary organic matter. Today, an especially problematic fungal genus sometimes found in buildings that have excess indoor moisture is Stachybotrys. Stachybotrys chartarum, commonly found in nature growing on celluloserich plant materials, has often been found in water-damaged building materials, such as ceiling tiles, wallpaper, sheet-rock and cellulose resin wallboard (fiberboard). Depending on the particular conditions of temperature, pH and humidity in which the mold is growing, Stachybotrys may produce mycotoxins, compounds that have toxic properties.

[0007] Other common fungi that can grow in residential and commercial buildings are Aspergillus species (sp.)., Penicllllum sp., Fusarium sp., AJternaria dianthicola, Aureobasidium pullulans (aka Pullularia pullulans), Phoma pigmentivora and Cladosporium sp. The moist indoor environment which promotes growth of these fungi can arise from water damage, excessive humidity, water leaks, condensation, water infiltration, or flooding, in some cases due to defects in building construction, faulty mechanical system design, and/or operational problems. Even modern homes and commercial buildings are not immune to fungal Invasion despite the use of technologically advanced building materials and more energy efficient construction and operation than in buildings of the past. Modem homes tend to be less well ventilated, and although the use of air conditioning reduces humidity making it harder for mold to grow, today's central air conditioning systems can also facilitate the spread of mold spores throughout a home. Increased use of paper products In homes and commercial buildings today further encourages mold growth. Heavy contamination of indoor or outdoor surfaces by dirt and/or oil can also provide a food source for a fungus. Vulnerable structures and materials that are difficult to access for cleaning, or for which cleaning is neglected, are particularly vulnerable to attack by fungi. Fungi are also known to contaminate stored paints, fuels, and many other industrial products.

[0008] Fungal colonies typically take on filamentous form, having long filament-like cells called hyphae. Under the right environmental conditions, hyphae grow into an intertwining network called the mycelium. A mycelium can be visible to the naked eye, appearing as . 2

Patent provided by Sughrue Mion, PLLC - http://www.sughrue.conn unsightly fuzzy green, bluish-gray or black spots, for example. When conditions for growth are less favorable, many varieties of fungi can respond by forming spores on specialized hyphal cells. Spores are the primary means for dispersal and survival of fungi, and can remain dormant for months or even years - even withstanding extremely adverse conditions, to germinate and flourish again when environmental variables such as light, oxygen levels, temperature, and nutrient availability again become favorable. Thickwalled spores are substantially more resistant to common disinfective agents than are the thinner-walled vegetative fungal cells. According to the U.S. Environmental Protection Agency, there is no practical way to eliminate all mold and mold spores in the indoor environment.

[0009] As mentioned above, paints and paint films or coatings are known to be vulnerable to mold contamination due to the presence of common organic components that act as cellulosic thickeners, surfactants and defoamers, and which can also serve as a source of food for fungus cells. Some of these components are casein, acrylic, polyvinyl and other carbon polymers. For example, latex is a water-dispersed binder comprising a carbon polymer. Inside the paint can, certain fungi (e.g., yeasts) can convert enough carboncontaining food sources to CO₂ to swell or even explode the can. Fungi can also discolor and reduce the viscosity of the paint, and produce foul odors. Both in-can preservation of paints and protection of the end use paint films, and the surfaces they cover, from mold, mildew and yeasts is necessary. To combat fungi, a variety of coating materials have been formulated which include organic or inorganic chemicals to discourage or prevent the growth of mildew חס the paint film. Ideally, these chemical fungicides or mildewcides slowly leach out of the paint to the surface, and maintain their inhibitory properties for the life of the paint film, causing little or no harm to the environment. In practice, however, the antifungal properties of most coating compositions in use today persist for variable lengths of time, depending on the amount of exposure to the elements, abrasion and erosion.

[0010] Most antifungal chemicals are non-specific as to the organism affected and can be detrimental to the environment, including toxicity to plant and animal life. It is more difficult to identify fungus-specific agents than it is to discover bacteria specific-agents because fungal cells share many similarities with the cells of higher organisms, whereas bacterial cells are distinctly different. For this reason, fungicides tend to be more toxic to humans and animals than are bactericides. U.S. Patent No. 5,882,731 (Owens) describes a number of common and proprietary chemical mlldewcide-containing products that have been investigated as additives for water-based latex mixtures. Some known antifungal agents that have been used in the coatings industry are: copper (II) 8quinolinolate (CAS No. 10380-28-6); zinc oxide (CAS No. 1314-13-2); zinc-dimethyl dithiocarbamate (CAS No. 137-30-4); 2-mercaptobenzothiazole, zinc salt (CAS No. 1553

Patent provided by Sughrue Mlon, PLLC - http://www.sughrue.com wo ״״»י־«. .,β.

OM); barium metaborate (CAS No. 13701-59-2); trlbutyl tin benzoate (CAS No. 4342-363). bis tributyl tin salicylate (CAS No. 22330-14-9), trlbutyl tin oxide (CAS No. 56-35-9); parabens: ethyl parahydroxybenzoate (CAS No. 120-47-8), propyl parahydroxybenzoate (CAS No. 94-13-3) methyl parahydroxybenzoate (CAS No. 99-76-3) and butyl parahydroxybenzoate (CAS No. 94-26-8); methylenebls(thiocyanate) (CAS No. 6317-186); 1,2-benzlsothiazoline-3-one (CAS No. 2634-33-5); 2-mercaptobenzo-thiazole (CAS No. 149-30-4); 5-chloro-2-methyl-3(2H)-isothiazolone (CAS No. 57373-19-0); 2-methyl3(2H)-isothiazolone (CAS No. 57373-20-3); zinc 2-pyridinethlol-N-oxide (CAS No. 1346341-7); tetra-hydro-3,5-di-methyt-2H-1,3,5-thiadiazine-2-thlone (CAS No. 533-74-4); Ntrlchloromethyl-thio-4-cyclohexene-1,2-dicarboximlde (CAS No. 133-06-2); 2-n-octyMlsothiazoline-3-ona (CAS No. 26530-20-1); 2,4,5,6-tetrachloro-isophthalonitrile (CAS No. 1897-45-6); 3-iodo-2-propynyl butylcarbamate (CAS No. 55406-53-6); diiodomethyl-ptolylsulfone (CAS No. 20018-09-1); N-(trlchloromethyl-thio)phthalimide (CAS No. 133-073) potassium N-hydroxy-methyl-N-methyl-dithlocarbamate (CAS No. 51026-28-9); sodium 2-pyridlnethiol-1-oxide (CAS No. 15922-78-8); 2-(thiocyanomethylthio) benzothiazole (CAS No. 21564-17-0); 2-4(-thlazolyl) benzimidazole (CAS No. 148-79-8). See V M King Bactericides, Fungicides, and Algicides, Ch. 29, pp. 261-267, and D.L. Campbell, Biological Deterioration of Paint Films, Ch. 54, pp. 654-661; both in Paint AND Coating Testing Manual, 14״ ed. of the Gardner-Sward Handbook, J.V. Koleske, Editor (1995), American Society for Testing and Materials, Ann Arbor, Ml. Currently, the Pesticide Action Network North America (PAN) lists In its Internet chemical database, www.oanna.oro. the above-mentioned chemicals plus more than 700 additional chemicals designated as pesticides having antifungal properties in soil and wood.

[00111 The mode of action of some of the metal-based antifungal agents Is thought to be chelation of metals that are necessary to growth of the organisms. Some of the nitrogenand/or sulfur-containing antifungal agents are thought to act by uncoupling oxidative phosphorylation in the fungal cells, or Inhibiting oxidation of glucose. The paraben compounds (aka hydroxybenzoate) are thought to affect membrane activity and Integrity.

[001Z1 Due to environmental and safety concerns, there Is Increasing pressure today on the coatings industry to eliminate some of the more effective but more toxic chemical preservatives from paints and other coating compositions. Yet at the same time, consumers wish to avoid purchasing spoiled or poorly performing products. Thus, there Is a great need in the industry today for safe and effective alternatives to conventional antifungal agents.

[00131 Various naturally occurring biological products that are said to possess antifungal activity are described in the background discussion of U.S. Patent Nos. 6,020,312;.;

602,097; and 5,885,782 [each incorporated in their entirety by reference herein]. In 4

Patent provided by Sughrue Mion, PLLC ־ http://www.sughrue.com many cases, the active component of those natural antifungal agents has not been identified nor completely characterized. Since most of the known naturally occurring antifungal agents are poorly characterized at best, the persistence and toxicity of such compounds in the environment is also unknown. Furthermore, the fact that many of those compounds are produced by microbes in the environment suggests that they may have a limited spectrum of antifungal activity. A drawback of most of the antifungal agents in use today is that they are as toxic to higher organisms as they are to the target fungi. The more target-specific antifungal agents tend to be very rare and/or costly.

[0014] Recently developed methods permit the preparation of synthetic peptide combinational libraries (SPCLs) that are composed of equimolar mixtures of free peptides that can be used with in vitro methods to determine bioactivity (Furka, A., et al. int. J. Pept. Protein Res. 37:487 (1991), Houghten, R. A., et al. Nature 354:84 (1991), Houghten, R. A., et al. BioTechniques 13:412 (1992). Libraries can consist of D- or Lamino acid stereoisomers or combinations of L- and D- and/or non-naturally-occurring amino acids. Other methods for synthesizing peptides of defined sequence are also known. Similarly, large-scale preparative methods are known. Certain recombinant methods for producing peptides have also been described, e.g., U.S. Pat. No. 4,935,351. While U.S. Patent Nos. 6,020,312; 5,602, 097; and 5,885,782 describe agricultural uses for certain synthetic antifungal peptides, none of those or any other peptidic agents have been previously investigated as additives for use in the paints and coatings industry.

[0015] Although significant advancement has been made in identifying various chemical agents and natural and synthetic peptides or proteins that demonstrate antifungal activity for certain uses (e.g., medical treatment or agricultural use), there is no indication that any such biomolecule could be used successfully in paints or other coating materials for protecting or treating non-living objects. Antifungal or fungus-resistant paints and other coating compositions are needed which do not suffer from the same limitations as conventional surface coating materials containing existing fungicides and antifungal agents. Ideally, an antifungal paint will contain fungus-specific fungus deterring/ inhibiting/ killing agents that are stable In paints and other coating mixtures during storage, persist in the resulting coat or film that is spread out over a surface in need of protection from fungal infestation, and which are safer to the environment Better antifungal materials would be especially welcomed by original equipment manufacturers (OEMs), and by the architectural, marine and industrial maintenance industries. In particular, antifungal and antibacterial additives to paints and coatings that work alone or synergistically with existing antifungal agents would be desirable.

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PCTVUS2004/021711

BRIEF SUMMARY OF THE PREFERRED EMBODIMENTS

[0016] The compositions and methods of the present Invention overcome some of the disadvantages of previous antifungal or fungus-resistant paints, coatings and other compositions such as elastomers, textile finishes, adhesives, and sealants. It was not previously known to combine a natural, synthetic or recombinant antifungal peptide, polypeptide or peptide with a paint or other coating material to provide a coated surface with sustainable antifungal activity that protects the recipient surface from fungal infestation and defacement, and which also provides fungus resistance to the composition itself. It Is now disclosed that antifungal peptidic agents offer a new tool in the arsenal of fungicidal and fungistatic chemicals. Accordingly, new antifungal and antibacterial paints, coatings, films and other compositions are provided which contain one or more bioactive peptides, polypeptides and/or proteins as antifungal and antibacterial peptidic agents. Methods of using the antifungal and antibacterial additives and compositions for treating existing fungal or bacterial colonies and/or for deterring or preventing fungal or bacterial infestations and inhibiting cell growth or proliferation on a variety of inanimate objects such as interior and exterior architectural surfaces and building materials are also provided. The compositions and methods disclosed herein avoid many of the drawbacks of existing methods and compositions which rely on non-specific chemical bacteriocides, fungicides, or antifungal agents. By application of a protective coating comprising one or more antifungal or antibacterial protein, polypeptide or peptide, one can prevent or deter or lessen the infestation and growth of fungus or bacterium. At the same time, the associated discoloration, disfiguration and/or degradation of the supporting substrate or surface can be avoided or reduced. The compositions of the invention are especially useful on surfaces where conditions are conducive to deposition and development of fungus or bacteria, and where control of fungal or bacterial growth is preferably accomplished with compositions which are not toxic to humans, pets and other animals or harmful to the environment.

[0017] In accordance with certain embodiments of the invention, an antifungal coating composition is provided that is effective for inhibiting the growth of Stachybotrys fungi. In some embodiments, an antifungal coating composition is provided that is effective for inhibiting the growth of one or more of the Aspergillus, Penicillium, Fusarium, Altemaria, and Cladosporium genera of fungi. In some embodiments, an antifungal coating composition is effective for inhibiting the growth of Rhizoctonia, Ceratocystls, Pythium, Mycosphaerella, and Candida genera of fungi, in certain instances, a coating is provided that is either antifungal, antibacterial, or both antifungal and antibacterial.

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WO 2005/007758 PCT7US2004/021711

[0018] In certain embodiments of the present invention, compositions are formulated for use as paints and the like, for coating surfaces. In certain embodiments, such coating includes impregnating porous or semi-porous materials that are capable of supporting fungal growth. These compositions contain various antifungal peptides or proteins, as described herein, and may also contain chemicals and other substances that are conventional and well known in the art. One of the important benefits of certain preferred embodiments of the present invention is that little or no formulation modification, other than the inclusion of the presently described antifungal peptide component, is needed to obtain substantial enhancement of fungus resistant, antifungal or fungicidal properties of the coating composition.

[0019] A further embodiment comprises using a film or coat comprising the coating composition, or the composition itself, to protect an object or material selected from the group consisting of wood, paint, adhesive, glue, paper, textile, leather, plastic, cardboard, caulking, from infestation and growth of a fungus.

[0020] A preferred coating material comprises a paint or coating. In some embodiments the paint or coating is applied prophylactically over a clean surface that is not contaminated by fungal spores. In other embodiments the paint or coating is applied to a surface already contaminated by fungal spores or growing fungus.

[0021] In certain embodiments of the present invention, a paint or other surface-coating composition is provided that contains an antifungal protein, polypeptide or peptide additive that retains its antifungal activity after being admixed with said paint or other surfacecoating composition, and retains antifungal activity after the paint or surface-coating composition is applied to a surface. Even after drying, the paint or other surface-coating composition renders the coated surface antifungal. More specifically, an antifungal paint or other surface-coating composition comprising antifungal protein, polypeptide or peptide additive is capable of biologically interacting with a susceptible fungus cell or spore In a manner that inhibits or prevents growth of the fungus, preferably for extended periods of time. Some additives of the present invention remained stable in the coating for an extended period of time (e.g., months) at ambient conditions. It is contemplated that with certain antifungal compositions, especially those containing microencapsulated antifungal peptides, the extended period of activity may comprise years. In a preferred embodiment, a polymer-based compound that prophalactically and continuously deters fungal infestation, inhibits or kills fungal cells is provided.

[0022] It is contemplated that in certain embodiments the compositions and methods of the present invention may be used to produce a fungal cell growth inhibitory surface, or a fungal cell killing surface, that remains active for extended periods. Such an antifungal surface may not need additional treatment with fungicide compositions, clean-up 7

Patent provided by Sughrtie Mion, PLLC - http://www.sughrue.com treatments to effect decontamination and cosmetic painting, thereby simplifying upkeep of the physical condition and appearance of fungus infestation prone surfaces such as building exteriors. It is contemplated that in some embodiments the compositions of the present invention may be easily applied to susceptible surfaces in advance of and/or during exposure to a fungus organism.

[0023] Isolated naturally occurring proteins, polypeptides and/or peptides are employed in some embodiments, and in preferred embodiments synthetic proteins, polypeptides and/or peptides are employed. In some embodiments, combinations of natural and/or synthetic proteins, polypeptides and peptides are employed. Still other embodiments employ at least one recombinant protein, polypeptide and/or peptide that is produced using specific expression vectors in a variety of host cells.

[0024] Antifungal peptides are chemically defined species that are easily synthesized and purified. They are not necessarily dependent upon the genetic stability or growth properties of microorganisms for their production. The methods and compositions of the present invention employ an array of different antibiotic compounds which are shown to have particular effectiveness in inhibiting the growth of or killing fungal cells. The compositions of the invention are effective in controlling the growth of fungi, and yet demonstrate a high degree of specificity to the target fungi, low toxicity and controlled persistence in the environment. Using the preferred methods it is possible to produce and identify desirable antifungal agents for use in paints and coatings in a much shorter time, and with a considerably higher-probability of success, than screening natural isolates for antifungal peptides. Since the preferred methods of production can control the chemical nature of the antifungal agents thus produced, synthesis and purification (if needed) of the peptides is much less problematic (e.g., cysteine is eliminated, which amino acid’s free sulfhydryl groups can cause unwanted cross linking). Thus, in some embodiments, the paint compositions of the present invention comprise peptides of precisely known chemical structure and characteristics. The use of D-amino acids increases the stability of certain of these compounds by being insensitive to common biological degradation pathways that degrade L-amino acid peptides. For instance, L-amino acid peptides may be stabilized by addition of D-amino acids at one or both of the peptide termini. However, biochemical pathways are available which will degrade even D-amino acids in these peptides so that long-term environmental persistence is not a problem. Of course, where the compositions of the Invention act rapidly or need not otherwise be stabilized, L-amino acids or mixtures of L-and D- amino acids may be useful. Unlike antifungal agents which only work as one or another stereoisomer, the compositions of the invention work well as either one or another stereoisomer or as a mixed stereoisomeric composition. Research leading to the current invention evaluated SPCLs for activity against fungal pathogens,

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WO 2005/007758 . PCT/US2004/021711 including pathogens of plants as well as those of animals. The library was composed of 52,128,400 six-residue peptides, each peptide being composed of D-amino acids and having non-acetylated N-termini and amidated C-termini. However, it is not necessary for a peptide composition of demonstrable antibiotic activity to be completely defined as to each residue. In fact, in certain instances, especially where the peptide compositions of the invention are being used to treat an array of fungal target organisms each with a different causative agent, mixed peptide compositions will be preferred. This is also likely to be the case where there is a desire to treat a fungal target with lower concentrations of numerous antifungal additives rather than a higher concentration of a single chemical composition. In other instances where, for instance, due to the increased cost of testing or producing a completely defined peptide antibiotic is prohibitive, the mixed peptide compositions of the invention having one or more variable amino acid residues may be preferred. In other instances, it may be possible to use peptide antibiotic compositions in coatings that have not been purified, that have not had their side chains de-blocked, and/or have not been cleared from the synthetic resin used to anchor the growing amino acid chains. Thus, antibiotic compositions comprising equimolar mixture of peptides produced in a synthetic peptide combinatorial library utilizing the methods of the invention have been derived and shown to have desirable antibiotic activity. In certain embodiments, these relatively variable compositions are specifically those based upon the sequences of one or more of the peptides disclosed in any of the U.S. Patent Nos. 6,020,312; 5,602,097; and 5,885,782.

[0025] The antibiotic compositions of the invention may also comprise a carrier. In certain instances, the carrier will be one suitable for permanent surface coating applications. In other instances, the carrier will be one suitable for use in applying the antibiotic compositions in semi-permanent or temporary coatings. In either instance, the earner selected should preferably be a carrier whose chemical and/or physical characteristics do not significantly interfere with the antibiotic activity of the peptide composition. It is known, for instance that certain microsphere carriers may be effectively utilized with proteinaceous compositions in order to deliver these compositions to a site of preferred activity such as onto a surface. Liposomes may be similarly utilized to deliver labile antibiotics. Saline solutions, coating-acceptable buffers and solvents and the like may also be utilized as carriers for the peptide compositions of the invention. Those peptides have been demonstrated to inhibit the growth of fungal cells from at least the Fusarium, Rhizoctonia, Ceratocystis, Pythium, Mycosphaerella and Candida species, and are believed to be active against additional genera, including at least some of those that are capable of infesting building materials and other inanimate objects.

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[0026] Similarly, processes for inhibiting growth of fungal cells comprise contacting the fungal cell with a paint or coating composition comprising at least one peptide. Using the techniques of the invention, selected pathogenic fungi, some pathogens of plants and others pathogens of animals, have been tested. The processes of the invention have, therefore, been specifically shown to be effective where the fungal cell is a fungal cell selected from the group of fungi consisting of Fusarium and Aspergillus. The processes of the invention are applied to fungal cells of a pathogen of an animal, such as a human. A method for selecting antibiotic compositions is also described. The method comprises first creating a synthetic peptide combinatorial library as described herein. Next, as further described in detail herein, a step of contacting a battery of fungal cells with aliquots of the synthetic peptide combinatorial library, each of which aliquots represents an equimolar mixture of peptides in which at least the two C-terminal amino acid residues are known and which residues are in common for each peptide in said mixture is accomplished. After allowing an appropriate period for growth, a next step is accomplished in which the growth of the battery of fungal cells as compared to untreated control cells is measured. Lastly, a determination is made of which of the aliquots most reduces the growth of fungal cells In a coating overall in the battery of fungal cells. Of course, the same method may be carried out in which each of the aliquots represents an equimolar mixture of peptides in which at least three, four, five or more C-terminal amino acid residues are known (depending upon the overall length of the ultimate peptide in the SPCL). Typically, such increasingly defined aliquots will be sequentially tested in order to select the succeeding best candidate peptides for testing. Thus, an additional step in the method entails utilizing the determination of which of the aliquots reduces the growth of fungal cells in a coating overall in said battery of fungal cells to select which aliquots to next test of a synthetic peptide combinatorial library where at least one additional Cterminal amino acid residue is known.

[0027] A method of treating or preventing growth of a fungus on a susceptible surface is also disclosed. These and other objects, features and advantages of the present invention will be readily apparent to one skilled in the art from the following detailed description and claims.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] The following detailed description, specific examples and claims, while Indicating the preferred embodiments of the invention, are given by way of illustration only and are considered representative of other embodiments. Accordingly, it will be readily apparent to one skilled in the artfrom this detailed description and the claims which follow that

Patent provided by Sughrue Mion, PLLC - hUp://www.sughrue.coin various changes, substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention.

[00291 Paints and other conventional protective or decorative coating materials typically contain polymeric substances such as casein, acrylic, polyvinyl and carbon polymers (e.g._r binders) which can serve as nutrients for fungal cells. As discussed above in the Background of the Invention, not only can these nutrient substances support the growth-of fungus on paint films or coated surfaces, fungus can also grow inside cans of liquid paints and coating compositions during storage. It was, therefore, an unexpected discovery that certain synthetic peptides, when added to a range of conventional paint and coating materials, render those compositions resistant to fungal infestation and growth. It was also surprising to find that such additives worked alone or in conjunction with existing biocides in a coating.

EXAMPLES

Example 1. Antifungal Peptide Additives for a Coating Composition

[0030] A group of preferred antifungal peptides that have either demonstrated activity as additives for coating mixtures, or that are expected to demonstrate such activity, are disclosed in U.S. Patent No. 6,020,312 (Edwards): U.S. Patent No. 5, 885, 782 (Edwards); and U.S. Patent No. 5,602,097 (Edwards), the disclosures of which are hereby incorporated in their entirety herein by reference. Preferred sequences that will be employed include one or more of SEQ ID Nos. 1-47, preferably SEQ ID Nos. 25-47. These and other peptides with antifungal activity are identified using methods and testing protocols like those described in the above-referenced patents. Additional peptides that are expected to demonstrate the desired activity in coatings are listed in Table I. The screening method generally Includes:

(a) creating a synthetic peptide combinatorial library using known methods and materials;

(b) testing a battery of fungal cells that are known to, or suspected of, infesting a building material or other object having a fungus-infestation susceptible surface with aliquots of the synthetic peptide library, wherein each aliquot comprises an equimolar mixture of peptides in which at least one of the C-terminal amino acid residues are known and which residues are in common for each peptide In the mixture;

(c) admixing said aliquots with a coating typically used on such building material and coating a surface with the admixture;

(d) allowing an appropriate period of time for growth, of the fungal cell under suitable culture conditions;

(e) comparing the growth of the treated fungal ceils with untreated control cells,

Patent provided by Sughrue Mion, PLLC http://www.sughrue.com (f) identifying optionally, assessing which of the aliquots reduced the growth of the fungal cells; and, the relative growth inhibitory activity of each aliquot compared to that of other aliquots (e.g., comparing IC50 data).

TABLE 1

<td> Name</td><td> Source</td><td> Seq. ID</td><td> Activity</td><td> Reference I</td>

<td></td><td> Synthetic_________ ..</td><td> 1</td><td> Fungi___</td><td> US</td>

<td> ______ _____— '— —</td><td> Synthetic___________</td><td> 2</td><td> Fungi____________</td><td> US</td>

<td></td><td> Synthetic____________</td><td> 3</td><td> Fungi</td><td> US</td>

<td></td><td> Synthetic__________״</td><td> 4</td><td> Fungi________{_}__</td><td> US</td>

<td> —-------------—¹</td><td> Synthetic___________</td><td> 5</td><td> Fungi__</td><td> us</td>

<td></td><td> Synthetic___</td><td> 6</td><td> Fungi__________</td><td> US</td>

<td> —--------</td><td> Synthetic__</td><td> 7</td><td> Fungi</td><td> US</td>

<td> ----—' —</td><td> Synthetic__________</td><td> 8</td><td> Fungi___</td><td> US</td>

<td> . -</td><td> Synthetic____________</td><td> 9</td><td> Fungi_______________</td><td> US</td>

<td> --</td><td> Synthetic___________</td><td> 10</td><td> Fungi</td><td> US</td>

<td> ——-- --</td><td> Synthetic ________</td><td> 11</td><td> -ungi__</td><td> US</td>

<td rowspan="2"> -</td><td> Svnthetic</td><td> 12</td><td> Fungi</td><td> US Patents,885,782</td>

<td> Synthetic_________</td><td> 13</td><td> Fungi</td><td> US</td>

<td> — -----</td><td> Synthetic____________</td><td> 14</td><td> Fungi ___________</td><td> us</td>

<td> —-- —</td><td> Synthetic____________</td><td> 15</td><td> Fungi _____</td><td> US</td>

<td> _ --</td><td> Synthetic____________</td><td> 16</td><td> Fungi____</td><td> US</td>

<td> — ----</td><td> Synthetic _________</td><td> 17</td><td> Fungi_____</td><td> US</td>

<td> ־--------- ------</td><td> Synthetic____________</td><td> 18</td><td> Fungi________________</td><td> US Patent.5,885,782</td>

<td></td><td> Synthetic</td><td> 19</td><td> Fungi_____________</td><td> US Patent.5,885,782</td>

<td> .— —</td><td> Synthetic</td><td> 20</td><td> Fungi__</td><td> US</td>

<td></td><td> Synthetic____________</td><td> 21</td><td> Fungi</td><td> US Patent.5,885,782</td>

<td></td><td> Synthetic____________</td><td> 22</td><td> Fungi ___________</td><td> US Patent.5,885,782</td>

<td> ———--—</td><td> Synthetic____________</td><td> 23</td><td> Fungi_____________</td><td> US Patent.5,885,782</td>

<td> --------</td><td> Synthetic____________</td><td> 24</td><td> Fungi_______________</td><td> US</td>

<td> —-----</td><td> Synthetic____________</td><td> 25</td><td> Fungi___</td><td> US</td>

<td> —*--------</td><td> Synthetic _________</td><td> 26</td><td> Fungi__</td><td> US</td>

<td> ------</td><td> Synthetic</td><td> 27</td><td> Fungi__</td><td> US</td>

<td></td><td> Synthetic____________</td><td> 28</td><td> Fungi_______________</td><td> US Patent.5,885,782</td>

<td> —--</td><td> Synthetic___________</td><td> 29</td><td> Fungi _____________</td><td> US Patent.5,885,782</td>

<td> --,״—</td><td> Synthetic _________</td><td> 30</td><td> Fungi___</td><td> US Patent.5,885,782</td>

<td></td><td> Synthetic ________</td><td> 31</td><td> Fungi__</td><td> US Patent.5,885,782</td>

<td> ----</td><td> Synthetic</td><td> 32</td><td> Fungi</td><td> US</td>

<td></td><td> Synthetic</td><td> 33</td><td> Fungi ________</td><td> US Patent.5,885,782</td>

<td></td><td> Synthetic</td><td> 34</td><td> Fungi________________</td><td> US Patent.5,885,782</td>

<td> — </td><td> Synthetic____</td><td> 35</td><td> Fungi___</td><td> US Patent.5,885,782</td>

<td></td><td> Synthetic</td><td> 36</td><td> Fungi__</td><td> us</td>

<td> —- -</td><td> Synthetic ________</td><td> 37</td><td> Fungi_______________</td><td> US</td>

<td></td><td> Synthetic___________</td><td> 38</td><td> Fungi_______________</td><td> US Patent.5,885,782</td>

<td> - --—</td><td> Synthetic__________</td><td> 39</td><td> Fungi__</td><td> US Patent.5,885,782</td>

<td> ——</td><td> Synthetic____________</td><td> 40</td><td> Fungi</td><td> US Patent.5,885,782</td>

<td> --— —</td><td> Synthetic__________</td><td> 41</td><td> Fungi________________</td><td> US Patent.5,885,782</td>

<td> —---</td><td> Synthetic___________</td><td> 42</td><td> Fungi ______________</td><td> US Patent.5,885,782</td>

<td></td><td> Synthetic___________</td><td> 43</td><td> Fungi______________</td><td> US Patent.5,885,782</td>

<td> ----------—, ..</td><td> Synthetic___________</td><td> 44</td><td> Fungi_______________</td><td> US Patent.5,885,782</td>

<td> ----—------</td><td> Synthetic ____</td><td> 45</td><td> Fungi_________________</td><td rowspan="2"> US US</td>

<td> ——--</td><td> Synthetic</td><td> 46</td><td> Fungi</td>

<td> -----—--- '</td><td> Synthetic</td><td> 47</td><td> Fungi</td><td> US Patent.5,885,782</td>

<td> Tachystatin A</td><td> Horseshoe Crab</td><td> 48</td><td> Gram+ &amp; Gram-, Fungi______________</td><td> Fujitani (2002)</td>

<td> | Androctonin________</td><td> Androctonus</td><td> 49</td><td> Gram+ &amp; Gram-,</td><td> Mandard ¢1999)______</td>

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<td> Name</td><td> Source</td><td> Seq. ID</td><td> Activity</td><td> Reference</td>

<td></td><td> Australis______________</td><td></td><td> Fungi _____</td><td></td>

<td> Tritrptlcin</td><td> Synthetic</td><td> 50</td><td> Gram+ &amp; Gram-, Fungi__</td><td> Schlbll (1999)</td>

<td> HNP-3 Defensin</td><td> Human</td><td> 51</td><td> Gram*־ &amp; Gram-, Virus, Fungi___________</td><td> Hill (1991)</td>

<td> Anti-fungal protein 1 (oafo-sl</td><td> Phytolacca Americana</td><td> 52</td><td> Fungi</td><td> Gao (2001)</td>

<td> Magalnin 2</td><td> Synthetic construct</td><td> 53</td><td> Gram.*־ &amp; Gram-, Fungi</td><td> Hara (2001)</td>

<td> Indoiicidin</td><td> Bos Taurus</td><td> 54</td><td> Gram.*. &amp; Gram-, Virus, Fungi__</td><td> Rozek (2000)</td>

<td> Defensin heliomicin</td><td> Heliothls virescens</td><td> 55</td><td> Fungi________________</td><td> Lamberty (2001)</td>

<td> Defensin heliomicin</td><td> Heliothis virescens</td><td> 56</td><td> Gram* &amp; Gram-, Fungi _____</td><td> Lamberty (2001)</td>

<td> Sativum defensin 1 fosdU</td><td> Seed of Pea</td><td> 57</td><td> Fungi</td><td> Almeida (2002)</td>

<td>¹ f ----— Gomesin</td><td> Synthetic</td><td> 58</td><td> Gram+ &amp; Gram-, Fungi, Mammalian cells__</td><td> Mandard (2002)</td>

<td> Lactoferricin B</td><td> Bovine</td><td> 59</td><td> Gram* &amp; Gram-, Virus, Fungi, Cancer cells _______</td><td> Hwang (1998)</td>

<td> PW2</td><td> Synthetic</td><td> 60</td><td> Fungi ____</td><td> Tinoco (2002)</td>

<td> Hepcldin 20_________</td><td> Human</td><td> 61</td><td> Fungi</td><td> Hunter (2002)________</td>

<td> Hepcldin 25_________</td><td> Human__________</td><td> 62</td><td> Fungi_______________</td><td> Hunter (2002)</td>

<td> AC-AMP2</td><td> Amaranthus caudatus</td><td> 63</td><td> Gram.*., Fungi</td><td> Martins (1996)</td>

<td> NK-Lysin</td><td> Sus scrofa</td><td> 64</td><td> Gram* &amp; Gram-, Fungi</td><td> Liepinsh (1997)</td>

<td> Magainin 2</td><td> African clawed frog</td><td> 65</td><td> Gram.* &amp; Gram-, Fungi, cancer cells</td><td> Gesell (1997)</td>

<td> Melittin B</td><td> Honey bee venom</td><td> 66</td><td> Gram.*- &amp; Gram-, Fungi, Mammalian cells ________________</td><td> Eisenberg</td>

<td> Thanatin</td><td> Podisus maculiventris</td><td> 67</td><td> Gram* &amp; Gram-, Fungi</td><td> Mandard (1998)</td>

<td> Antimicrobial peptide 1 _________</td><td> Common ice plant</td><td> 68</td><td> Gram+ &amp; Gram-, Fungi</td><td> Michalowski (1998)</td>

<td> Melanotropin alpha (Alpha-MSH)_______</td><td> Bovine</td><td> 69</td><td> Gram +, Fungi</td><td> Cutuli (2000)</td>

<td> CORTICOSTATIN III (MCP-1)__________</td><td> Rabbit</td><td> 70</td><td> Gram+ &amp; Gram-, Virus, Fungi_______״</td><td> Selsted (1988)</td>

<td> CORTICOSTATIN 111 (MCP-1)</td><td> Rabbit</td><td> 71</td><td> Gram+ &amp; Gram-, Virus, Fungi___________</td><td> Selsted (1988)</td>

<td> Cecropin B</td><td> Chinese oak silk moth</td><td> 72</td><td> Gram+ &amp; Gram-, Fungi__</td><td> Qu (1982)</td>

<td> Semlnalplasmin</td><td> Bovine</td><td> 73</td><td> Gram+ &amp; Gram-, Fungi, Mammalian cells____________________</td><td> Theii (1983)</td>

<td> NP-3A defensin</td><td> Rabbit</td><td> 74</td><td> Gram+ &amp; Gram-, Virus, Fungi</td><td> Zhu (1992)</td>

<td> HNP-1 Defensin</td><td> Human</td><td> 75</td><td> Gram+ &amp; Gram-, Virus, Fungi ....</td><td> Zhang (1992)</td>

<td> HNP-2 Defensin</td><td> Human</td><td> 76</td><td> Gram+ &amp; Gram-, Virus, Fungi</td><td> Selsted (1989)</td>

<td> HNP-4 Defensin</td><td> Human</td><td> 77</td><td> Gram+ &amp; Gram-, Fungi _____</td><td> Wilde (1989)</td>

<td> Histatin 5</td><td> Human</td><td> 78</td><td> Gram+ &amp; Gram-, Fungi ______</td><td> Raj (1998)</td>

<td> Histatin 3__</td><td> j Human</td><td> _ ⁷θ</td><td> Gram+ &amp; Gram-,</td><td> Oppenheim (1988)</td>

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WO 2005/007758 ____________ PCT/US2004/021711

<td> Name</td><td> Source</td><td> Seq. ID</td><td> Activity</td><td> Reference</td>

<td></td><td></td><td></td><td> Fungi</td><td></td>

<td> Histatin 8</td><td></td><td> 80</td><td> Gram+ &amp; Gram-, Fungi</td><td> Yin (2003)</td>

<td> Tracheal antimicrobial peptide</td><td> Bovine</td><td> 81</td><td> Gram+ &amp; Gram-, Fungi___</td><td> Zimmermann (1995)</td>

<td> AMP1 (MJ-AMP1)</td><td> Garden four-o’clock</td><td> 82</td><td> Gram+, Fungi__</td><td> Cammue (1992)</td>

<td> AMP2 (MJ-AMP2)</td><td> Garden four-o’clock</td><td> 83</td><td> Gram+, Fungi</td><td> Carrimue (1992)</td>

<td> MBP-1</td><td> Maize</td><td> 84</td><td> Gram+ &amp; Gram-, Fungi</td><td> Duvick (1992)</td>

<td> AFP2</td><td> Rape</td><td> 85</td><td> Fungi</td><td> Terras (1993)</td>

<td> AFP1</td><td> Turnip</td><td> 86</td><td> Fungi __________</td><td> Terras (1993)</td>

<td> AFP2</td><td> Turnip</td><td> 87</td><td> Fungi ________</td><td> Terras (1993)</td>

<td> ADENOREGULIN</td><td> Two coloured leaf frong</td><td> 88</td><td> Gram+ &amp; Gram-, Fungi</td><td> Mor (1994)</td>

<td> Protegrin 2</td><td> Pig</td><td> 89</td><td> Gram+ &amp; Gram-, Virus, Fungi ___</td><td> Kokryakov (1993)</td>

<td> Protegrin 3</td><td> Pig</td><td> 90</td><td> Gram+ &amp; Gram-, Virus, Fungi</td><td> Kokryakov (1993)</td>

<td> Histatin 1</td><td> Crab eating macaque</td><td> 91</td><td> Gram+ &amp; Gram-, Fungi ___</td><td> Xu (1990)</td>

<td> Peptide PGQ</td><td> African clawed frog</td><td> 92</td><td> Gram+ &amp; Gram-, Fungi</td><td> Moore (1991)</td>

<td> Ranalexin</td><td> Bull frog</td><td> 93</td><td> Gram+ &amp; Gram-, Fungi ___</td><td> Halverson (2000)</td>

<td> GNCP-2</td><td> Guinea pig</td><td> 94</td><td> Gram+ &amp; Gram-, Virus, Fungi</td><td> Nagaoka (1991)</td>

<td> Protegrin 4</td><td> Pig</td><td> 95</td><td> Gram+ &amp; Gram-, Virus, Fungi</td><td> Zhao (1994)</td>

<td> Protegrin 5</td><td> Pig</td><td> 96</td><td> Gram+ &amp; Gram-, Virus, Fungi</td><td> Zhao (1995)</td>

<td> BMAP-27</td><td> Bovine</td><td> 97</td><td> Gram+ &amp; Gram־, Fungi</td><td> Skerlavaj (1996)</td>

<td> BMAP-28</td><td> Bovine</td><td> 98</td><td> Gram+ &amp; Gram-, Fungi</td><td> Skerlavaj (1996)</td>

<td> Butorin I</td><td> Asian toad</td><td> 99</td><td> Gram+ &amp; Gram-, Fungi</td><td> Park (1996)</td>

<td> Buforin II</td><td> Asian toad</td><td> 100</td><td> Gram+ &amp; Gram-, Fungi __</td><td> Yi (1996)</td>

<td> BMAP-34</td><td> Bovine</td><td> 101</td><td> Gram+ &amp; Gram-, Fungi</td><td> Scocchi (1997)</td>

<td> Tricholongin</td><td> Trichoderma longibrachiatum</td><td> 102</td><td> Gram+ &amp; Gram-, Fungi</td><td> Rebuffat (1991)</td>

<td> Dermaseptin 1</td><td> Sauvage's leaf frog</td><td> 103</td><td> Gram+ &amp; Gram-, Fungi</td><td> Mor (1994)</td>

<td> pseudo-hevein (Minor hevin)__</td><td> Para rubber tree</td><td> 104</td><td> Fungi</td><td> Soedjanaatmadja (1994)</td>

<td> Gaegurin-1</td><td> Wrinkled frog</td><td> 105</td><td> Gram+ &amp; Gram-, Fungi</td><td> Park (1994)</td>

<td> Skin peptide tyrosine-tyrosine</td><td> Two-colored leaf frog</td><td> 106</td><td> Gram+ &amp; Gram-, Fungi</td><td> Mor (1994)</td>

<td> Penaeidin-1</td><td> Penoeid shrimp</td><td> 107</td><td> Gram+ &amp; Gram-, Fungi</td><td> Destoumieux (2000)</td>

<td> Neutrophil defensin 1 (HANP-1)________</td><td> Golden hamster</td><td> 108</td><td> Gram+, Fungi</td><td> Mak (1996)</td>

<td> Neutrophil defensin 3 (HANP-3)________</td><td> Golden hamster</td><td> 109</td><td> Gram+, Fungi</td><td> Mak (1996)</td>

<td> Misgurin</td><td> Oriental weatherfish</td><td> 110</td><td> Gram+ &amp; Gram-, Fungi.</td><td> Park (1997)</td>

<td> PN-AMP</td><td> Japenese morning glory________________</td><td> 111</td><td> Gram+, Fungi</td><td> Koo (1998)</td>

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<td> Name</td><td> Source</td><td> Seq. ID</td><td> Activity</td><td> Reference</td>

<td> Histone H2B-1 tHLP-1) (Fragment)</td><td> Rainbow trout</td><td> 112</td><td> Gram+ &amp; Gram־, Fungi___</td><td> Robinette (1998)</td>

<td> Histone H2b-3 fHLP-3) (Fragment)</td><td> Rainbow trout</td><td> 113</td><td> Fungi</td><td> Robinette (1998)</td>

<td> Neutrophil defensin 2 (RMAD-2)_______</td><td> Rhesus macaque</td><td> 114</td><td> Gram+ &amp; Gram-, Fungi</td><td> Tang (1999)</td>

<td> Termicln</td><td> Pseudacanthotermes spiniger</td><td> 115</td><td> Gram*־, Fungi</td><td> Lamberty (2001)</td>

<td> Splngerin</td><td> Pseudacanthotermes spiniger__</td><td> 116</td><td> Gram+ &amp; Gram-, Fungi__________________</td><td> Lamberty (2001)</td>

<td> Aurein 1.1</td><td> Southern bell frog</td><td> 117</td><td> Gram+ &amp; Gram-, Fungi___________</td><td> Rozek(2000)</td>

<td> Ponerlcin Gl</td><td> Ponerine ant</td><td> 118</td><td> Gram+ &amp; Gram-, Fungi_______________</td><td> Drivel (2001)</td>

<td> Brevinin-1 BB</td><td> Rio Grande leopard frog ____________</td><td> 119</td><td> Gram+ &amp; Gram-, Fungi</td><td> Goraya (2000)</td>

<td> Ranalexin-1 CB</td><td> Gree frog</td><td> 120</td><td> Gram+ &amp; Gram-, Fungi________________</td><td> Halverson (2000)</td>

<td> Ranatuerin-2CA</td><td> Green frog</td><td> 121</td><td> Gram+ &amp; Gram-, Fungi . _______</td><td> Halverson (2000)</td>

<td> Ranatuerin-2CB</td><td> Green frog</td><td> 122</td><td> Gram+ &amp; Gram-, Fungi_____________</td><td> Halverson (2000)</td>

<td> Glnkbllobin</td><td> Ginkgo</td><td> 123</td><td> Gram+ &amp; Gram-, Virus, Fungi___________</td><td> Wang (2000)</td>

<td> Alpha-basrubrln (Fraament)</td><td> Malabar spinach</td><td> 124</td><td> Virus, Fungi</td><td> Wang (2001)</td>

<td> Pseudin 1</td><td> Paradoxical frog</td><td> 125</td><td> Gram+ &amp; Gram-, Fungi________________</td><td> Olson (2001)</td>

<td> Parabutoporin</td><td> Scorpion</td><td> 126</td><td> Gram+ &amp; Gram-, Fungi, Mammalian cells __________________</td><td> Moerman (2002)</td>

<td> Opistoporin 1</td><td> African yellow leg scorpion</td><td> 127</td><td> Gram+ &amp; Gram-, Fungi, Mammalian cells</td><td> Moerman (2002)</td>

<td> Oplstoporin 2</td><td> African yellow leg scorpion</td><td> 128</td><td> Gram+ &amp; Gram-, Fungi, Mammalian cells ______________</td><td> Moerman (2002)</td>

<td> Histone H2A (fraament)</td><td> Rainbow trout</td><td> 129</td><td> Gram+, Fungi</td><td> Fernandes (2002)</td>

<td> Dolabellanln B2</td><td> Sea hare</td><td> 130</td><td> Gram+ &amp; Gram-, Fungi ____________</td><td> lijima (2002)</td>

<td> Cecropin A</td><td> Nocutuld moth</td><td> 131</td><td> Gram.*־ &amp; Gram-, Fungi</td><td> Bulet (2002)</td>

<td> HNP-5 Defensin</td><td> Human</td><td> 132</td><td> Gram+ &amp; Gram-, Fungi</td><td> Jones(1992)</td>

<td> HNP-6 Defensin</td><td> Human</td><td> 133</td><td> Gram+ &amp; Gram-, Fungi________________</td><td> Jones(1993)</td>

<td> Holotricln 3</td><td> Holotrichia diomphalia</td><td> 134</td><td> Fungi</td><td> Lee (1995)</td>

<td> Lingual antimicrobial peptide_____________</td><td> Bovine</td><td> 135</td><td> Gram+ &amp; Gram-, Fungi</td><td> Schonwetter (1995)</td>

<td> RatNP-3</td><td> Rat</td><td> 136</td><td> Gram+ &amp; Gram-, Virus, Fungi</td><td> Yount (1995)</td>

<td> GNCP-1</td><td> Guinea pig</td><td> 137</td><td> Gram+ &amp; Gram-, Virus, Fungi__________</td><td> Nagaoka(1993)</td>

<td> Penaeldln-4a</td><td> Penoeld shrimp</td><td> 138</td><td> Gram+ &amp; Gram-, Fungi</td><td> Desioumieux (2000)</td>

<td> Hexapeptide</td><td> Bovine</td><td> . .1.39</td><td> Gram+ &amp; Gram״, Virus, Fungi, Cancer I cells</td><td> Vogle (2002)</td>

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<td> Name</td><td> Source</td><td> Seq. ID</td><td> Activity</td><td> Reference</td>

<td> P-18</td><td></td><td> 140</td><td> Gram+ &amp; Gram-, Fungi, Cancer cells</td><td> Lee (2002)</td>

<td> MUC7 20- Mer</td><td> Human</td><td> 141</td><td> Gram+ &amp; Gram-, Fungi</td><td> Bobek (2003)</td>

<td> Nigrocln 2</td><td> Rana nigromacuiata</td><td> 142</td><td> Gram+ &amp; Gram-, Fung!</td><td> Park (2001)</td>

<td> Nigrocln 1</td><td> ^ana nigromacuiata</td><td> 143</td><td> Gram+ &amp; Gram־, Fungi</td><td> Park (2001)</td>

<td> Lactoferrin (Lf) peptide 2</td><td></td><td> 144</td><td> Fungi</td><td> Ueta(200l) '</td>

<td> ib-AMP3</td><td> Impatlens balsam in a</td><td> 145</td><td> Gram+, Fungi</td><td> Ravi (1997)</td>

<td> lb-AMP4</td><td> m patlens balsamlna</td><td> 146</td><td> Gram* Fungi________</td><td> Ravi (1997)</td>

<td> Dhvar4</td><td> Synthesis</td><td> 147</td><td> Gram+ &amp; Gram-, Fungi</td><td> Ruissen (2002)</td>

<td> DhvarS</td><td> Synthesis</td><td> 148</td><td> Gram+ &amp; Gram-, Fungi</td><td> Ruissen (2002)</td>

<td></td><td> Synthetic</td><td> 149</td><td> Fungi</td><td rowspan="43"></td>

<td></td><td> Synthetic</td><td> 150</td><td> Fungi</td>

<td></td><td> Synthetic</td><td> 151</td><td> Fungi</td>

<td></td><td> Synthetic</td><td> 152</td><td> Fungi</td>

<td></td><td> Synthetic</td><td> 153</td><td> Fungi</td>

<td></td><td> Synthetic</td><td> 154</td><td> Fungi</td>

<td></td><td> Synthetic</td><td> 155</td><td> Fungi</td>

<td></td><td> Synthetic</td><td> 156</td><td> Fungi_________________</td>

<td></td><td> Synthetic</td><td> 157</td><td> Fungi</td>

<td></td><td> Synthetic</td><td> 158</td><td> Fungi</td>

<td></td><td> Synthetic</td><td> 159</td><td> Fungi_______________</td>

<td></td><td> Synthetic</td><td> 160</td><td> Fungi________________</td>

<td></td><td> Synthetic</td><td> 161</td><td> Fungi_________________</td>

<td></td><td> Synthetic</td><td> 162</td><td> Fungi</td>

<td></td><td> Synthetic</td><td> 163</td><td> Fungi</td>

<td></td><td> Synthetic</td><td> 164</td><td> Fungi</td>

<td></td><td> Synthetic</td><td> 165</td><td> Fungi</td>

<td></td><td> Synthetic</td><td> 166</td><td> Fungi</td>

<td></td><td> Synthetic</td><td> 167</td><td> Fungi</td>

<td></td><td> Synthetic</td><td> 1B8</td><td> Fungi</td>

<td></td><td> Synthetic</td><td> 169</td><td> Fungi</td>

<td></td><td> Synthetic </td><td> 170</td><td> Fungi</td>

<td></td><td> Synthetic</td><td> 171</td><td> Fungi</td>

<td></td><td> Synthetic</td><td> 172</td><td> Fungi_______________</td>

<td></td><td> Synthetic</td><td> 173</td><td> Fungi</td>

<td></td><td> Synthetic .</td><td> 174</td><td> Fungi</td>

<td></td><td> Synthetic</td><td> 175</td><td> Fungi</td>

<td></td><td> Synthetic</td><td> 176</td><td> Fungi</td>

<td></td><td> Synthetic</td><td> Ϊ77</td><td> Fungi</td>

<td></td><td> Synthetic</td><td> 178</td><td> Fungi</td>

<td></td><td> Synthetic</td><td> 179</td><td> Fungi</td>

<td></td><td> Synthetic</td><td> 180</td><td> Fungi</td>

<td></td><td> Synthetic</td><td> 181</td><td> Fungi</td>

<td></td><td> Synthetic</td><td> 182</td><td> Fungi</td>

<td></td><td> Synthetic</td><td> 183</td><td> Fungi</td>

<td></td><td> Synthetic</td><td> 184</td><td> Fungi</td>

<td></td><td> Synthetic</td><td> 185</td><td> Fungi</td>

<td></td><td> Synthetic</td><td> 186</td><td> Fungi</td>

<td></td><td> Synthetic</td><td> 187</td><td> Fungi</td>

<td></td><td> Synthetic</td><td> 188</td><td> Fungi</td>

<td></td><td> Synthetic ..</td><td> 189</td><td> Fungi</td>

<td></td><td> Synthetic</td><td> 190</td><td> Fungi</td>

<td></td><td> Synthetic</td><td> 191</td><td> Fungi</td>

<td> Name</td><td> Source</td><td> Seq. ID</td><td> Activity</td>

<td></td><td> Synthetic</td><td> 192</td><td> Fungi</td>

<td></td><td> Synthetic</td><td> 193</td><td> Fungi</td>

<td></td><td> Synthetic</td><td> 194</td><td> Fungi</td>

<td></td><td> Synthetic</td><td> 195</td><td> Fungi</td>

<td></td><td> Synthetic</td><td> 196</td><td> Fungi</td>

<td></td><td> Synthetic</td><td> 197</td><td> Gram+ &amp; Gram-, Fungi</td>

<td></td><td> Synthetic</td><td> 198</td><td> Gram+ &amp; Gram-, Fungi</td>

<td></td><td> Synthetic</td><td> 199</td><td> Gram+ &amp; Gram-, Fungi</td>

[0031] In the above-referenced U.S. Patent Nos. 6,020,312; 5,885,782; and 5,602,097 an iterative process was used to identify active peptide sequences with broad spectrum antifungal activity. A representative method employs a hexapeptide library with the first two amino acids in each peptide chain individually and specifically defined and with the last four amino acids consisting of equimolar mixtures of 20 amino acids. Four hundred (400) (20²) different peptide mixtures each consisting of 130,321 (19⁴)(cysteine was eliminated) individual hexamers were evaluated. In such a peptide mixture, the final concentration for each peptide was 9.38 ng/ml, in a mixture composed of 1.5 mg (peptide mix)/ml solution. This mixture profile assumed that an average peptide has a molecular weight of 785. This concentration was sufficient to permit testing for antifungal activity. Both D- and L- amino acid containing peptides may be constructed and tested to identify peptide compositions that can inhibit or kill fungi that can grow on the surfaces of inanimate objects. Peptide compositions comprising substantially homogeneous peptide compositions, as well as mixtures of peptides derived from amino acids that are between 3 to 25 residues in length (a length readily accomplished using standard peptide synthesis procedures), especially six residues in length, are disclosed in U.S. Patent Nos. 6,020,312; 5,885,782; and 5,602,097. A preferred antifungal peptide that inhibits or kills one or more fungus that infests and grows on the surfaces of inanimate objects is a hexapeptide having the amino acid sequence Phe Arg Leu Lys Phe His (SEQ ID No. 41).

[0032] Homogeneous peptide compositions are chiefly composed □f a single active peptide species of a well-defined sequence. Minor amounts (less than 20% by moles) of impurities may coexist with the peptide in these compositions so long as they do not interfere with the growth inhibitory properties of the active peptide(s). Target fungi include but are not limited to those fungi that can infest indoor and outdoor structures and building materials causing defacement (e.g., deterioration or discoloration), odor, environment hazards, and other undesirable effects. Alternatively to using one or more isolated antifungal peptides as the antifungal peptidic agent, the agent may instead be a peptide library aliquot containing a mixture of peptides in which at least two (and preferably three or four) of the N-terminal amino acid residues are known. If the peptidic agent is a mixture of peptides, at least one will have antifungal activity. As will be apparent in examples which follow, for ease of production and lower cost, in many instances it will be preferred to use a peptide library aliquot that contains at least one antifungal peptide, preferably the hexapeptide of SEQ ID No. 41, but is impure to the extent that it may also include peptides of unknown exact sequence which may or may not have antifungal activity. In addition, the peptide or peptide library may be one that has side chains blocked, is attached to the synthetic resin or both blocked and attached.

Example 2. Identifying Antifungal Peptides that Inhibit Target Organisms

[0033] The testing methods described in U.S. Patent Nos. 6,020,312; 5,885,782; and 5,602,097 may be employed to screen the peptide library for antifungal activity against a wide variety of fungus genera and species. Preferably the methods are modified to screen against fungal organisms that are known to, or suspected of, infesting construction materials or other vulnerable materials and surfaces. More preferably, fungal cells used for screening the peptide library include members of the genera Stachybotrys (especially Stachybotrys chartarum), Aspergillus species (sp.), Penicillium sp., Fusarium sp., Memaria dianthicola, Aureobasidium pullulans (aka Pullularia pullulans). Phoma plgmentivora and Cladosporium sp. Cell culture conditions may also be modified appropriately to provide favorable growth and proliferation conditions, as is within the capability of one of ordinary skill in the art. The above-mentioned methods will be used to identify peptides or groups of peptides that demonstrate broad-spectrum antifungal activity. Similar methods will be used to identify particular peptides or groups of peptides that target specific fungus genera or species. Alternatively, but less preferred, any other suitable peptide/polypeptide/pratein screening method could be used instead to identify antifungal peptide candidates for testing as active antifungal agents in paints and other coating materials.

[0034] It is known that certain of the peptides of particular usefulness in the coatings of the invention, as disclosed in US Patent Nos. 6,020,312; 5,602,097, and 8,885,782, exhibit variable abilities to inhibit fungai growth as adjudged by the minima! inhibitory concentrations (MIC mg/ml) and/or the concentrations necessary to inhibit growth of fifty percent of a population of fungal spores (IC50 mg/ml). MICs may range depending upon peptide additive and target organism from about 3 to about 300 mg/ml, while IC50’s may range depending upon peptide additive and target organisms from about 2 to about 100 mg/ml. Target organisms susceptible to these amounts include Fusarium oxysporum, Fusariam Sambucinum, Rhizoctonia Solani, Ceratocystis Fagacearum, Pphiostoma ulmi,

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WO 2005/0077» PCT/BS200W21711

Pythlum ultlmum, Magaporthe Aspergillus nidulans, Aspergillus fumigatus, end

Aspergillus Parasiticus.

[0035] The mode of action of antifungal peptides, polypeptides and proteins, by which they exert their inhibitory or fungicidal effects, can be varied. For instance, certain peptides may operate to destabilize fungal cell membranes, while the modes of action of others could include disruptions of macromolecular synthesis or metabolism. While the modes of action of some known antifungal peptides have been determined (see, e.g., Fiedler, Η. P., et al. 1982. Nikomyclns: microbial inhibitors of chitin synthase. J. Chem. Technol. Biotechnol. 32:271-280; Isono, K. and S. Suzuki. 1979. The polyoxins: pyrimidine nucleoside peptide antibiotics inhibiting fungal cell wall biosynthesis. Heterocycles 13:333-351), mechanisms which explain their modes of action and specificity have typically not yet been determined. Initial studies to elucidate antifungal mode of action of peptides involves a physical examination of mycelia and cells to determine if the peptides can perturb membrane functions responsible for osmotic balance, as has been observed for other peptides (Zasloff, M. 1987. Proc. Natl. Acad. Sci. USA 84:5449-5453)- Disruption of appressorium formation may also be the mechanism by which some peptides inhibit fungal growth (see e.g., published US Patent Application 10/601,207, expressly incorporated herein by reference in its entirety). For the purposes of preparing and using antifungal peptides, polypeptides and/or proteins as active antifungal agents in paints and other coating compositions, it is not necessary to understand the mechanism by which the desired antifungal effect is exerted on fungus cells.

Example 3. Varying the Amino Acid Sequence of Antifungal Peptides

[0036] For the purposes of preparing antifungal paints and other coating compositions containing antifungal peptidic agents, it should be appreciated that it is not necessary for the amino acid sequence of a peptide having demonstrable antifungal activity to be completely defined. In certain situations, especially where an antifungal peptide is being used to target an array of fungal genera or species, mixed peptide additives may be preferable. This is also likely to be the case where there is a desire to treat or prevent infestation by a particular species of fungus using lower concentrations of numerous antifungal peptides rather than a higher concentration of a single peptide. In other situations where, for instance, due to the increased cost of testing or producing a completely defined peptide antifungal peptide is prohibitive, the mixed peptide compositions having one or more variable , amino acid residues may be preferred.

. Similarly, It may be preferable to leave synthetic peptides of the invention blocked and/or covalently attached to the synthetic resin so Song as sufficient antifungal activity is 19

Patent provided by Sughrue Mion. PLLC - http://www.sughnje.com exhibited in the costing. Thus, antifungel additive compositions comprising equimolar mixtures of peptides produced in a synthetic peptide combinatorial library utilizing the methods described herein and/or in U.S. Patent No. 6,020,312, U.S. Patent No. 5, 885, 782, or U.S. Patent No. 5,602,097 may be employed as antifungal agents in paints, coatings and films.

[0037] The antifungal peptide additives for the coatings of the invention may be constructed using a variety of amino acid precursors. Of course, the peptides may be homogenous compositions containing only D-, L- or cyclic (non-racemic) amino acids. The chemical structure of such amino acids (which term is used herein to include imino acids), regardless of stereoisomeric configuration, may be based upon that of the nineteen or twenty naturally-occurring amino acids: alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartate (Asp; D), glutamine (Gin; Q), glutamate (Glu; E), glycine (Gly; G), histidine (His; H), isoleucine (He; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), proline (Pro; P), phenylalanine (Phe; F), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V). Cysteine (Cys; C) is preferably excluded to prevent disulfide linkage problems in the products. The compositions of the invention may also be non-homogenous, containing for instance both D-, L- and/or cyclic amino acids. The peptide compositions may also contain amino acids which are other than the naturally-occurring amino acids (e.g., norleucine), as are known to those of skill in the art. The peptides may also be constructed as retroinversopeptidomimetics of any of the peptides shown to be active in either the D- or L- configurations. It is known, for instance, that the retroinversopeptidomimatic of SEQ ID No. (41) is inhibitory (albeit less so than either the D- or L- configurations) against certain household fungi such as Fusarium and Aspezg/7/us (Guichard, 1994).

[0038] Preferred antifungal paints and coatings will comprise one or more of the peptides disclosed in SEQ ID Nos. 1-199, more preferably SEQ ID Nos. 1-47. These sequences establish a number of precise chemical compositions which have been shown to have antifungal activity against a spectrum of fungi, but which were not previously known to be useful for treating and/or protecting building materials and other non-living objects from infestation by fungi. A highly preferred antifungal peptide is the hexapeptide of SEQ ID

No. 41.

[0039] In certain instances, the peptides will have completely defined sequences. In other instances, the sequence of the antifungal peptide will be defined for only certain of the Cterminal amino acid residues leaving the remaining amino acid residues defined as equimolar ratios. For example, certain of the peptides of SEQ ID Nos. 1-199 have somewhat variable amino acid compositions. Thus, in each aliquot of the SPCL containing a given SEQ ID Nos. having a variable residue, the variable residues will each 20

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W0 2005M775S . Ρ&lt;™«־»״״*’.

be uniformly represented in equimolar amounts by one of nineteen different naturally occurring amino acids In one or the other stereoisomeric form. However, the variable residues may be rapidly defined using the method described in one or more of U.S.

Patent Nos. 6,020,312; 5,602, 097; and 5,885,782 to identify peptides that possess . activity for controlling fungal growth. In the cited patents it was demonstrated that peptides encompassed by the C-terminal sequence ״XXXXRF” (SEQ ID No. 1) exhibited antifungal activity for a wide spectrum of fungi. For ease of reference, peptides herein are written in the C-terminal to N-terminal direction to denote the sequence of synthesis. However, the conventional N־termlnal to C-terminal manner of reporting amino acid sequences is utilized in the Sequence Listings. This relatively variable composition, therefore, is described as an antifungal peptide even though it is likely that not every peptide encompassed by that general sequence will possess the same or any antifungal activity.

[0040] In the next round of identification of antifungal peptides encompassed by the general sequence ״XXXXRF״ (SEQ ID No. 1) parent composition of known antifungal activity, XXXLRF (SEQ ID No. 9) peptides mixtures were found to exhibit significant antibiotic activity (also disclosed in U.S. Patent Nos. 6,020,312; 5,602, 097; and 5,885,782). Similarly to the parent composition XXXXRF (SEQ ID No. 1), the XXXLRF (SEQ ID No. 9) peptides will have a mixed equimolar array of peptides representing the same nineteen amino acid residues, some of which may have antifungal activity and some of which may not have such activity. Overall, however, the XXXLRF (SEQ ID No. 9) peptide composition is itself an antifungal agent. This process is carried out to the point where completely defined peptides are produced and tested for their antifungal activity, as described in Example 2 or using any suitable method that would be known to one of skill In the art. As a result, and as was accomplished for the representative peptide FHLRF (SEQ ID No. 31), all amino acid residues in a six residue peptide will be known.

[0041] It will be recognized by those of skill in the art that the peptides to be employed as antifungal agents for paints, coatings arid other compositions, once selected, may be modified to contain functionally equivalent amino acid substitutions and yet retain the same or similar antifungal characteristics. The Importance of the hydropathic index of amino acids in conferring biological function on a protein has been discussed generally by Kyte and Doolittle, J. Mol. Biol., 157:105-132, 1982. It is well known that certain amino acids may be substituted for other amino acids having a similar hydropathic index or score and still retain similar if not identical biological activity. As displayed in Table 2 below, amino acids are assigned a hydropathic, index on the basis of their hydrophobicity and charge characteristics, it is believed that the relative hydropathic character of the amino acid determines the secondary structure of the resultant protein, which in turn defines the 21

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PCTAJS2004/021711 protein with the substrate molecule. Similarly, in peptides whose secondary structure is not a principal aspect of the Interaction of the peptide, positton within the peptide and the characteristic of the amino acid residue determine the interactions the peptide has In a biological system. It is proposed that biological functional equivalence may typically be maintained where amino acids having no more than a +/-1 to 2 difference in the index value, and more preferably within a +M difference, are exchanged.

TABLE 2

AMINO ACID Isoleucine

Valine Leucine Phenylalanine Cysteine/Cystine Methionine

Alanine Glycine Threonine Tryptophan

Serine Tyrosine

Proline Histidine

Glutamic Acid Glutamine

Aspartic Acid Asparagine

Lysine Arginine hydropathic index

4.5

4.2

3.8

2.8

2.5

1.9

1.8 .4 .7 .9 .8 .3 .6 .2 .5 .5 .5 .5 .9 .5

[0042] Thus, it is expected that isoleucine, for example, which has a hydropathic index of +4.5, can be substituted for valine (+4.2) or leucine (+3.8), and still obtain a protein having similar biologic activity. Alternatively, at the other end of the scale, lysine (-3.9) can be substituted for arginine (-4.5), and so on. Accordingly, these amino acid substitutions are generally based on the relative similarity of R-group substituents, for example, in terms of size, electrophilic character, charge, and the like. In general, although these are not the only such substitutions, the preferred substitutions which take various of the foregoing characteristics into consideration include the following:

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PCT/US2004/02I711

TABLES

<td> Originally</td><td> Exemplary</td>

<td> Residue</td><td> Substitutions</td>

<td> alanine</td><td> gly; ser</td>

<td> arginine</td><td> lys</td>

<td> asparagines</td><td> gin; his</td>

<td> aspartate</td><td> glu</td>

<td> cysteine</td><td> ser</td>

<td> glutamate</td><td> asp</td>

<td> glutamine</td><td> asn</td>

<td> glycine</td><td> ala</td>

<td> histidine</td><td> asn; gin</td>

<td> isoleucine</td><td> leu; val</td>

<td> leucine</td><td> ile; val</td>

<td> lysine</td><td> arg; gin; glu</td>

<td> methionine</td><td> met; leu; tyr</td>

<td> serine</td><td> thr</td>

<td> threonine</td><td> ser</td>

<td> tryptophan</td><td> tyr</td>

<td> tyrosine</td><td> trp; phe</td>

<td> vaiine</td><td> Ile; leu</td>

Example 4. Stabilized Antifungal Peptide Compositions

[0043] A variety of modifications can be made to the peptides as long as antifungal activity is retained. Some modifications may be used to increase the intrinsic antifungal potency of the peptide. Other modifications may facilitate handling of the peptide. Peptide functional groups that may typically be modified include hydroxyl, amino, guanidinlum, carboxyl, amide, phenol, imidazol rings or sulfhydryl. Typical reactions of these groups include but are not limited to acetylation of hydroxyl groups by alkyl halides. Carboxyl groups may be esterified, amidated or reduced to alcohols. Carbodiimides or other catalysts may be used to catalyze the amidation of carboxyl groups. The amide groups of asparagine or glutamine may be deamidated under acidic or basic conditions. Acylation, alkylation, arylatlon or amidation reactions readily occur with amino groups such as the primary amino group of the peptide or the amino group of lysine residues. The phenolic group of tyrosine can be halogenated or nitrated. Examples where solubility of a peptide

Patent provided by Sughrue Mion, PLLC - http://www.sughrue.com could be decreased include acylating charged lysine residues or acetylating the carboxyl groups of aspartic and glutamic acids. Techniques and materials that are suitable for carrying out each of these modifications are well known in the art and have been described in the literature.

[0044] Another way in which the antifungal activity of the peptides may be stabilized in paints and other coatings and compositions is by linking or conjugation to another molecule. Peptides may be conjugated to soluble or insoluble carrier molecules to modify their solubility properties as needed. Examples of soluble carrier molecules include polymers of polyethyleneglycol and polyvinylpyrrolidone. Alternatively, a peptide may be chemically linked or tethered to an insoluble molecule. Examples of insoluble polymers include sand or other silicates, and polystyrene, cellulose and polyvinylchloride. Such polymers are often employed in coatings. The molecular size of the conjugated polymer chosen for conjugating with an antifungal peptide is preferably suited for carrying out the desired additional function in the coating. Techniques and materials for conjugating peptides to other molecules are well known in the art and have been described in the literature.

[0045] Still another way in which the antifungal activity may be controlled or stabilized is by microencapsulating the peptides to enhance their stability in liquid coating compositions and in the final paint film or coat. For example, polyester microspheres may be used to encapsulate and stabilize the peptides in a paint composition during storage, or to provide for prolonged, gradual release of the peptide after it is dispersed in a paint film covering a surface that is vulnerable to attachment and growth of fungal cells or spores. Any suitable microencapsulation technique as would be known to one of ordinary skill in the art may be employed. Such encapsulation may enhance, or confer a particulate nature to, one or more antifungal peptide. The encapsulating membrane may provide protection to the peptide from peptidases, proteases, and other peptide bond or side chain modifying substances, it may serve to increase the average particle size of the antifungal peptidic agent to a desired range, and it may allow controlled release of the peptide(s) from the encapsulating material, alter surface charge, hydrophobicity, hydrophilicity, solubility and/or dispersability of the particulate material, or any combination of those functions. Examples of microencapsulation (e.g., microsphere) compositions and techniques are described in Wang, Η. T. et al., J. of Controlled Release 17.23-25, 1991, and U.S. Patent Nos. 4,324,683; 4,839,046; 4,988,623; 5,026,650; 5,153,131; 6,485,983; 5,627,021 and 6,020,312). Other microencapsulation methods which may be employed are those described in U.S. Patent Nos. 5,827,531; 6,103,271; and 6,387,399.

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Example 5. Large-scale Production of Antifungal Peptides

[0046] An antifungal peptide sequence identified as described above may be grown in bacterial, insect, or other suitable cells employing techniques and materials that are well known In the art, except DNA encoding the antifungal peptides described herein will be used instead of a previous DNA sequence. For example, an expression vector will include a DNA sequence encoding SEQ ID No. 1 in the correct orientation and reading frame with respect to the promoter sequence to allow translation of the DNA encoding the SEQ ID No. 1. Examples of the cloning and expression of an exemplary gene and DNAs are known. Either batch culture production methods or continuous fed-batch culture methods may be employed to produce commercial-scale quantities of antifungal peptides.

Example 6. Antifungal Additives Isolated from Microorganisms

[004η Although synthetically obtained antifungal peptidic agents {i.e., peptides, polypeptides and proteins) that are identified and produced as described above are highly preferred, it is also possible to employ suitable naturally occurring antifungal peptidic agents, and microbes that produce such agents, as additives in paints and other coatings. A number of such naturally occurring peptide additives are listed in Table 1. A drawback to this is the time-consuming process of searching for naturally produced antifungal agents with very low-probability of success. The use of natural antifungal products isolated in commercial quantity from microorganisms is also limited in usefulness due in large part to purification problems. Large-scale cell culture of the antifungal agentproducing microorganism is required for the purification of the antifungal product. In many instances, the cultural isolate responsible for the production of the antifungal agent is not an isolate which is easily batch-cultured or it is entirely incapable of batch culturing. Furthermore, complicated purification strategies are often required to purify the active product to a reasonable level of homogeneity. A substantial disadvantage to the use of naturally derived antifungal agents is the potential for co-purification of unwanted microbial byproducts, especially byproducts which are undesirably toxic. In many cases, these factors lead to high production costs and make large-scale isolation of antifungal products from natural isolates impractical. Purifications may be even more difficult where racemized mixtures are possible where only a single stereoisomer is active, or where disulfide linkages are possible between peptide monomers. Even when desirable naturally occurring antifungal proteins or polypeptides are isolated, for example, and their amino acid sequences at least partially identified, synthesis of the native molecule, or portions thereof, may be problematic due to the need for specific disulfide bond formation, high

Patent provided by Sughrue Mion, PLLC - http://www.sughrue.com histidine requirements, and so forth. Nonetheless, natural sources provide additional sequences to be explored as coating additives.

Example 7. Coating Formulations Containing Antifungal Peptide Additives

[00481 One or more of the antifungal peptides or peptide compositions, prepared as described in any of the foregoing examples, is mixed with a base paint or other coating, which may be any suitable commercially available product, a wide variety of which are well known in the art. Preferably the base composition is free of chemicals and other additives that are toxic to humans or animals, and/or that fail to comply with applicable environmental safety rules or guidelines. In some instances, it may be preferred to custom blend a paint or coating mixture using any combination of various naturallyoccurring and synthetic components and additives that are known in the art and are also described in U.S. 2004-0175407 A1 or U.S. 2004-0109853 A1.

s

[0049] Coating components generally include a binder, a liquid component, a colorizing agent, □ne or more additive, or a combination of any of those. A coating typically comprises a material often referred to as a “binder, which is the primary material in a coating capable of producing a film. In most embodiments, a coating will comprise a liquid component (e.g., a solvent, a diluent, a thinner), which often confers and/or alters the coating’s rheological properties (e.g., viscosity) to ease the application of the coating to a surface. Usually a coating (e.g., a paint) will comprise a colorizing agent (e.g., a pigment), which usually functions to alter an optical property of a coating and/or film. A coating will often comprise an additive, which is a composition incorporated into a coating to (a) reduce and/or prevent the development of a physical, chemical, and/or aesthetic defect in the coating and/or film; (b) confer some additional desired property to a coating and/or film; or (c) a combination thereof. Examples of an additive include an accelerator, an adhesion promoter, an antifloating agent, an antiflooding agent, an antifoaming agent, an antioxidant, an antiskinning agent, a buffer, a catalyst, a coalescing agent, a corrosion Inhibitor, a defoamer, a dehydrator, a dispersant, a drier, an electrical additive, an emulsifier, a film-formation promoter, a fire retardant, a flow control agent, a gloss aid, a leveling agent, a light stabilizer, a marproofing agent, a matting agent, a neutralizing agent, a preservative, a rheology modifier, a slip agent, a viscosity control agent, a wetting agent, or a combination thereof. The content for an individual coating additive in a coating generally Is 0.0001% to 20.0%, including all intermediate ranges and combinations thereof. However, In many Instances it is preferred if the concentration of a single additive

WO 2005/007758 PCT7US2004/021711 in a coating comprises between 0.0001% and 10.0%, including all intermediate ranges and combinations thereof.

[0050] Some of the usual types of components of paints and coatings are summarized as follows:

[0051] Binders: oil-based (e.g., oils, alkyd resins, oleoresinous binders, and fatty acid epoxy esters; polyester resins; modified cellulose; polyamide; amidoamine; amino resins; urethanes; phenolic resins; epoxy resins; polyhydroxyether; acrylic resins; polyvinyl binders; rubber resins; bituminous; polysulfide and silicone.

[0052] Liquid Components: solvents; thinners; diluents; plasticizers; and water (e.g., hydrocarbons; oxygenated solvents; chlorinated hydrocarbons, nitrated hydrocarbons, other organic liquids)

[0053] Colorants: pigments and dyes.

[00541 Additives: preservatives (e.g., blocides/bactericides/fungicides/algaecides); wetting agents; buffers (e.g., ammonium bicarbonate, both monobasic and dibasicphosphate buffers, Trizma base and zwitterionic buffers); rheology modifiers; defoamers; catalysts (e.g., driers, acids, bases, urethane catalysts); antiskinning agents; light stabilizers; corrosion inhibitors; dehydrators; electrical additives; and anti-insect additives.

[0055] Preservatives serve to reduce or prevent the deterioration of a coating and/or film by a microorganism, by acting as a biocide, which kills an organism, a biostatic, which reduces or prevents the growth of an organism, or a combination of effects. Examples of a biocide include, for example, a bactericide, a fungicide, an algaecide, or a combination thereof.

[0056] A preferred paint or coating composition contains, as a preservative, an antifungal peptidic agent (i.e., one or more peptides, polypeptides or proteins), according to any of Examples 1-6. An antifungal peptidic agent may be used as a partial or complete substitute (״replacement) for another fungicide and/or fungistatic that is typically used in a fungus prone composition. It is contemplated that 0.0001% to 100%, including all intermediate ranges and combinations thereof, of a conventional antifungal component in a coating formulation may be substituted by an antifungal peptidic agent. In some formulations, the concentration of antifungal peptidic agent may exceed 100%, by weight or volume, of the non-peptidic antifungal component (fungicide or fungistat) that is being replaced. A conventional non-peptidic antifungal component may be replaced with an antifungal peptidic agent equivalent to 0.001% to 500% (by weight, or by volume), including all intermediate ranges and combinations thereof, of the substituted antifungal component. For example, to produce a coating with similar fungal resistance .properties as a non-substituted formulation, it mav require that 20% (e.g., 0.2 kg) of a chemical 27

Patent provided by Sughrue Mion, PLLC . http://www.s11ghrije.com fungicide may be replaced by 10% (e.g., 0.1 kg) of an antifungal peptidic agent. In another exemplary formulation, to produce a coating with similar fungal resistance as a non-substituted formulation, it may require replacing 70% of a chemical fungicide (e.g., 0.7 kg) with the equivalent of 127% (e.g., 1.27 kg) of antifungal peptidic agent. The various assays described herein, or as would be known to one of ordinary skill in the art in light of the present disclosure, may be used to determine the fungal resistance properties of a composition (e.g., a coating, a film) produced by direct addition of an antifungal peptidic agent and/or substitution of some or all of a non-peptidic or chemical antifungal component by an antifungal peptidic agent. Such additives may be directly admixed with the coating, applied as a primer coating, applied as an overcoat, or any combination of these application techniques.

[005η A preservative may comprise an in-can preservative, an in-film preservative, or a combination thereof. An in-can preservative is a composition that reduces or prevents the growth of a microorganism prior to film formation. Addition of an in-can preservative during a water-borne coating production typically occurs with the introduction of water to a coating composition. Typically, an In-can preservative is added to a coating composition for function during coating preparation, storage, or a combination thereof. An in-film preservative is a composition that reduces or prevents the growth of a microorganism after film formation. Oftentimes an in-film preservative is the same chemical as an in-can preservative, but added to a coating composition at a higher (e.g., two-fold) concentration for continuing activity after film formation.

[0058] Examples of preservatives that have been used in coatings include a metal compound (e.g., an organo-metal compound) biocide, an organic biocide, or a combination thereof. Examples of a metal compound biocide include barium metaborate (CAS No. 13701-59-2), which is a fungicide and bactericide; copper (II) 8-quinolinolate (CAS No. 10380-28-6), which is a fungicide; phenylmercuric acetate (CAS No. 62-38-4), tributyltin oxide (CAS No. 56-35-9), which is less preferred for use against Gram-negative bacteria; tributyltin benzoate (CAS No. 4342-36-3), which is a fungicide and bactericide; tributyltin salicylate (CAS No. 4342-30-7), which is a fungicide; zinc 2-pyridinethiol-Noxide (CAS No. 13463-41-7), which is a fungicide; zinc oxide (CAS No. 1314-13-2), which is a fungistatic/fungicide and algaecide; a combination of zinc-dimethyldithiocarbamate (CAS No. 137-30-4) and zinc 2-mercaptobenzothiazole (CAS No. 155-04-4), which acts as a fungicide; zinc 2-pyridinethio!-N-oxide (CAS No. 13463-41-7), which is a fungicide; a metal soap; or a combination thereof. Examples of metals comprised in a metal soap biocide include copper, mercury, tin, zinc, or a combination thereof. Examples of an organic acid comprised in a metal soap biocide include a butyl oxide, a laurate, a naphthenate, an octoate, a phenyi acetate, a phenyl oleate, or a combination thereof. It is 28

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WO2W0775S PCT/US2004«״״ anticipated that they peptide additives of the present invention will work in combination with or synergistically with such preservative.

[0059] An example of an organic biocide that acts as an algaecide includes 2-methylthio-

4- tert-butylamino-6-cyclopropylamino-s-triazine (CAS No. 28159-98-0). Examples of an organic biocide that acts as a bactericide include a combination of 4,4-dimethyloxazoiidine (CAS No. 51200-87-4) and 3,4,4-trimethyloxazolidine (CAS No. 75673-43-7);

5- hydroxy-methyl-l-aza-SJ-dloxablcylco (3.3.0.) octane (CAS No. 59720-42-2);

2(hydroxymethyl )-aminoethanol (CAS No. 34375-28-5); 2-(hydroxymethyl)-amino-2methyl-1-propanol (CAS No. 52299-20-4); hexahydro-1,3,5-triethyl-s-triazine (CAS No. 108-74-7); 1-(3-chloroallyl)-3,5,7-triaza-1-azonia-adamantane chloride (CAS No. 5122978-8); 1-methyl3,5,7־-triaza-1-azonia-adamantane chloride (CAS No. 76902-90-4), pchloro-m-cresol (CAS No. 59-50-7); an alkylamine hydrochloride; 6-acetoxy-2,4-dimethyl1,3-dioxane (CAS No. 828-00-2); 5-chloro-2-methyl-4-lsothiazolin-3-one (CAS No. 2617255-4); 2-methyl-4-isothiazolin-3-one (CAS No. 2682-20-4); 1,3-bis(hydroxymethyl)-5,5dimethylhydantoin (CAS No. 6440-58-0); hydroxymethyl-5,5-dimethylhydantoin (CAS No. 27636-82-4); or a combination thereof. Examples of an organic biocide that acts as a fungicide include a parabens; 2-(4-thiazolyl)benzimidazole (CAS No. 148-79-8); Ntrichloromethyl-thio-4־cyclohexene-1,2-dicarboxlmide (CAS No. 133-06-2); 2-n-octyl-4isothiazoline-3-one (CAS No. 26530-20-1); 2.4,5,6-tetrachloro-isophthalonitrile (CAS No. 1897-45-6); 3-iodo-2-propynyl butyl carbamate (Cas No. 55406-53-6); N- (trichloromethylthio)phthalimide (Cas No. 133-07-3); tetrachloroisophthalonitrile (Cas No. 1897-45-6); potassium N-hydroxy-methyl-N-methyl-dithiocarbamate (Cas No. 51026-28-9); sodium 2pyridinethiol-1-oxide (Cas No. 15922-78-8); or a combination thereof. Examples of a parbens include butyl parahydroxybenzoate (Cas No. 94-26-8); ethyl parahydroxybenzoate (Cas No. 120-47-8); methyl parahydroxybenzoate (Cas No. 99-763); propyl parahydroxybenzoate (Cas No. 94-13-3); or a combination thereof. Examples of an organic biocide that acts as an bactericide and fungicide include 2-mercaptobenzothiazole (Cas No. 149-30-4); a combination of 5-chloro-2-methyl-3(2H)-isothiazoline (Cas No. 26172-55-4) and 2-methyl-3(2H)־isothiazolone (Cas No. 2682-20-4); a combination of 4-(2-nitrobutyi)-morpholine (Cas No. 2224-44-4) and 4,42)-׳-ethylnitrotrimethylene dimorpholine (Cas No. 1854-23-5); tetra-hydro-3,5-di-methyl-2H-1,3,5-thladiazine-2-thione (Cas No. 533-74-4); potassium dimethyldithiocarbamate (Cas No. 128-03-0); or a combination thereof. An example □f an organic biocide that acts as an algaecide and fungicide includes diiodomethyl-p-tolysulfone (Cas No. 20018-09-1). Examples of an organic biocide that acts as an algaecide, bactericide and fungicide include glutaraldehyde (CAS No. 111-30-8); methylenebis(thiocyanate) (Cas No. 6317-18-6); 1,2dibromo-2,4-dicyanobutane (CAS No. 35691-65-7); 1,2-benzlsothiazoline-3-one (CAS No.

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2634-33-5); 2-(thiocyanomethyl-thio)benzothiazole (CAS No. 21564-17-0), or a combination thereof. An example of an organic biocide that acts as an algaecide, bactericide, fungicide and molluskicide includes 2-(thiocyanomethyl-thio)benzothiozole (CAS No. 21564-17-0) and methylene bis(thiocyanate) (CAS No. 6317-18-6).

[0060! In certain situations of use, an applicable environmental law or regulation may encourage the selection of an organic biocide such as a benzisothlazolmone derivative. An example of a benzisothiazolinone derivative is Busan™ 1264 (Buckman Laboratories, Inc.), Proxel™ GXL (Avecia Inc.), or Preventol® VP OC 3068 (Bayer Corporation), which comprises 1,2-benzisothiazoiinone (CAS No. 2634-33-5). In the case of Busan™ 1264, the primary use is a bactericide and/or fungicide at 0.03% to 0.5% in a water-borne coating.

[0061] Often, a preservative is a proprietary commercial formulation and/or a compound sold under a tradename. Examples include organic biocides under the tradename Nuosept® (international Specialty Products), which are typically used in a water-borne coating. Specific examples of a Nuosept® biocide includes Nuosept® 95, which comprises a mixture of bicyclic oxazolidines, and is typically added to 0.2% to 0.3% concentration to a coating composition; Nuosept® 145, which comprises an amine reaction product, and is typically added to 0.2% to 0.3% concentration to a coating composition; Nuosept® 166, which comprises 4,4-dimethyloxazolidine (CAS No. 5120087-4), and Is typically added to 0.2% to 0.3% concentration to a basic pH water-borne coating composition; or a combination thereof. A further example is Nuoclde® (International Specialty Products) biocides, which are typically used fungicides and/or algaecides. Examples of a Nuocide® biocide is Nuocide® 960, which comprises 96% tetrachlorisophthalonitrile (CAS No. 1897-45-6), and is typically used at 0.5% to 1.2% in a water-borne or solvent-borne coating as a fungicide; Nuocide® 2010, which comprises chlorothalonil (CAS No. 1897-45-6) and IPBC (CAS No. 55406-53-6) at 30%, and is typically used at 0.5% to 2.5% in a coating as a fungicide and algaecide; Nuocide® 1051 and Nuocide® 1071, each which comprises 96% N-cyclopropyl-N-(1-dimethylethyl)-6(methylthio)-1,3,5-triazine2,4־-diamine (CAS No. 28159-98-0), and is typically used as an algaecide in antifouling coatings at 1.0% to 6.0% or water-based coatings at 0.05% to 0.2%, respectively; and Nuocide® 2002, which comprises chlorothalonil (CAS No. 189745-6) and a triazine compound at 30%, and is typically used at 0.5% to 2.5% in a coating and/or a film as a fungicide and algaecide.

[0062] An additional example of a tradename biocide for coatings includes Vancide® (R.

T. Vanderbilt Company, Inc.). Examples of a Vancide® biocide include Vancide® TH, which comprises hexahydro-1,3,5-triethyl-s-triazine (CAS No. 108-74-7), and is generally used in a water-borne coating; Vancide® 89, which comprises N-trichloromethylthio-430

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PCT/US2004/021711 cTdohexene-H-dicarboxImide (CAS No. 133-06-2) and related compounds such as captan (CAS No. 133-06-2), and is used as a fungicide In a coating composition; or a combination thereof. A bactericide and/or fungicide for coatings, particularly^ waterborne coating, Is a Dowicil™ (Dow Chemical Company). Examples of a DowiCil biocide include Dowicil™ QK-20, which comprises 2,2-dlbromo-3-nitrilopropionamlde (CAS No. 10222-01-2), and is used as a bactericide at 100 ppm to 2000 ppm in a coating; Dowtal™ 75 which comprises 1-(3-chloroallyl)-3,5,7-triaza-1-azoniaadamantane chloride (CAS No. 51229-78-8). and Is used as a bactericide at 500 ppm to 1500 ppm in a coating; Dowicil™ 96 which comprises 7-ethyl bicydooxazolidine (CAS No. 7747-35-5), and is used as a bactericide at 1000 ppm to 2500 ppm in a coating; Bioban™ CS-1135, which comprises 4 4-dimethyioxazolldine (CAS No. 51200-87-4), and is used as a bactencide at 100 ppm to 500 ppm in a coating; or a combination thereof. An additional example of a tradename biodde for coatings Indudes Kathon® (Rohm and Haas Company). An example of a Kathon® biocide Includes Kathon® LX, which typically comprises 5-chloro-2-methyl-4jsofhlazolin-3-one (CAS no 26172-55-4) and 2-methyM-isothlazolin-3-one (CAS no 268220-4) at 1 5%, and is added from 0.05% to 0.15% In a coating. Examples of tradename fungicides and algaeddes include those described for Fungitrd® (International Specialty Products) which are often formulated for solvent-bome and water-borne coatings, and incan and film preservation. An example is Fungitrol® 158, which comprises 15% tnbutyltm benzoate (CAS No. 4342-36-3) (15%) and 21.2% alkylamine hydrochlorides, and is typically used at 0.35% to 0.75% in a water-borne coating for In-can and film preservation. An additional example is Fungitrol® 11. which comprises N-(trichloromethylthio) phthalimide (CAS No. 133-07-3). and is typically used at 0.5% to 1.0% as a fungicide for solvent-bome coating. A further example is Fungitrol® 400, which comprises 98% 3-iodo2-prcpynl N-butyl carbamate CIPBC־) (Cas No. 55406-53-6), and is typically used at 0.15% to 0.45% as a fungicide for a water-borne or a solvent-bome coating. See Table 4.

TABLE 4

<td> Company________________ Arch Chemicals, Inc. 800.344.9168/Fax:</td><td> Product Roster__——-----— Zinc Omadine (Zinc Pyrithione/fungicide/algaecide) Sodium Omadine (Sodium Pyrlthione/fungicide־׳</td>

<td> 203.271.4060 E-mail: sales@archbioddes.com. www.archbiocldes.com</td><td> algaecide) Copper Omadine (Copper Pyrithione/algaecide) Triadine 174 (Triazine/bactericide) Omaclde IPBC (lodopropynyl-butyl carbomate/fungiclde) Other: antifouling agents--</td>

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PC17US2004/021711

<td> Company</td><td> Product Roster</td>

<td> Avecia Protection &amp; Hygiene Wilmington, DE 800.523.7391/Fax: 302.477.8120 E-mail: biocides@avecia.com www.avecia.com/biocldes</td><td> Proxel GXL (BIT) Proxel BDZO (BIT) Proxel BZ (BIT/ZPT) Proxel TN (BIT/Triazine) Proxel XL2 (BIT) Densil C404 (Chlorthalonil) Densil P (Densil P) Densil DN (BUBIT) Vantocil IB (PHMB)</td>

<td> BASF Corp. Mount Olive, NJ 973.426.4358/Fax: 973.426.3863 www.biocides.basf-corp.com</td><td> Myacide AS Technical (Bronopol, solid) Myacide AS 2, 30 and 15 (Bronopol, solutions) Myacide GDA Technical (50% Glutaraldehyde) Myacide GA 42, 26 &amp; 15 (Glutaraldehyde, solut.) Protectol PE (Phenoxyethanol, liquid) Daomet Technical (Dazoment, solid) Myacide HT Technical (Triazine, liquid)</td>

<td> Buckman Laboratories, Inc. Memphis, TN E-mail: knetix@buckman.com</td><td> Busan (wood preservative/pkg. pres.) Butrol (corrosion inhibitor/rust Inhibitor) Busan (bactericide; mold inhibitor and biocide)</td>

<td> Cognis Corp. Ambler, PA 800.445.2207/Fax: 215.628.1111 E-mail: shruti.sinahal@cognis.com www.coanis.com</td><td> Nopcoclde N400 (Cholorthalonil-40% solution) Nopcocide N-98 (Chlorothalonil-100%) Nopcoclde P-20 (IPBC-20% solution) Nopcoclde P-40 (IPBC-40% solution) Nopcocide P-100 (IPBC-100% active)</td>

<td> International Specialty Products aka ISP Wayne, NJ 800.622.4423/Fax: 973.628.4001 E-mail: info@isDcorp.com www.ispcorD.com</td><td> Fungitrol (fungicides) Biotrend (biocides) Nuocide (fungicides/algaecides) Nuosept (antimicrobial agents)</td>

<td> Rohm and Haas Company Philadelphia, PA www.rohmhaas.com</td><td> Katon LX 1.5% (preservative) Rocima 550 (preservative) Rocima 607 (preservative) Rozone 2000 (dry film fungicide) Skane M-8 (dry film fungicide)</td>

<td> Troy Corp. Florham Park, NJ Fax: 973.443.0843 E-mail: marketinq@trovcorp.com www.trovcorD.com</td><td> Polyphase 678 Polyphase 663 Polyphase OST Polyphase 641 Troysan 680 Mergal K10N</td>

[0063] As would be known to one of ordinary skill in the art, determination of whether damage to a coating and/or film is due to microorganisms (e.g., film algal defacement, film fungal defacement), as well as the efficacy of addition of a preservative to a coating and/or film composition in reducing microbial damage to a coating and/or film, may be

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ZiriZdXrmined by techniques ־״־b as those that are described in ־ASTM Book of Standards Volume 06.01, Paint - Tests for Chemical, Physical, and Optical Properties Appearance,. D3274-95, 04610-08, □2574-00, □327340. D3456-86, □5589-97 and D5590-00. 2002; and in ־Paint and Coating Testing Manual, Fourteenth Edit,on of the Gardner-Sward Handbook,. (Koleske, J. V. Ed.), pp. 654-661. 1995. Examples 0 microorganisms typically selected In such procedures as positive controls of a coa ,״g and/or fiim damaging microorganism indude, for exampie, Asperg,^ (ATCC 10196) Aspergillus fleeus (ATCC 9643). Aspers ״/ger (ATCC 9642), Pseudomonas aerog/nosa (ATCC 10145), Auroobasid/um P״M־״־ (ATCC 9348). Pe״«_U״r ״״4־™ (ATCC 9849), Penicillium funlculosum (ATCC 9644), or a combination thereof.

[00641 in general, a preserve, and use of a preservative in a coating, is known to those of skill in the art, and all such materials and techniques for using a preservative ,־ ״ coating may be applied In the practice of the present invention (see, for example, Flick, . W .Handbook of Paint Raw Materials, Second Edition,263-285 ־ and 879-998. 1989; in “Paint and Coating Testing Manual, Fourteenth Edition of the Gardner-Sward Handboo . (Koleske, J. V. Ed.), pp 261-267 and 664-661, 1995; in “Paint and Surface Coatings. Theory and Practice. Second Edition.־ (Lamboume, R. and Striven־, T. A״ Eds.), pp. 193194 371-382 and 543-547,1999; Wicks, Jr. Z. W. Jonas, F. N״ Pappas, S. P. Organic Coatings, Science and Technology, Volume 1: Film Fonnatio״. Components, and Appearance,” pp. 318-320,1992; Wicks. Jr״ Z. W״ Jones. F. N״ Pappas. S. P. Organic Coatings, Science and Technology. Volume 2; Applications. Properties and Performance PO 145 309 319-323 and 340-341, 1992; In “Paints, Coatings and Solvents, Seoon , Completely Revised Edition,“ (Stoye, D. and Freitag, W. Eds.) pp 6.127 and 165.1998; and in “Handbook of Coatings Additives', pp. 177-224,1987).

roossi It is contemplated that any previously described formulation of a fungal-prone composition may be modified to Incorporate an antifungal peptldio agent. Examples of described coating compositions include over 200 industrial water-borne coating formulation־ (e.g., air dry coatings, air dry or force air dry coatings, anti-skid of non-slip coating־, bake dry coatings, dear coatings, coil coatings, concrete coatings, dipping enamels, lacquers, primers, protective coatings, spray enamels, traffic and airfte coatings) described in “Industrial water-based paint formulations, 1988, over 550 architectural water-borne coating formulations (e.g. exterior paints, exterior ename ־, exterior coatings, Interior paints, interior enamels, interior coatings, extenor/intenor pain s, exterior/interlor enamels, exterlor/lnterior primers, exterlor/lntedor stains), descnbed m Water-based trade paint formulations.“ 1988, the over 400 solvent borne coating formulations (e.g. exterior paints, exterior enamels, exterior coatings, exterior sealers, exterior ״Ilers, exterior primers, interior paints, interior enamels, interior coatings, interior

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WO 2005/007758 PCI7US2004/Q21711 primers, exterior/interior paints, exterior/interior enamels, exterior/interior coatings, exterior/interior varnishes) described in “Solvent-based paint formulations, 1977, and the over 1500 prepaint specialties and/or surface tolerant coatings (e.g., fillers, sealers, rust preventives, galvanizers, caulks, grouts, glazes, phosphatizers, corrosion inhibitors, neutralizers, graffiti removers, floor surfacers) described in Prepaint Specialties and Surface Tolerant Coatings, by Ernest W. Flick, Noyes Publications, 1991.

[0066] An exemplary exterior gloss alkyd house paint that comprises an antifungal peptidlc agent is as follows in Table 5:

TABLES

Component

Grind:

first alkyd second alkyd aliphatic solvent: duodecane lecithin

TiO₂ micron silica bentonite clay second alkyd first alkyd antifungal peptidic agent > optionally, in combination with a conventional mildewcide

Letdown:

aliphatic solvent: dudecane) first drier: 12% solution cobalt) second drier: 18% solution Zr) third drier: 10% solution Ca) Anti skinning agent:

methyl ethyl ketoxime Aliphatic solvent

Weight or Volume

232.02 lb or 29.9 gallons

154.2 lb or 20 gallons

69.55 lb or 1.7 gallons

7.8 lb or 0.91 gallons

185.25 lb or 5.43 gallons

59.59 lb or 2.7 gallons

18.00 lb or 1.44 gallons

97.22 lb or 12.61 gallons

69.84 lb or 9.00 gallons effective amount/ up to

7.8 lb or 0.82 gallons

19.50 lb or 3.00 gallons 2.00 lb or 0.23 gallons 2.92 lb or 0.32 gallons 8.00 lb or 0.98 gallons

3.22 lb or 0.42 gallons 9.75 lb or 1.50 gallons

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[006η A preferred exterior flat latex house paint comprising an antifungal peptidic agent will contain the following components, listed in typical order of addition in Table 6:

TABLE 6

Component water hydroxyethyl cel lul ose glycols polyacrylate dispersant Antifungal Peptidic Agent optionally, other biocide(s) non-ionic surfactant titanium dioxide silicate mineral calcined clay acrylic latex, @ 60% coalescent defoamers ammonium hydroxide 2.5% HEC solution antifungal peptidic agent

Weight or Volume

244.5 lb or 29.47 gallons 3 lb or 0.34 gallons 60 lb or 6.72 gallons 6.8 lb or 0.69 gallons effective amount up to lb or 1 gallons lb or 0.11 gallons 225 lb or 6.75 gallons 160 lb or 7.38 gallons 50 lb or 2.28 gallons

302.9 lb or 34.42 gallons

9.3 lb or 1.17 gallons lb or 0.26 gallons

2.2 lb or 0.29 gallons 76 lb or 9.12 gallons 1.8 lb or 0.82 gallons

[0068! From these representative formulations, it will be readily appreciated that a wide variety of paints and other coating compositions may be improved by addition of an antifungal peptidic agent. Some of these include industrial water-borne coating formulations (e.g., air dry coatings, air dry or force air dry coatings, anti-skid of non-slip coatings, bake dry coatings, clear coatings, coil coatings, concrete coatings, dipping enamels, lacquers, primers, protective coatings, spray enamels, traffic and airfield coatings); architectural water-borne coating formulations (e.g., exterior paints, exterior enamels, exterior coatings, interior paints, interior enamels, interior coatings, exterior/interior paints, exterior/interior enamels, exterior/interior primers, and exterior/interior stains); solvent borne coating formulations (e.g., exterior paints, exterior enamels, exterior coatings, exterior sealers, exterior fillers, exterior primers, interior paints, interior enamels, interior coatings, interior primers, exterior/interior paints, exterior/interior enamels, exterior/interior coatings, and exterior/interior varnishes); and prepaint specialties and/or surface tolerant coatings (e.g., fillers, sealers, rust preventives, 35

Patent provided by Sughrue Mion, PLLC http://www.sughrue.com galvanizers, caulks, grouts, glazes, phosphatizers, corrosion inhibitors, neutralizers, graffiti removers and floor surfacers).

[00691 An antifungal paint or coating containing an antifungal peptidic agent may then be tested and used as described elsewhere herein, or the product may be employed for any other suitable purpose as would be recognized by one of skill In the art in light of this disclosure. For instance, the physical properties (e.g., purity, density, solubility, volume solids and/or specific gravity, rheology, viscometry, and particle size) of the resulting antifungal liquid paint or other coating product, can be assessed using standard techniques that are known in the art and/or as described in Paint and Coating Testing Manual, 14^th ed. of the Gardner-Sward Handbook, J.V. Koleske, Editor (1995), American Society for Testing and Materials (ASTM), Ann Arbor, Ml, and applicable published ASTM test methods. Alternatively, any other suitable testing method as would be known to one of ordinary skill in the art in light of the present disclosures, may be employed for assessing physical properties of the paint or coating mixture containing an abovedescribed antifungal peptide additive.

Example 8. Inhibition of In-Can Mold Growth by Antifungal Peptides

[0070] As mentioned in the background discussion, the quality of a liquid coating mixture may suffer markedly if microorganisms degrade one or more of the components during storage. Since many of the coating products in use today contain Ingredients that make it susceptible or prone to fungal infestation and growth, it is common practice to include a preservative. Although bacterial contamination may be a contributing factor, fungi are typically a primary cause of deterioration of a liquid paint or coating. Foul odor, discoloration, thinning and clumping of the product, and other signs of deterioration of components render the product commercially unattractive and/or unsatisfactory for the intended purpose. If the container will be opened and closed a number of times after its initial use, in some instances over a period of several months or years, it will inevitably be inoculated with ambient fungus organisms or spores subsequent to purchase by the consumer.

[0071] To avoid spoilage, it is especially desirable to ensure that the product will remain stable and usable for the foreseeable duration of storage and use by enhancing the longterm antifungal properties of the paint or coating with an antifungal peptide agent. The incan stability and prospective shelf life of a paint or coating mixture containing an abovedescribed antifungal peptide agent may be assessed using any appropriate testing method as would be known to one of skill In the art using conventional microbiological techniques. A fungus known to infect paints or other coatings is preferably employed as the test organism.

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Example 9. Testing Protocols for Evaluating Antifungal Coatings

[0072] One suitable assay protocol for evaluating coatings containing an antifungal peptide is described by the American Society for Testing and Materials (ASTM) in D5590-94 (Standard Test Method for Determining the Resistance of Paint Films and Related Coatings to Fungal Defacement by Accelerated Four-Week Agar Plate Assay), which is hereby incorporated herein by reference. The test method is modified as indicated below, and generally comprises:

(a) preparing a set of four 1 x 10 cm aluminum coupons approximately 1/32 in thick are prepared as follows: (1) blank Al coupon; (2) Al coupon coated with an aqueous solution of a peptide produced and identified as described in the preceding examples, and allowed to dry; (3) Al coupon coated on both sides with a base paint composition, allowed to dry, and then the paint film is coated with a like amount of the same test peptide solution as applied to coupon 2; and (4) Al coupon painted with a paint mixture containing the same base paint composition as for coupon 3 and a like amount of the peptide, as for coupons 2 and 3. Preferably duplicate or triplicate sets of these specimens are prepared. Optionally, a conventional biocide may be included as a positive control. The base paint composition may be any suitable water-based latex paint, without biocides, which is available from a number of commercial suppliers.

(b) Each of the specimens from (a) is placed on a bed of nutrient agar and uniformly innoculated with a fungal suspension. A preferred test organism is Fusarium oxysporum. The fungal suspension may be applied by atomizer or by pipet, however a thin layer of nutrient agar mixed with the fungal innoculum Is preferred.

(c) The specimens are incubated at about 28°C under 85 to 90% relative humidity for 4 weeks.

(d) Fungal growth on each specimen is preferably rated weekly as follows:

None = 0; traces of growth (&lt;10% coverage) = 1; light growth (10-30%) = 2; moderate growth (30-60%) = 3; and heavy growth (60% to complete coverage) = 4.

[0073] Another suitable assay protocol for testing the antifungal properties of a coating or paint film containing an antifungal peptide is described by the ASTM in D-5590-94 (Standard Test Method for Resistance to Growth of Mold on the Surface of Interior Coatings in an Environmental Chamber), which is hereby incorporated herein by _ref_erence> The testing protocol generally includes

Patent provided by Sughrue Mion, PLLC ־ http://www.sughnje.com (a) Preparation of the Coated Surface. Duplicate or triplicate sets of approximately 1/2 in. thick, 3 x 4 in. untreated wooden or gypsum board panels are prepared as follows:

(1) blank panel; (2) coated with an aqueous solution of a peptide produced and identified as described in the preceding examples, and allowed to dry; (3) coated on both sides with a base paint composition, allowed to dry, and then the paint film is coated with a like amount of the same test peptide solution as applied to panel 2; and (4) painted with a paint mixture containing the same base paint composition as for panel 3 and a like amount of the peptide, as for panels 2 and 3. Optionally, a conventional biocide may be included as a positive control.

(b) Contamination. The panels are randomly arranged and suspended in an environmental cabinet above moist soil that has been inoculated with the desired fungus, preferably Fusarium oxysporum. Enough free space is provided to allow free circulation of air and avoiding contact between the panels and the walls of the cabinet.

(c) Incubation. The panels are incubated for two weeks at 30.5 - 33.5°C and 95 98% humidity.

(d) Scoring. A set of panels (test, control, and, optionally, a positive control) are removed for analysis at intervals, preferably weekly. The mold growth on the specimen panels is rated as described above.

[0074! Alternatively, one or more equivalent testing protocols may be employed, and field tests of coating compositions containing laboratory-identified antifungal peptides or candidate peptides may be carried out in accordance with conventional methods as would be known to those of skill in the art.

Example 10 - Latex Paints with Antifungal Peptidic Agents

[0075] Both the interior latex (Olympic Premium, flat, ultra white, 72001) and acrylic paints (Sherwin Williams DTM, primer/finish, white, B66W1; 136-1500) appeared to be toxic to both Fusarium and Aspergillus. Therefore, eight individual wells (48-well microtito plate) of each paint type were extracted on a dally basis with 1 ml of phosphate buffer for 5 days (14 &amp; 6) and then allowed the plates were allowed to dry before running the assay. Each well contained 16ul of respective paint.

Extract testing:

[0076] The extract from two wells each of the two paints for each day was tested for toxicity by mixing the extract 1:1 with 2X medium and inoculating with spores (10E4) of Aspergillus or Fusarium. The extracts had no affect on growth of either test fungus.

Well testing:

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PCT/US2004/0217U

[0077! The extracted and non-extracted wells for each of the paints were tested with a range of Inoculum levels in growth medium using the two different fungi. For Fusarium the range was 10E1-10E4 and for Aspergillus 10E2-10E5.

Well Testing of Acrylic Paint Plates:

[0078] Both Fusarium and Aspergillus grew in all extracted wells at all inoculum levels.

Only Aspergillus grew in non-extracted wells at the 10E5 level and not at lower levels indicative of an Inherent biocidal capability.

Well Testing of Latex Paint Plates:

[0079] Fusarium grew in the extracted wells only at the 10E4 inoculum level but not at 10E1 - 10E3. Aspergillus grew in all extracted wells showing an inoculum level effect. No growth was observed for either Fusarium or Aspergillus in non-extracted wells.

Conclusion:

[0080] Extraction of the toxic factor(s) found in both paints was possible. However, it appeared that it may be less extractable from the latex paint.

Evaluation of peptide activity in presence of acrylic and latex paints

[00811 It was established that it was possible to extract both acrylic and latex paints dried in a 48-well format to make them non-toxic to the test microorganisms - Fusarium and Aspergillus. Using that information an experiment was designed to determine the effect the paint has on peptide activity against two test organisms.

Experimental design:

[0082] 1. Coat 48-well plastic plates with 16μΙ of acrylic or latex paint. Dry for two days under hood.

[0083] 2. Extract designated wells with 1-ml phosphate buffer changing the buffer on a daily basis for 7days. Control wells were not extracted to confirm paint toxicity.

[00841 3. Add 20μΙ of peptide series in duplicate to designated dry paint coated wells. Peptide, SEQ ID No. 41, series were added in a two-fold dilution series to wells and allowed to dry. The concentration of peptide added ranged from 200pg/20pl to 1.5pg/20pL Inoculated paint-coated plates as follows:

[0085] 1. Extracted control wells received 180μΙ of medium + 20μ! of spore suspension (10⁴ spores/20pl of medium). Inoculum was either Fusarium or Aspergillus in each case.

[0086] 2. Non-extracted control wells received 180μΙ of medium + 20μΙ of spore suspension (10⁴ spores/20pl of medium).

[0087] 3. Extract wells with dried peptide series received 180μΙ of medium + 20μΙ of spore suspension (10⁴ spores/20pl of medium). In duplicate.

[0088] 4. Extract wells that did not have dried peptide series received 160μΙ of medium + 20μΙ of spore, suspension (10⁴/20μΙ of medium) + 20μΙ peptide series as above. In duplicate.

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[0089] 5. Plates were observed for growth over a 5-day period.

Growth and peptide controls:

[0090] 1. Use sterile non-paint coated 48 well plastic plates

[0091] 2. Growth control wells for each test fungus received 180pl of medium + 20pl of spore suspension (10⁴ spores/20pl of medium).

[0092] 3. Peptide activity controls received 160pl of medium + 20pl of spore suspension (10⁴ spores/20pl of medium) + 20pl peptide series as above. Peptide series were added in a two-fold dilution series to wells and range from 200pg/20pl to 1.5pg/20pl. Therefore, the range of peptide tested was 200pg/200pl or 1 .Opg/pl (1000pg/ml) to 0.0075pg/pl (7.5pg/ml).

[0093] 4. Uninoculated medium served as blank for absorbance readings taken at 24,48,72,96 and 120h.

Results:

[0094] Unextracted wells containing either latex or acrylic paint Inhibited growth of both Fusarium and Aspergillus. Extracted wells containing either latex or acrylic paint allowed growth of both Fusarium and Aspergillus.

[0095] The calculated MIC for Fusarium in peptide activity control experiments was 15.62 pg/ml. For Aspergillus the calculated MIC was 61.4 pg/ml.

[0096] For extracted acrylic-coated plates the following results were obtained.

[0097] Controls as stated in above.

[0098] For Fusarium with dried peptide, inhibition was seen at 1000 and 500 pg/ml after 5 days. Spores exposed to liquid peptide added to dry paint wells were inhibited at 1000, 500 and 250 pg/ml after 4 days, and 1000 and 500 pg/ml after 5 days.

[0099] For Aspergillus with dried peptide, inhibition was seen at 1000 pg/ml after 5 days. Spores exposed to liquid peptide added to dry paint wells were inhibited at 1000 and 500pg/ml after 5 days.

[0100] For extracted latex-coated plates the following results were obtained.

[0101] Controls as stated above.

[0102] For Fusarium with dried peptide, inhibition was seen at 1000 pg/ml after 5 days. Spores exposed to liquid peptide added to dry paint wells were inhibited at 1000 pg/ml after 5 days.

[01031 For Aspergillus with dried peptide, inhibition was seen at 1000 pg/ml after 5 days. Spores exposed to liquid peptide added to dry paint wells were inhibited at 1000 pg/ml after.5 days.

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Example 11. Coating a Surface to Inhibit Fungus Infestation and Growth

[0104] When anchorage, food and moisture are available, fungus microorganisms are able to survive where temperatures permit Particularly susceptible surfaces include porous materials such as stone, brick, wall board (sheetrock) and celling tiles; and semiporous materials, including concrete, unglazed tile, stucco, grout, painted surfaces, roofing tiles, shingles, painted or treated wood and textiles. Any type of indoor or outdoor object, structure or material that is capable of providing anchorage, food and moisture to fungal cells is potentially vulnerable to infestation with mold, mildew or □ther fungus. Moisture generally appears due to condensation on surfaces that are at or below the dew point for a given relative humidity. To inhibit or prevent fungus infestation and growth, one or more antifungal peptidic agents from Example 1 or 3-6, preferably approximately 250 -1000 mg/L of the hexapeptide of SEQ ID No. 41, is dissolved or suspended in water and applied by simply brushing or spraying the solution onto a pre-painted surface such as an exterior wall that is susceptible to mold infestation. Conventional techniques for applying or transferring a coating material to a surface are well known in the art and are suitable for applying the antifungal peptide composition. The selected peptides have activity for inhibiting or preventing the growth of one or more target fungi. The applied peptide solution is then dried on the painted surface, preferably by allowing it to dry under ambient conditions. If desired, drying can be facilitated with a stream of warm, dry air. Optionally, the application procedure may be repeated one or more times to increase the amount of antifungal peptide that is deposited per unit area of the surface. As a result of the treatment, when the treated surface is subsequently subjected to the target mold organisms or spores and growth promoting conditions comprising humidity above about typical indoor ambient humidity, presence of nutrients, and temperature above about typical indoor ambient temperature and not exceeding about 38°C, the ability of the surface to resistance fungal infestation and growth is enhanced compared to its prepainted condition before application of the antifungal peptide.

[0105] A simple spray-coated surface may not provide sufficient durability for certain applications such as surfaces that are exposed to weathering. Longer-term protection may be provided against adhesion and growth of mold by mixing one or more of the antifungal peptides with a base paint or other coating composition, which may be any suitable, commercially available product as are well known in the art. Preferably the base composition is free of chemicals and other additives that are toxic to humans or animals, and/or that fall to comply with applicable environmental safety rules or guidelines. The -typical components, additives and properties of conventional paints and coating materials, and film-forming techniques, are well known in the art and are also described in U.S.

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2004-0175407 A1 and U.S. 2004-0109853 A1.

[0106] If additional, long-term protection against growth and adhesion of mold, mildew and fungus is desired, the paint or other coating composition may Include a barrier material that resists moisture penetration and also prevents or deters penetration and adhesion of the microorganisms and the airborne contaminants which serve as food for the growing organisms. Some typical water repellent components are acrylic, siliconates, metal-stearates, silanes, siloxane and paraffinic waxes. The user will preferably take additional steps to deter mold infestation include avoiding moisture from water damage, excessive humidity, water leaks, condensation, water Infiltration and flooding, and taking reasonable steps to avoid buildup of organic matter on the treated surface.

Example 12. Method of Treating a Fungus-Infested Surface

[010η Although it is preferred that In situations where existing fungal growth Is present, the mold colonies and spores are first removed or substantially eliminated before application of one of the present antifungal coatings, it is expected that in some situations an antifungal compositions will be applied to existing mold infected surfaces. In this case, the composition, containing one or more antifungal peptides, may inhibit, arrest the growth of, or substantially eradicate the mold. Early detection and treatment is highly preferred in order to minimize the associated discoloration or other deterioration of the underlying surface due to mold growth. The treatment procedure may consist of simply applying one or more coats of an antifungal peptide solution, paint or other coaling composition as described above In Example 11.

Example 13. Method of Impregnating a Porous Substrate to Inhibit Fungus Growth [0108] Porous or semi-porous objects or materials such as paper, fabrics, carpet, some types of stone, and many other Items that are employed indoors or outdoors, have internal surface areas that can be susceptible to Infestation by mold and are very difficult to treat effectively by conventional methods. It is within the scope of the present invention to impregnate such porous objects with a coating material containing one or more antifungal peptide, as described in one or more of the preceding examples. The liquidity of the composition is such that It is capable of penetrating Into the pores of the object. In this way, an effective amount of the antifungal peptide is deposited on the internal surfaces as well as the exterior ones. Circumstances requiring treatment of a porous surface may benefit from using a relatively thin coating material rather than a thick, pigmented paint, in order to facilitate penetration of the pores.

WO 2005/007758 PCT7US2004/021711

Example 14 Coating a Fruit or Grain Storage Vessel to Inhibit Mold

[0109] The interior walls of grain silos or other fruit or grain storage or transportation tanks are coated with a peptldic antifungal composition of Example 12 or 13 to deter the attachment and growth of mold organisms inside the container. By selecting antifungal peptides that target specific organisms, and are non-toxic to humans or animals, mold contamination of a wide variety of agricultural products may be deterred.

Example 15. Use of Antifungal/Anti-Bacterial Peptides

[0110] Over the past decade, outbreaks of food poisoning and hospital-acquired infections by so-called super bugs” have become increasingly frequent. These are strains of bacteria that are resistant to conventional antibiotics, such as Methicillin-resistant Staphylococcus aureus (MRSA) and Vero-cytotoxin producing variants of Escherichia coli. Worldwide public concern about hygienic surfaces have also been heightened today due to the emergence and spread of new viral infections such as SARS. The current proliferation of antimicrobial cleaners, utensils, food preparation surfaces and coating systems aimed at fulfilling the demands of an increasingly hygiene conscious public are a testament to those widespread concerns.

[0111] Some of the antifungal peptides, particularly the 810־ amino acid residue long peptides also have the property of inhibiting the growth of bacteria, including diseasecausing bacteria such as Staphalococcus and Streptococcus. Thus, it is known that peptides such as 41,197,198, and 199, can inhibit growth of E. amylovora, E. carotovora, E. coli, R. solanocerum, S. aureus, and S. faecalis in standard media at IC50׳s of between 10-1100 mg/ml and MiG’s of between 20-1700 mg/ml. Staphalococcus and Streptococcus bacteria are of special concern in hospital environments where antibiotic resistance is increasingly common. A multipurpose paint or coating is prepared by combining one or more antifungal peptide selected as described in any of Examples 1-6 with one or more antibacterial peptide. One such combination is the peptide of SEQ ID No. 41 and the peptide of SEQ ID No. 41. Alternatively a peptide is selected with both antifungal and antibacterial peptides 6-10. Paints and other coatings containing the antifungal/antibacterial peptides will be applied to surfaces to lend antifungal and antibacterial properties to those surfaces. It is expected that the use of these and other antifungal/antibacterial peptidic agents will avoid the problem human toxicity that is associated with conventional biocidal compounds in today's paints and coatings. The advantage of combined antifungal and antibacterial activity will find particular usefulness in hospital environments and other health care settings.

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WO 2005/007758 ״ ״ , . ,

Example 16. Combined Use of Antifungal Peptides and Other Antimicrobials [0112] A paint composition containing one or more conventional antifungal substance may be modified by addition of one or more of the antifungal peptides described herein. As described in preceding examples, the antifungal peptidic agent may be a single peptide of precisely known sequence, preferably the hexapeptide of SEQ ID No. 198 or an antifungal/antibacterial peptide as described in Example 15. Alternatively, the peptidic agent may be a peptide library aliquot containing a mixture of peptides in which at least two (and preferably three or four) of the N-termlnal amino acid residues are known. If the peptidic agent is a mixture of peptides, one or more peptide will have antifungal activity. [0113] Combining a non-peptidic antifungal agent with one or more antifungal peptide may provide antifungal activity over and above that seen with either the peptide(s) or the non-peptidic agent alone. The expected additive inhibitory activity of the combination is calculated by summing the inhibition levels of each component alone. The combination is then tested on the test organism to derive an observed additive inhibition. If the observed additive inhibition is greater than that of the expected additive inhibition, synergy is exhibited. More specifically, a synergistic combination of an antifungal peptide, or an aliquot of a peptide library containing at least one antifungal peptide, occurs when two or more fungal cell growth-inhibitory substances distinct from the peptide or peptide library aliquot are observed to be more inhibitory to the growth of a test organism than the sum of the inhibitory activities of the individual components alone.

[0114] A testing method for determining additive or synergistic combinations comprises first creating a synthetic peptide combinatorial library. Each aliquot of the library represents an equimolar mixture of peptides in which at least the two C-terminal ammo acid residues are known. Using the testing methods described in one or more of U.S. Patent No. 6,020,312. U.S. Patent No. 5,885,782. and U.S. Patent No. 5.602,097 it is possible to determine for each such aliquot of the synthetic peptide combinatorial library, a precisely calculated concentration at which it will inhibit a test fungus in a coating. Next, the aliquot of the synthetic peptide combinatorial library is mixed with at least one nonpeptide antifungal compound to create a test mixture. As with the peptide component of the mixture, the baseline ability of the non-peptide antifungal substance to inhibit the test fungus Is determined initially. Next, the test fungus is contacted with the test mixture, and the inhibition of growth of the test organism is measured as compared to at least one untreated control. More controls are desirable, such as a control for each individual component of the mixture. Similarly, where there are more than two components being tested, the number of controls to be used must be increased in a manner well known to those of skill in the art of growth inhibition testing. From the separate test results for the peptidic and non-peptidic agents the expected additive effect on inhibition of growth is 44

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2005/007758 PCTTUS2004/021711 detemlned using standard techniques. After the growth Inhibition tests are complete for the combination of peptidic and non-peptldlc agents, the actual or observed effect on the inhibition of growth is determined. The expected additive effect and the observed effect are then compared to determine whether a synergistic Inhibition of growth of the test fungus has occurred. The methods used to detect synergy may utilize non-peptide antimicrobial agents in combination with the inhibitory peptides described above.

[011 SI As described above, an antifungal peptidic agent may be used in combination with one or more existing fungicides and/or fungistatics identified herein or as would be known to one of ordinary skill in the art. It is expected that some such combinations of the antifungal peptidic agent with another fungicide and/or fungistatic may provide advantages such as a broader range of activity against various organisms, a synergistic antifungal or preservative effect, or a longer duration of effect.

[01161 Another potentially advantageous combination includes an antifungal peptidic agent and a preservative that acts against non-fungal organisms (e.g., a bactericide, an algaecide), as it is contemplated that many fungal prone compositions and surfaces coated with such compositions are also susceptible to damage by a variety of organisms. Examples of preservatives that an antifungal peptidic agent may substitute for and/or be combined include, but are not limited to those non-peptidic antimicrobial compounds (i.e., biocides, fungicides, algaecides and mildewcides) have been shown to be of utility and are currently available and approved for use in the U.S./NAFTA, Europe, and the Asia Pacific region. These antimicrobial agents are listed in Table 3, together with the name of a supplier.

[0117] Certain peptides contemplated for use as described herein have been shown (in one or more of U.S. Patent Nos. 6,020,312; 5,602,097; and 5,885,782) to involve synergy between antifungal peptides and non-peptide antifungal agents that is useful in controlling growth of the Fusarium, Rhizoctonia, Ceratocystis, Pythium, Mycosphaere/ia, Aspergillus and Candida genera of fungi. In particular, synergistic combinations have been described and successfully used to inhibit the growth of Aspergillus fumigatus and A. parasiticus, and also Fusarium oxysporum with respect to agricultural applications. It is expected that these and other synergistic combinations of peptide and non-peptide agents will be useful as additives In paints, coatings and other compositions for deterring, preventing, or treating a fungal infestations.

Example 17. Combined Use of Antifungal Peptides and OP Degrading Agents

[01181 Beyond the concerns about food poisoning and hospital acquired infections by antibiotic-resistant ״super bugs, and worries about SARS-like outbreaks, there is also a need to prevent or protect against the possibility of contamination of public facilities and 45

Patent provided by Sughroe Mion, PLLC - http://www.sugr1rue.com surfaces by toxic chemicals due to accidental spills, improper application of certain insecticides, or as a result of deliberate criminal or terroristic acts. In particular, organophosphorus compounds (״organophosphate compounds or ״OP compounds) and organosulfur (“OS”) compounds, which are used extensively as insecticides, are highly toxic to many organisms, including humans. OP compounds function as nerve agents, and some of the most toxic OP compounds are known to have been used as chemical warfare agents. As discussed in more detail in U.S. 20040109853־ A1, some OP chemical warfare agents can be taken up through skin contact and can remain on material, equipment and terrain for long periods of time (e.g., weeks). By addition of a thickener (e.g., a variety of carbon polymers), even volatile OP agents may be rendered less volatile and more persistent on a contaminated surface.

[0119] Thus, it can be readily appreciated that in some situations a multifunctional surface treatment that combines antifungal properties with the ability to degrade organophosphorus compounds would be desirable. Such composition may be In the form of a coating, a paint, a non-film forming coating, an elastomer, an adhesive, an sealant, a material applied to a textile, or a wax, and may be modified by addition of one or more antifungal peptide selected as described in Examples 1-6 and an organophosphorus compound detoxifying agent such as an OP degrading enzyme or cellular material containing such activity. Suitable OP degrading agents are described in

US 2004-0175407 A1 and U.S. 2004-0109853 A1.

Example 18. Adhesives, Sealants and Elastomers Containing Antifungal Peptides

[0120] The antifungal additives described above are expected to be additionally useful for coating or mixing into sealants and elastomers such as grouts and caulks, especially those that are in frequent contact with, or constantly exposed to fungal nutrients and/or moisture. Examples of adhesives and sealants (e.g., caulks, acrylics, elastomers, phenolic resin, epoxy, polyurethane, anaerobic and structural acrylic, high-temperature polymers, water-based industrial type adhesives, water-based paper and packaging adhesives, water-based coatings, hot melt adhesives, hot melt coatings for paper and plastic, epoxy adhesives, plastisol compounds, construction adhesives, flocking adhesives, industrial adhesives, general purpose adhesives, pressure sensitive adhesives, sealants, mastics, urethanes) for various surfaces (e.g., metal, plastic, textile, paper), and techniques of preparation and assays for properties, have been described in Skelst, I., ed., Handbook of Adhesives, 3rd Ed., Van Nostrand Reinhold, New York, 1990;. Satriana, M.J, Hot Melt Adhesives: Manufacture and Applications, Noyes Data 46

Cotton,’New Jersey, 1974; Petrie, E. M, Handbook of Adhesives and Sealants, McGraw-Hill New York, 2000; Hartshorn, S. R. ed., Structural Adhesives-Chemistry and Technology. Plenum Press. New York, 1986; Flick, E. W. Adhesive and Sealant Compound Formulations, 2nd Ed., Noyes Publications, New Jersey, 1984; Flick, E״ Handbook of Raw Adhesives 2nd Ed., Noyes Publications, New Jersey, 1989; Flick, E״ Handbook of Raw Adhesives, Noyes Publications, New Jersey, 1982; Dunning, H. R-, Pressure Sensitive Adhesives- Formulations and Technology, 2nd Ed. Noyes Data Corporation^ New Jersey, 1977; and Flick, E. W, Constraction and Structural Adhesives and Sealants, Noyes Publications, New Jersey, 1988. An adhesive, sealant or elastomer composition containing one or more conventional antifungal substance may be modified by addition of one or more of the antifungal peptides described In Examples 1-6. The antifungal peptidic agent may be a single peptide of precisely known sequence, preferably the hexapeptide of SEQ ID No. 41 or an antifungal/antlbacterial peptide as described in Example 14. Alternatively, the peptidic agent may be a peptide library aliquot containing a mixture of peptides in which at least two (and preferably three or four) of the N-termmal amino acid residues are known. If the peptidic agent is a mixture Of peptides, at least one peptide will have antifungal activity.

Example 19. Antifungal Textile Finish

10121] An antifungal peptidic agent may also be incorporated Into a material applied to a textile, such as, for example, a textile finish. Textile finishes (e.g., soil-resistant finishes, stain-resistant finishes) and related materials for application to a textile are described, for example in Johnson, K., Antistatic Compositions for Textiles and Plastics, Noyes Data Corporation. New Jersey, 1976; Rouette, H.K., Encycloped״ of Textile Wishing, Springer, Veriag, 2001; TEXTILE Finishing Chemicals: An Industrial Guide, by Ernest W. Flick Noyes Publications, 1990; and Handbook of Fiber Finish Technology, by Philip E. Slade, Marcel Dekker, 1998. One type of water repellent and/or oil repellent textile finish is Scotchguard™ (3M Corporate Headquarters, Maplewood, Minnesota, U.S.A.). A textile finish may be modified by addition of one or more of the antifungal peptides described in Examples 1-6. The antifungal peptidic agent may ba a single peptide of precisely known sequence, preferably the hexapeptide of SEQ ID No. 41 or an antifungal/antlbacterial peptide as described In Example 15. Alternatively, the pepbdic agent may be a peptide library aliquot containing a mixture of peptides in which at least two (and preferably three or four) of the N-termlnal amino acid residues are known. If the peptidic agent is a mixture of peptides, at least one peptide will have antifungal activity.

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Example 20. Polymer-Llnked Antifungal Peptides

[0122] In Example 4, above, conjugation of a peptide to a polymer carrier molecule or insoluble substrate is described for stabilizing the antifungal activity in the paint film or coating. That capability may also be used to advantage by chemically linking or otherwise associating one or more antifungal peptides to a polymeric material or plastic fabric which would otherwise be more susceptible to infestation, defacement or deterioration by fungus. Conventional techniques for linking the N- or C- terminus of a peptide to a longchain polymer may be employed. The antifungal peptide may include additional amino acids on the linking end to facilitate linkage to the PVC polymer. A PVC-membrane such as a flexible or retractable roof or covering for an outdoor stadium, is treated to chemically link antifungal peptides to at least a portion of the outer surface of the membrane prior to its installation. Where an installed polymer membrane covering' is already infested by mold, and it is not practical for it to be removed and replaced by a antifungal peptidelinked polymer membrane, it may be feasible to clean the existing infestation or discoloration, and then apply or bond a suitable antifungal coating containing a stabilized antifungal peptide. PVC Is only one of many well-known types of plastic or polymercontaining materials that could be linked to an antifungal peptide in this manner.

Example 21. Kit for Preparing an Antifungal Coating

[0123] For ease of production, in most instances an antifungal paint or coating product containing antifungal peptidic agents will be provided to the consumer as a single premixed formulation. Alternatively, in order to optimize the initial activity and extend the useful lifetime of the antifungal coating, the antifungal peptidic agent may instead be packaged separately from the paint or coating product into which the antifungal agent is to be added. For increased stability, the peptidic agent may also contain a suitable solid or liquid carrier. As In preceding examples, the antifungal peptidic agent may comprise one or more pure antifungal peptides of defined sequence, or it may include a peptide library aliquot containing a mixture of peptides in which at least two (and preferably three or four) of the N-terminal amino acid residues are known (as in SEQ ID Nos. 1-24). If the peptidic agent is a mixture of peptides, at least one will have antifungal activity.

[0124] In some situations it may also be preferred to store a fungal-prone material in a separate container (pot) prior to application, in order to minimize the occurrence of fungal contamination prior to use and for other reasons. Separation of conventional coating components is typically done to reduce film formation during storage for certain types of coatings. Accordingly, some or all of the different components of the antifungal composition are stored in a plurality of containers, or as a multi-pack kit, and the components are admixed prior to and/or during application. For example, 0.001% to

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100%, including all intermediate ranges and combinations thereof, of the antifungal peptidic agent may be stored in a separate container from one or more fungai-prone materials of the final composition. A multi-pack kit may include one or more pots of a fungal-prone material, preferably including 2- to 5-packs of fungal-prone material. A new antifungal composition may be prepared at or near the time of use by combining a fungal-prone material (e.g., carbon polymer-containing binder) with other coating components, including an antifungal peptide, polypeptide or protein, as described herein.

EXAMPLE 22 Identification of Non-Peptide Appressorlum Inhibitors

This example describes the isolation and identification of additional inhibitors of appressorium formation (Thines, E. etal.,J. Antibiotics 51:117-122, 1998). It is contemplated that the described inhibitors are capable of being able to be combined with the peptides of the present invention.

Extracts obtained from submerged cultures of fungi were screened for the ability to inhibit formation of appressoria in germinating conidiospores of Magnaporthe grisea on different inductive and non-inductive surfaces, in the presence and absence of inducers.

A. Identification of Glisoprenln Appressorium Inhibitors

Magnaportha grisea P2 was cultivated as previously described (Thines, E. et al., J. Antibiotics 51:117-122, 1998). Appressorium formation was followed as reported previously (Thines, E. et al., FEMS Microbiol. Lett. 151:219-224, 1997).

From the 300 extracts obtained from submerged cultures of fungi tested, four exhibited inhibitory activity. The active compounds from all four fungi were isolated and characterized. The fungi producing inhibitors of appressorium formation were identified as Giiociadium roseum Bainier, strain HA 190-95, Dasyscyphus sp. A23-96, Hypocrea sp. A20-96 and Hericium ramosum TA76020. Bioactivity-guided fractionation yielded glisoprenins A, C, D and E from HA190-95, all four of which were active only on a hydrophobic surface (Thines, E. et al., J. Antibiotics 51:117-122,1998: Thines, E. et al., FEMS Microbiol. Lett. 151:219-224, 1997],

Whereas glisoprenin A had been already known from a Giiociadium species (Tomoda, H. et al., J. Antibiotics 45:1202-1206, 1992), compounds C, D and E were new (Thines, E. et al., J. Antibiotics 51:117-122, 1998; Thines, E. et al., FEMS Microbiol. Lett. 151:219-224, 1997;

48a

Tomoda, H. et al., J. Antibiotics 45:1202-1206, 1992; Sterner, 0. etal., J. Antibiotics

51:228-231, 1998).

The effects of glisoprenins could be reversed by dioctanoylglycerol but not by 1-oleyl-2-acetyl-glycerol (Thines, E. et al., FEMS Microbiol. Lett. 156:91-94, 1997), both known activators of protein kinase C in mammalian cells (In .”Lipid Second Messengers”, Eds. R. M. Bell, J. H. Exton &amp; S. M. Prescott. Plenum Press, New York and London, pp.1-58, 1996), indicating that there are differences between fungal and animal cells in the signal pathway via PKC. It has been described that PKC plays a role in appressorium formation (Hamer, J. E. and Holden, D. W״ Fungal Gen. 8/0/21:111997 ,16־).

It is contemplated that the differences observed between fungal signaling via PKC and the pathway used in mammalian cells may be used for identifying new and selective fungicides for plant diseases (e.g., rice blast disease). For example, the inhibition by glisoprenins could be reversed competitively by 1,2-dioctanoylglycerol but not by i-oleoyl-2-acetyl-glycerol, both effective activators of protein kinase C in mammalian cells. This would be in agreement with the two separated signal pathway model. It is further contemplated that protein kinase C might be a selective target for fungicides.

B. Identification of Fatty Acid Appressorium Inhibitors

The active principle in the mycelial extracts from the other fungi was oleic acid. Other fatty acids and their derivatives were tested (Thines, E. et al., J. Antibiotics 51:117-122, 1998). Fatty acids were added dissolved in methanol at a final solvent concentration of &lt; 20 ml litre'¹. No effects of solvent on germ tube elongation or appressorium formation were observed at this concentration. Experiments were performed in triplicate and 100 conidia were evaluated in each test. Compounds were tested at 0.5, 1, 2, 4, 6, 8, 10, 15, 20, 25, 40, 50, 80 and 100 mg litre . Saturated fatty acids with chain lengths of 16, 18 or 20 carbon atoms were not active. Mono-unsaturated acids, irrespective of the configuration of the double bond, were inhibitory. The corresponding methyl esters had no effect. Linoleic acid, with two double bonds, was not active.

In Table 7 the concentrations of glisoprenins and fatty acids that reduced appressorium formation by 80(±5)% are given.

TABLE 7

Inhibition of Appressorium Formation in Magnaporthe grisea by Glisoprenins A, C, D and E and Fatty Acids on a Hydrophobic (A) and Hydrophilic (B, C) Surface

48b

<td></td><td colspan="3"> Al C_eo^e(mg litre*¹) __________</td>

<td> Compound</td><td> ΖΓ</td><td> EF</td><td> C°</td>

<td> Glisoprenin A or C or D</td><td> 4</td><td> &gt;100</td><td> &gt;100</td>

<td> Glisoprenin E</td><td> 40</td><td> &gt;100</td><td> &gt;100</td>

<td> Palmitoleic acid</td><td> 10</td><td> 10</td><td> ־100&lt;</td>

<td> Petroselinic acid</td><td> 10</td><td> 20</td><td> &gt;100</td>

<td> Petroselaidic acid</td><td> 20</td><td> 20</td><td> &gt;100</td>

<td> Oleic acid</td><td> 20</td><td> 2</td><td> &gt;100</td>

<td> Elaidic acid</td><td> 20</td><td> 1</td><td> &gt;100</td>

<td> c/s-Vaccenic acid</td><td> 20</td><td> 20</td><td> &gt;100</td>

<td> trans-Vaccenic acid</td><td> &gt;100</td><td> 10</td><td> &gt;100______</td>

<td colspan="3">^aAIC₈o: Concentration wherein appressorium formation inhibition was 80(± 5</td><td> %.</td>

¹⁵Control GelBond sheet: 95-6(±2-4)% of the germinating conidia formed appressoria.

“Induction with 0-2 mg litre1,16 ¹־-hexadecanedioi: 91-4(±3.6)% of the germinated conidia formed appressoria.

“induction with 25 mg litre'¹ chlorophenyithio-cAMP: 94-6(±2-1)% of the germinated conidia formed appressoria.

Oleic acid and elaidic acid interfered with the induction by 1,16-hexadecanediol but not with cAMP (i.e., 8-(4־chlorophenylthio)adenosine-3',5'-monophosphate). It has been reported that the induction by chemical signals proceeds via cAMP (Dean, R. A. Annu. Rev. Phytopathol. 35:211-234, 1997; Choi, W. and Dean, R. A., The Plant Cel! 9:1973-1983, 1997), and it is contemplated that the fatty acids interfere upstream of protein kinase A.

EXAMPLE 23 Lipid Anti-Fungal Agents

Certain lipid molecules have been described as possessing anti-fungal activity. In certain embodiments, an additional anti-fungal agent that may be combined with a peptide of the present invention comprises a lipid may comprise a fatty acid, a glisoprenin, or a combination thereof. In preferred embodiments, an anti-fungal agent comprises a fatty acid, a glisoprenin, or a combination thereof. In some preferred aspects, a glisoprenin is a glisoprenin A, B, C, D, E, F, or a combination thereof. In certain preferred aspects, a fatty acid is a monounsaturated fatty acid. In some particularly preferred aspects, a fatty acid is a free fatty acid. In other preferred aspects, a fatty acid is not a methyl ester fatty acid analog. In other facets, a fatty acid may be an elaidic acid, an oleic acid, a palmitoleic acid, a petroselinic acid, a petroselaidic acid, a c/s-Vaccenic add, a trans-Vaccenic acid or a combination thereof. However, other fatty acids are contemplated, and are described herein.

48c

As used herein a lipid refers to any hydrophobic or amphipathic organic compound obtainable from a biological material, and any analog thereof, excluding such molecules obtainable from a biological material consisting essentially of a nucleic acid, a carbohydrate or a proteinaceous molecule. It is contemplated that an anti-fungal composition of the present invention can comprise one or more lipids.

A lipid usually comprises at least ten carbon atoms including one or more chemical moieties such as a hydrocarbon, an alcohol, an aldehyde, an acid, an amine, a giucide or a combination thereof. Non-limiting examples of lipids include a molecule comprising a fatty acid (e.g., a triacylglycerol, a phospholipid, a glycolipid), a surfactant, an aliphatic alcohol, an aliphatic wax, a terpene, a steroid, a sterol, a far soluble vitamin (e.g., A, D, E, K), a coenzyme (e.g., ubiquinone), a pigment (e.g., a carotenoid), a phenolic, an analog of the forgoing, or a combination thereof. As used herein an “analog of a lipid refers to a chemically modified lipid or synthetic variant of a lipid, including a structural isomer, as would be known to one of skill in the art.

In certain embodiments, a lipid may be anionic (/.©., comprising a net negative charge), cationic (i.e., comprising a net positive charge) or neutral in net charge at pH 7. Non-limiting examples of anionic lipids include a palmitic acid, a stearic acid, an arachidonic acid, an oleic acid, a linolenic acid, a linoleic acid, a myristic acid, a phosphatidyl glycerol, a diphosphatidyl glycerol a phosphatidyl serine, a phosphatidyl inositol or a combination thereof. Non-limiting examples of cationic lipids include, a phosphatidyl ethanolamine, a phosphatidyl choline or a combination thereof. In certain facets, a cationic counter ion (e.g., Ca⁺²) may be combined with an anionic lipid, or an anionic counter ion may be combined with a cationic lipid.

A. Lipid Preparation

As is known to those of ordinary skill in the art, one may obtain various lipids and their derivatives from a commercial source (e.g., Arista Industries, Inc., 557 Danbury Road, Wilton, CT 06897 U.S.A.; Avantt Polar Lipids, Inc., 700 Industrial Park Drive, Alabaster, AL 35007 U.S.A.) or, in certain cases, a biological material or chemical synthesis. Generally, a lipid may be obtained from a biological material through extraction with a non-aqueous solvent. Methods for preparation of a lipid from a biological source are well known in the art, and generally comprise tissue sample homogenation, solvent extraction, fractionation and/or drying protocols. For example, a lipid may be obtained using a chloroform/methanol extraction protocol (Folch et aL, J Biol Chem 1957, 226:497, 1957). In other cases, a particular lipid may be obtained using a more specialized protocol, as would be known by one of ordinary skill in the art. For example, hexane/isopropanol extraction has been used for obtaining nervous tissue lipids (Hara et al., Anal

48d

Biochem 90:420, 1978; Eder, K. etal., Clin Chim Acta 219:93, 1993). tn another example, solvent elution of a dry column comprising an animal tissue has been used for obtaining animal lipids (Marmer, W. N, etal., Lipids 16:365, 1981; Elmer-Frohlich, K. etal., JAOCS69:243, 1992). In a further example, a method for extracting highly polar lipids has been described (Hajra, lipids 1974, 9:502, 1974; Dreyfus et a/. Anal Biochem 249:67-78, 1977). In an additional example, methods for obtaining plasma lipids and acylglycerol lipids have been described (J Clin Invest 37:350, 1956). In a further example, phosphoinositides extraction has been described (Biochem 7212:489, 1983; Br J Haematol23:571-585, 1972), etc.

Once a lipid has been extracted, it can be fractionated by any technique known to one of ordinary skill in the art. For example, a non-polar lipid may be fraction eluted from phospholipids and glycospingolipids using a sephadex G-25 binding column (Dreyfus H et al., Anal Biochem 249:67, 1997). In another example, a lipid may be obtained from a plant material by fractionation using low pressure chromatography (Christie, W. W. et al, J High Resol Chromatogr 18:97, 1995). In a further example, a lipid may be obtained from a plant material through fractionation by high performance liquid chromatography (El-Hamdy, A. H. et al J High Resol Chromatogr 16:55, 1993). In an additional example, a lipid obtained from a plant material by fractionation using a silica column (Liu, J. et a! JAOCS 70:343, 1993).

In certain embodiments, an anti-fungal agent is a fatty acid. A fatty acid generally is a component of a larger lipid structure, such as an acylglycerol. An acylglycerol (a.k.a. a neutral fat, a fat, a neutral glyceride, a glyceride, an oil) generally comprises a glycerol esterified to 1, 2 or 3 fatty acid(s). A monacylglycerol (a.k.a. monoglyceride) comprises glycerol and one fatty acid ester, a diacylglycerol (a.k.a. diglyceride) comprises glycerol and two fatty acid esters, while a triacylglycerol (a.k.a. a triglyceride) comprises a glycerol and with three fatty acid esters. Free fatty acids are often obtained from chemical or enzymatic release from lipids such as an acylglycerol.

Methods for preparing free fatty acids are known in the art, and include, for example, acid elution of lipids in conjunction with various fractionation methods, as well as a solid phase micro-extraction technique (Tomaino, R. M. et ai., J Agric Food Chem 49:3993, 2001). However, preparation of a fatty acid as a methyl ester analog rather than a free acid is common (Morrison et al J Lipid Res 5:600, 1964; Piretti, Μ. V. et al Chem Phys Lipids 47:149, 1988; Lewis, T. et al, J. Microbiol Meth. 43:107, 2000; Ichihara, K. etal., Lipids 37:523, 2002).

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B. Fatty Acids

As used herein, a fatty acid refers to an organic compound comprising a hydrocarbon chain that includes a terminal carboxylate group or an analog thereof. Non-limiting types of fatty acids include a short chain fatty acid, a medium chain fatty acid, a long chain fatty acid, a saturated fatty acid, a monounsaturated fatty acid, a polyunsaturated fatty acid, a branched chain fatty acid, a ring fatty acid, an acetylenic fatty acid, or a combination thereof.

A fatty acid comprising a 2 to 7 carbon atom chain is known herein as a “short-chain organic acid”, and generally possesses considerable aqueous solubility, A fatty acid comprising an 8 to 14-carbon atom chain is known herein as a “medium chain fatty acid, while a fatty acid comprising a 15 or more carbon atom chain is known herein as a long chain fatty acid”. In certain aspects, a carbon backbone chain of a fatty acid may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10,

11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 35,

36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, 60, 61,

62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79 or 80 carbons in length, including all intermediate ranges. Non-limiting examples of fatty acid carbon backbone lengths Include a fatty acid of 12 carbons (e.g., iaurate), 14 carbons (e.g., myristate), 16 carbons (e.g., palmitate, palmitoleate), 18 carbons (e.g., stearate, oleate, tarirate, linoleate, linolenate, petroselinate, ricinoleate), 20 carbons (e.g., arachidate, arachidonate), 22 carbons (e.g., behenate, erucate) or 24 carbons (e.g., lignocerate). In certain embodiments, a fatty acid may be unsaturated, monounsaturated or polyunsaturated, referring to whether the hydrocarbon chain possess no carbon double bonds, one carbon double bond, or a plurality of carbon double bonds (e.g., 2, 3, 4, 5, 6, Z or 8 double bonds), respectively.

1. Monounsaturated fatty acids

Monounsaturated fatty acids acid (a.k.a. monoenoic fatty acids) include, but are not limited to, an obtusilic acid [a.k.a. a cis-4-decenoic acid, a 10:1(n-6) acid]; a caproleic acid [a.k.a. a cis-9-decenoic acid, a 10:1 (n-1) acid]; an undecylenic acid (a.k.a. a 10c-11:1 acid); a lauroleic acid [a.k.a. a cis-5-lauroleic acid, a 12:1(0-7) acid]; a linderic acid [a.k.a. a cis-4-dodecenoic acid, a 12:1(n-8) acid]; a myristoleic acid [a.k.a. a cis-9-tetradecenoic acid, a 14:1(n-5) acid]; a physeteric acid [a.k.a. a cis-5-tetradecenoic acid, a 14:1(n-9) acid]; a tsuzuic acid [a.k.a. a cis-4-tetradecenoic acid, a 14:1(n-10) acid]; a palmitoleic acid [a.k.a. a cis-9-hexadecenoic acid, a 16:1(n-7) acid]; a trans-3-hexadecenoic acid (a.k.a. a trans-16:1 n-13 acid); a petroselinic acid [a.k.a. a cis-6־octadecenoic acid, a 18:1(0-12) acid]; a petroselaidic acid (a.k.a., a 6/-18:1 acid] an oleic acid [a.k.a. a cis-9־octadecenoic acid, a 18:1(n-9) acid]; an elaidic acid (a.k.a. a trans-9־octadecenoic acid); a vaccenic acid [a.k.a. a cis-11 -octadecenoic acid, a 18:1(n-7) acid]; a trans-vaccenic acid (a.k.a. a trans-11-octadecenoic acid, a trans-11-18:1 acid); a gadoleic acid

48f

[a.k.a. a cis-9-eicosenoic acid, a 20:1(n-11) acid]; a gondoic acid [a.k.a. a cis-11-eicosenoic acid, a 20:1(n-9) acid]; a cetoleic acid [a.k.a. a cis-11-docosenoic acid, a 22:1(n11־) acid]; an erucic acid [a.k.a. a cis-13-docosenoic acid, a 22:1 (n-9) acid]; a nervonic acid (a.k.a. a cis-15-tetracosenolc acid, a 24:1(n-9) acid]; or a combination thereof. As with many lipids, monounsaturated fatty acids may be obtained from commercial providers known to those of ordinary skill in the art or may be obtained from a biological material. For example, myristoleic acid and oleic acid may be obtained from olive oil. In another example, palmitoleic acid may be obtained from fish fats. In a further example, trans-3-hexadecenoic acid may be obtained from green algae photosynthetic membranes. In an additional example, petroselinic acid may be obtained from parsley seeds. In a supplemental example, elaidic acid and trans-vaccenic acid may be obtained from butter, cheese, milk or beef. In another example, vaccentc acid may be obtained from animal fats or butter. In a further example, gadoleic acid may be obtained from cod liver oil. In a final example, erucic acid and nervonic acid may be obtained from cruciferae (e.g., Lunaria annua) seed oil.

2. Fatty acid analogs

Various fatty acid analogs are known in the art. As used herein, a “fatty acid analog refers to any chemically modified free fatty acid. Preferred fatty acid analog(s) are analogs of monounsaturated fatty acid(s). Examples of fatty acid analogs contemplated for use in the present invention include adding a charged ion to a fatty acid produce a salt, replacing the acid carboxyl group with an ester, isomerizing a cis double bond in a fatty acid hydrocarbon backbone chain into a trans double bond, as well as fatty acid analogs such as a hydroxy fatty acid, a thia fatty acid, an amide fatty acid, an aldehyde fatty acid, a methoxy fatty acid, a keto fatty acid, a dicarboxy fatty acid, or a combination thereof.

In certain embodiments, a fatty acid analog is a thia fatty acid, wherein a carbon of the carbon backbone is substituted by a sulfur. Non-limiting examples of a thia fatty acid include a dodeca thia acetic acid, a tetradeca thia acetic acid or a combination thereof. In other embodiments, a fatty acid analog is an amide fatty acid, wherein a carboxyl group hydroxyl moiety is substituted by an amino moiety. Non-limiting examples of amide fatty acids include the amide analog of a 13-cis-docosenoic acid (a.k.a. an erucamide); the amide analog of a 13-t-docosenoic acid; the amide analog of a 9-octadecenoic acid (a.k.a. an oleamide). In certain embodiments, a fatty acid analog is an aldehyde analog of a fatty acid (e.g., a pristanic acid). In other embodiments, a fatty acid analog may comprise a keto fatty acid (e.g., a t9-keto-2-decenoic acid).

In further embodiments, a fatty acid analog is a hydroxy fatty acid, wherein the carbon backbone chain comprises a hydroxyl moiety. Non-h’miting examples of hydroxy fatty acids include a

48g

2-hydroxyl in oleic acid, which may be obtained from Thymus vulgaris seed oil; a 2-hydroxytetracosanoic acid (a.k.a. a cerebronic acid), which may be obtained from cerebrosides of animals; a 2-hydroxy-15-tetracosenoic acid (a.k.a. a hydroxynervonic acid), which may be obtained from cerebrosides of animals; a ricinoleic acid (a.k.a. a 12-hydroxy-9-octadecenoic acid), which can be obtained from castor bean oil; a lesquerolic acid (a.k.a. a 14־hydroxy-11-eicosenoic acid), which can be obtained from Lesquerella: or a combination thereof. In specific facets, a hydroxy fatty acid is an estolide, which comprise a dimer of a fatty acid esterified to a hydroxy fatty acid, and may be produced by heating oleic acid with sulfuric or perchloic acid (Cermak, S.C. et al., JAOCS 78:557, 2001).

In other embodiments a fatty acid analog comprises a methoxy fatty acid (e.g., a a-methoxy fatty acid). Non-limiting examples of methoxy fatty acids include a 2-methoxy hexadecanoic acid; a 2-methoxy-5-hexadecenoic acid; a 2-methoxy-6-hexadecenoic acid (Carballeira, N.

M. et al., Lipids, 27:72, 1992; Carballeira N. M. et al., J. Nat. Prod. 61:675, 1998); a 7-methoxy-4-tetradecenoic acid (Cardellina J. H. et al., Phytochemistry 17:2091, 1978); a 9-methoxypentadecanoic acid and/or a 15-methoxytricosanoic acid, which may be obtained from Schizymenia dubyi (Barnathan, G. et al., Phytochemistry 47:761, 1998), a

11-methoxyheptadecanoic acid and/or a 11-methoxynonadecanoic acid, which may be obtained from Helicobacter pylori (Inamoto, Y. et al., J Gastroenterol, 30:315, 1995), or a combination thereof.

A dicarboxylic acid is the same in structure as a fatty acid, with the exception of comprising a carboxylic acid moiety at both ends of the hydrocarbon chain. Examples of dicarboxylic acids include an adipic acid (a.k.a. a hexanedioic acid), a pimeiic acid (a.k.a. a heptanedioic acid), a suberic acid (a.k.a. an octanedioic acid), an azelaic acid (a.k.a. a nonanedioic acid), a sebacic acid (a.k.a. a decanedioic acid), a thapsic acid, a brassylic acid, and any combination of the forgoing. An adipic acid may be obtained by cyclohexane or cyclohexanol oxidation. A pimeiic acid may be obtained from oxidized castor oil. A suberic acid may be obtained as a product of ricinoleic acid oxidation. An azelaic acid may be obtained from oxidation and cleavage of an oleic acid with chromic acid. A sebacic acid may be produced by alkali cleavage of castor oil.

3. Glisoprenins

Certain types of terpenes are contemplated as anti-fungal agents. As used herein, a terpene” refers to an organic compound comprising a hydrocarbon chain that comprises one or more isoprene units. An isoprene unit is a four-carbon hydrocarbon chain, wherein and additional carbon is branched off as a methyl moiety from the second carbon of the chain. Isoprene units have two double bonds, and are generally viewed as a five carbon structural unit. Terpenes

48h generally possess chemical formulas comprising multiples of five carbons based on the number of isoprene units in their chemical structure. However, one or more of hydrocarbon chains of the isoprene units of a terpene is chemically modified, such as by alterations in the positions of double bonds, additions or subtraction of chemical moieties, cyclizations of chemical structures, and the like, thus giving each terpene its structural distinctiveness.

Glisoprenin A, glisoprenin B, glisoprenin C, glisoprenin D, glisoprenin E and glisoprenin F are each 45-carbon terpenes (i.e., a 9 isoprene terpene), which is unusual as few terpenes of greater than 8 isoprene subunits have been identified from biological sources (Sterner, O., J. Antibiot. (Tokyo), 51(2):228-231, 1998). Glisoprenin A and glisoprenin B may be isolated as liquid lipids from Gliocladium species FO-1513 using acetone extraction, vacuum drying, dissolving in a chloroform, and silica gel fractionation and elution with chloroform and chloroform methanol solvents as has been described [Tomoda, H.etal., J. Antibiot. (Tokyo), 45(8):1202-1206, 1992], Glisoprenin C, glisoprenin D and glisoprenin E may be isolated as liquid lipids from Gliocladium roseum species HA190-95 using ethyl acetate extraction, addition of sodium sulfate and vacuum drying, dissolving in methanol, silica gel chromatography and H.P.L.C. water-methanol separation as has been described [Thines, E. etal.,J. Antibiot. (Tokyo), 51(2):117-122, 1998; Nishida, H. et al., J. Antibiot. (Tokyo), 45(10):1669-1676, 1992). Glisoprenin F may be isolated as a liquid lipid from Gliocladium catenulatum strain (NRRL 22970) using ethanol extraction, partitioning between hexane and methanol, silica gel fractionation of the methanol fraction, followed by H.P.L.C. separation using methanol and CH₂CI2 as has been described [Joshi, B. et al., J. Nat. Prod. 62(5):730-733, 1999], Additional methods for producing glisoprenins, and using one or more glisoprenins as anti-fungal agent(s), are described in specific example 22.

One skilled in the art will readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned as well as those inherent therein. It should be understood, however, that the enzyme compositions, enzymes, microorganism-based particulate materials, compounds, coatings, paints, films, methods, procedures, and techniques described herein are presently representative of preferred embodiments. These techniques are intended to be exemplary, are given by way of illustration only, and are not intended as limitations on the scope. Other objects, features, and advantages of the present invention will be readily apparent to one skilled in the art from the following detailed description; specific examples and claims; and various changes, substitutions, other uses and modifications that may be made to the invention disclosed herein without departing from the scope and spirit of the invention or as defined by the scope of the appended claims.

As used herein other than the claims, the terms a, an, “the, and “said means one or more. As used herein in the ciaim(s), when used in conjunction with the words “comprises” or

48i “comprising, the words “a, an, the, or “said may mean one or more than one. As used herein another may mean at !east a second or more.

All patents and publications mentioned in this specification are indicative of the levels of those skilled in the art to which the invention pertains. All patents and publications so referenced are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

As would be known to one of ordinary skill in the art, many variations of nomenclature are commonly used to refer to a specific chemical composition. Accordingly, several common alternative names may be provided herein in quotations and parentheses/brackets, or other grammatical technique, adjacent to a chemical composition’s preferred designation when referred to herein. Additionally, many chemical compositions referred to herein are further identified by a Chemical Abstracts Service registration number. As would be known to those of ordinary skill in the art, the Chemical Abstracts Service provides a unique numeric designation, denoted herein as “CAS No., for specific chemicals and some chemical mixtures, which unambiguously identifies a chemical composition's molecular structure.

in various embodiments described herein, exemplary values are specified as a range. Examples of such ranges cited herein include, for example, a size of a biomolecule, a tempature for growth and/or preparation of a microorganism, a chemical moiety's content in a coating component, a coating component's content in a coating composition and/or film, a coating component’s mass, a glass transition temperature (T_B), a temperature for a chemical reaction (e.g., film formation, chemical modification of a coating component), the thickness of a coating and/or film upon a surface, etc. It will be understood that herein the phrase including all intermediate ranges and combinations thereof’ associated with a given range is all integers and sub-ranges comprised within a cited range. For example, citation of a range 0.03% to 0.07%, including all intermediate ranges and combinations thereof is specific values within the sited range, such as, for example, 0.03%, 0.04%, 0.05%, 0.06%, and 0.07%, as well as various combinations of such specific values, such as, for example, 0.03%, 0.06% and 0.07%, 0.04% and 0.06%, or 0.05% and 0.07%, as well as sub-ranges such as 0.03% to 0.05%, 0.04% to 0.07%, or 0.04% to 0.06%, etc. Additionally, example 12 provides additional descriptions of specific numeric values within a cited range.

48j

A.

Biomolecules

As used herein, a “biomolecule composition” of the present invention refers to a composition comprising a biomolecule. As used herein, a biomoiecule” refers to a compound comprising of one or more chemical moieties typically synthesized in living organisms, including but not limited to, an amino acid, a nucleotide, a polysaccharide or simple sugar, a lipid, or a combination thereof. A preferred biomolecule of the present invention comprises a proteinaceous molecule. As used herein a proteinaceous molecule comprises a polymer formed from amino acids, such as a peptide or a polypeptide. Examples of proteinaceous molecules include an enzyme, an antibody, a receptor, a transport protein, structural protein, or a combination thereof. Examples of a peptide include inhibitory peptides of 3-15 amino acids.

In addition to the sources described herein for biomolecules, reagents, living cells, etc., one of ordinary skill in the art may obtain such materials and/or chemical formulas thereof for use in the present invention from convenient source such as a public database, a biological depository, and/or a commercial vendor. For example, various nucleotide sequences, including those that encode amino acid sequences, may be obtained at a public database, such as the Entrez Nucleotides database found at:

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Nucleotide, which includes sequences from other databases including GenBank, RefSeq, and PDB. In another example, various amino acid sequences may be obtained at a public database, such as the Entrez databank found at: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Protein, which includes sequences from other databases including SwissProt, PIR, PRF, PDB, GenBank, and RefSeq. Additional examples of such databases are listed at: http://www.rcsb.0rg/pdb/links.html#Databases, and numerous nucleic acid sequences and/or encoded amino acid sequences can be obtained from such sources. In a further example, biological materials that comprise, or are capable of comprising such biomolecules (including living cells), may be obtained from a depository such as the American Type Culture Collection (“ATCC”), P.O. Box 1549 Manassas, VA 20108, USA. In an additional example, biomolecules, chemical reagents, biological materials, and equipment may be obtained, as is well known to those of ordinary skill in the art, from commercial vendors such as Amersham Biosciences®, 800 Centennial Avenue, P.O. Box 1327, Piscataway, NJ 08855-1327 USA; BD Biosciences®, including Clontech®, Discovery Labware®, Immunocytometry Systems® and Pharmingen®, 1020 East Meadow Circle, Palo Alto, CA 94303-4230 USA; Invitrogen™, 1600 Faraday Avenue, PO Box 6482, Carlsbad, California 92008 USA; New England Biolabs®, 32 Tozer Road, Beverly, MA 01915-5599 USA; Merck®, One Merck Drive, P.O. Box 100, Whitehouse Station, NJ 08889-0100 USA; Novagene®, 441 Charmany Dr,, Madison, Wi 537191234 USA; Promega®, 2800 Woods Hollow Road, Madison WI 53711 USA; Pfizer®, including

48k

Pharmacia®, 235 East 42nd Street, New York, NY 10017 USA; Quiagen®, 28159 Avenue

Stanford, Valencia, CA 91355 USA; Sigma-Aldrich®, including Sigma, Aldrich, Fluka, Supelco and

Sigma-Aldrich Fine Chemicals, RO Box 14508, Saint Louis, MO 63178 USA; Stratagene®, 11011

N. Torrey Pines Road, La Jolla, CA 92037 USA, etc.

In addition to those techniques specifically described herein, one of ordinary skill in the art may manipulate a cell, nucleic acid sequence, amino acid sequence, and the like, in light of the present disclosures, using standard techniques known in the art [see, for example, In ״Molecular Cloning” (Sambrook, J״ and Russell, D.W., Eds.) 3rd Edition, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press, 2001; In Current Protocols in Molecular Biology (Chanda, V. B. Ed.) John Wiley &amp; Sons, 2002; In “Current Protocols in Nucleic Acid Chemistry (Harkins, E. W. Ed.) John Wiley &amp; Sons, 2002; In “Current Protocols in Protein Science” (Taylor, G. Ed.) John Wiley &amp; Sons, 2002; In Current Protocols in Cell Biology (Morgan, K, Ed.) John Wiley &amp; Sons, 2002; In “Current Protocols in Pharmacology (Taylor, G. Ed.) John Wiley &amp; Sons, 2002; In Current Protocols in Cytometry” (Robinson, J. P. Ed.) John Wiley &amp; Sons, 2002; In Current Protocols in Immunology (Coico, R. Ed.) John Wiley &amp; Sons,

2002].

B. Enzymes

The selection of a biomolecule for use in the present invention depends on the desired property that is to be conferred to a composition of the present invention. A preferred biomotecule of the present invention comprises an enzyme, as enzymatic activity is a preferred property to be conferred to a biomolecule composition, coating and/or paint in the present invention. As used herein, the term “enzyme refers to a molecule that possesses the ability to accelerate a chemical reaction, and comprises one or more chemical moieties typically synthesized in living organisms, including but not limited to, an amino acid, a nucleotide, a polysaccharide or simple sugar, a lipid, or a combination thereof. As used herein, the term bioactive” refers to the ability of an enzyme to accelerate a chemical reaction differentiating such activity from a like ability of a composition, and/or a method that does not comprise an enzyme to accelerate a chemical reaction.

In preferred embodiments, an enzyme comprises a proteinaceous molecule. It is contemplated that any proteinaceous molecule that functions as an enzyme, whether identical to the wild-type amino acid sequence encoded by an isolated gene, a functional equivalent of such a sequence, or a combination thereof, may be used in the present invention. As used herein, a wild-type enzyme refers to an amino acid sequence that functions as an enzyme and is identical to the sequence encoded by an isolated gene from a natural source. As used herein, a functional equivalent״ to the wild-type enzyme is a proteinaceous molecule comprising a sequence and/or a structural analog of a wild-type enzyme’s sequence and/or structure and functions as an enzyme. The functional equivalent enzyme may possess similar or the same enzymatic properties, such as catalyzing chemical reactions of the wild-type enzyme's EC classification, or may possess other desired enzymatic properties, such as catalyzing the desirable chemical reactions of an enzyme that is related to the wild-type enzyme by sequence and/or structure. Examples of a functional equivalent of a wild-type enzyme are described herein, and include mutations to a wild-type enzyme sequence, such as a sequence truncation, an amino acid substitution, an amino acid modification, a fusion protein, or a combination thereof, wherein the altered sequence functions as an enzyme.

In certain embodiments, an enzyme.may comprise a simple enzyme, a complex enzyme, or a combination thereof. As known herein, a “simple enzyme is an enzyme wherein the chemical properties of moieties found in its amino acid sequence is sufficient for producing enzymatic activity. As known herein, a complex enzyme is an enzyme whose catalytic activity functions only when an apo-enzyme is combined with a prosthetic group, a co-factor, or a combination thereof. An apo-enzyme is a proteinaceous molecule and is catalytically inactive without the prosthetic group and/or co-factor. As known herein, a prosthetic group” or coenzyme is non-proteinaceous molecule that is attached to the apo-enzyme to produce a catalytically active complex enzyme. As known herein, a “holo-enzyme is a complex enzyme that comprises an apo-enzyme and a co-enzyme. As known herein, a “co-factor is a molecule that acts in combination with the apo-enzyme to produce a catalytically active complex enzyme. In some aspects, a prosthetic group is one or more bound metat atoms, a vitamin derivative, or a combination thereof. Examples of metal atoms that may be used as a prosthetic group and/or a co-factor include Ca, Cd, Co, Cu, Fe, Mg, Mn, Ni, Zn, or a combination thereof. Usually the metal atom is an ion, such as Ca^Zt, Cd^z+, Co^zt, Cu^Zt, Fe*^z, Mg²*, Mn²’, Ni²*, Zn^z+, or a combination thereof. As known herein, a “metaHoenzyme is a complex enzyme that comprises an apoenzyme and a prosthetic group, wherein the prosthetic group comprises a metal atom. As known herein, a metal activated enzyme is a complex enzyme that comprises an apo-enzyme and a co-factor, wherein the co-factor comprises a metal atom.

A chemical that binds a proteinaceous molecule is known herein as a ligand. As used herein, “bind or binding” refers to a physical contact between the proteinaceous molecule at a specific region of the proteinaceous molecule and the ligand in a reversible fashion. Examples of binding interactions are well known in the art, and include such interactions as a ligand known as an “antigen binding an antibody, a ligand binding a receptor, and the like. A portion of the proteinaceous molecule wherein substrate binding occurs is known herein as a “binding site. A

48m ligand that is acted upon by the enzyme in the accelerated chemical reaction is known herein as a substrate. A contact between the enzyme and a substrate in a fashion suitable for the accelerated chemical reaction to proceed is known herein as “substrate binding. A portion of the enzyme involved in the chemical interactions that contributed to the accelerated chemical reaction is known herein as an active site.

A chemical that slows or prevents the enzyme from conducting the accelerated chemical reaction is known herein as an inhibitor.” A contact between the enzyme and the inhibitor in a fashion suitable for slowing or preventing the accelerated chemical reaction to proceed upon a target substrate is known herein as inhibitor binding. In some embodiments, inhibitor binding occurs at a binding site, an active site, or a combination thereof. In some aspects, an inhibitor’s binding occurs without the inhibitor undergoing the chemical reaction. In specific aspects, the inhibitor may also be a substrate such as in the case of an inhibitor that precludes the enzyme from catalyzing the chemical reaction of a target substrate for the period of time inhibitor binding occurs at an active and/or binding site. In other aspects, an inhibitor undergoes the chemical reaction at a rate that is slower relative to a target substrate.

In some embodiments, enzymes may be described by the classification system of The International Union of Biochemistry and Molecular Biology (IUBMB”). The IUBMB classifies enzymes by the type of reaction catalyzed and enumerates each sub-class by a designated enzyme commission number (EC). The IUBMB classification of various enzymes may be obtained using the computerized database at http://www.chem.qmw.ac.uk/iubmb/enzyme/. Based on these broad categories, an enzyme may comprise an oxidoreductase (EC 1), a transferase (EC 2), a hydrolase (EC 3), a lyase (EC 4), an isomerase (EC 5), a ligase (EC 6), or a combination thereof. Often, an enzyme may be able to catalyze multiple reactions, and thus have multiple EC classifications.

Generally, the chemical reaction catalyzed by an enzyme alters a moiety of a substrate. As used herein, a moiety or group, in the context of the field of chemistry, refers to a chemical sub-structure that is a part of a larger molecule. Examples of moiety include an acid halide, an acid anhydride, an alcohol, an aldehyde, an alkane, an alkene, an alkyl halide, an alkyne, an amide, an amine, an arene, an aryl halide, a carboxylic acid, an ester, an ether, a ketone, a nitrile, a phenol, a sulfide, a sulfonic acid, a thiol, etc.

An oxidoreductase catalyzes an oxido-reduction of a substrate, wherein the substrate is either a hydrogen donor and/or an electron donor. An oxidoreductase is generally classified by the substrate moiety that is the donor or acceptor. Examples of oxidoreductases include an

48n oxidoreductase that acts on a donor CH-OH moiety, (EC 1.1); an donor aldehyde or a donor oxo moiety, (EC 1.2); a donor CH-CH moiety, (EC 1.3); a donor CH-NH_Z moiety, (EC 1.4); a donor CH-NH moiety, (EC 1.5); a donor nicotinamide adenine dinucieotide (“NADH) or a donor nicotinamide adenine dinucleotide phosphate (NADPH”), (EC 1.6); a donor nitrogenous compound, (EC 1.7); a donor sulfur moiety, (EC 1.8); a donor heme moiety, (EC 1.9); a donor diphenol or a related moiety as donor, (EC 1.10); a peroxide as an acceptor, (EC 1.11), a donor hydrogen, (EC 1.12); a single donor with incorporation of molecular oxygen (“oxygenase), (EC 1.13); a paired donor, with incorporation or reduction of molecular oxygen, (EC 1.14); a superoxide radical as an acceptor, (EC 1.15); an oxidoreductase that oxidises a metal ion, (EC 1.16); an oxidoreductase that acts on a donor CH_Z moiety, (EC 1.17); a donor iron-suifur protein, (EC 1.18); a donor reduced flavodoxin, (EC 1.19); a donor phosphorus or donor arsenic moiety, (EC 1.20); an oxidoreductase that acts on an X-H and an Y-H to form an X-Y bond, (EC 1.21); as well as a other oxidoreductase, (EC 1.97)(13 this the right number?]; or a combination thereof.

A transferase catalyzes the transfer of a moiety from a donor compound to an acceptor compound. A transferase is generally classified based on the chemical moiety transferred. Examples of transferases include an transferase that catalyzes the transfer of a one-carbon moiety, (EC 2.1); an aldehyde or a ketonic moiety, (EC 2.2); an acyl moiety, (EC 2.3); a glycosyl moiety, (EC 2.4); an alkyl or an aryi moiety other than a methyl moiety, (EC 2.5); a nitrogenous moiety, (EC 2.6); a phosphorus-containing moiety, (EC 2.7); a sulfur-containing moiety, (EC 2.8); a selenium-containing moiety, (EC 2.9); or a combination thereof.

A hydrolase catalyses the hydrolysis of a chemical bond. A hydrolase is generally classified based on the chemical bond cleaved or the moiety released or transferred by the hydrolysis reaction. Examples of hydrolases include a hydrolase that catalyzes the hydrolysis of an ester bond, (EC 3.1); a glycosyl released/transferred moiety, (EC 3.2); an ether bond, (EC 3.3); a peptide bond, (EC 3.4); a carbon-nitrogen bond, other than a peptide bond, (EC 3.5); an acid anhydride, (EC 3.6); a carbon-carbon bond, (EC 3.7); a halide bond, (EC 3.8); a phosphorusnitrogen bond, (EC 3.9); a sulfur-nitrogen bond, (EC 3.10); a carbon-phosphorus bond, (EC 3.11); a sulfur-suifur bond, (EC 3.12); a carbon-sulfur bond, (EC 3.13); or a combination thereof.

A lyase catalyzes the cleavage of a chemical bond by reactions other than hydrolysis or oxidation. A lyase is generally classified based on the chemical bond cleaved. Examples of lyases include a lyase that catalyzes the cleavage of a carbon-carbon bond, (EC 4,1); a carbonoxygen bond, (EC 4.2); a carbon-nitrogen bond, (EC 4.3); a carbon-sulfur bond, (EC 4.4); a carbon-halide bond, (EC 4.5); a phosphorus-oxygen bond, (EC 4.6); a other lyase, (EC 4.99) [Is this the right number?]; or a combination thereof.

An isomerase catalyzes a change within one molecule. Examples of isomerases include a racemase or an epimerase, (EC 5.1); a c/s-trans-isomerases, (EC 5.2); an intramolecular isomerase, (EC 5.3); an intramolecular transferase, (EC 5.4); an intramolecular lyase, (EC 5.5); a other isomerases, (EC 5.99) [Is this the right number?]; or a combination thereof.

A ligase catalyses the formation of a chemical bond between two substrates with the hydrolysis of a diphosphate bond of a triphosphate such as ATP. A ligase is generally classified based □n the chemical bond created. Examples of lyases include a ligase that form a carbon— oxygen bond, (EC 6.1); a carbon—sulfur bond, (EC 6.2); a carbon—nitrogen bond, (EC 6.3); a carbon—carbon bond, (EC 6.4); a phosphoric ester bond, (EC 6.5); or a combination thereof.

1. Preferred Enzymes

A preferred enzyme for use in the present invention comprises a hydrolase. A preferred hydrolase comprises an esterase. A preferred esterase comprises an esterase that catalyzes the hydrolysis of an organophosphorus compound. Examples of such preferred esterases are those identified by enzyme commission number EC 3.1.8, the phosphoric triester hydrolases. As used herein, a phosphoric triester hydrolase catalyzes the hydrolytic cleavage of an ester from a phosphorus moiety. Examples of a phosphoric triester hydrolase include an afyldialkylphosphatase, a diisopropyl-fluorophosphatase, or a combination thereof.

An aryldialkylphosphatase (EC 3.1.8.1) is also known by its systemic name aryltriphosphate dialkylphosphohydrolase, and various enzymes in this category have been known in the art by names such as “organophosphate hydrolase; “paraoxonase; A-esterase; aryltriphosphatase; organophosphate esterase; “esterase B1; “esterase E4; paraoxon esterase; pirimiphos-methyloxon esterase; OPA anhydrase”; “organophosphorus hydrolase; phosphotriesterase; PTE; paraoxon hydrolase; “OPH”; and organophosphorus acid anhydrase. An aryldialkylphosphatase catalyzes the following reaction: aryl dialkyl phosphate + H_ZO = an aryl alcohol + dialkyl phosphate. Examples of an aryl dialkyt phosphate include an organophosphorus compound comprising a phosphonic acid ester, a phosphinic acid ester, or a combination thereof.

A diisopropyl-fluorophosphatase (EC 3.1.8.2) is also known by its systemic name diisopropyt-fluorophosphate fluorohydrolase, and various enzymes in this category have been known in the art by names such as “DFPase”; tabunase; “somanase”; “organophosphorus acid anhydrolase; organophosphate acid anhydrase; “OPA anhydrase; diisopropylphosphofluoridase; “diaikylfiuorophosphatase”; “diisopropyl phosphorofluoridate

48p hydrolase”; isopropylphosphorofluoridase; and “diisopropylfluorophosphonate dehalogenase.

A diisopropyl-fluorophosphatase catalyzes the following reaction: diisopropyl fluorophosphate +

H₂O = fluoride + diisopropyl phosphate. Examples of a diisopropyl fluorophosphates include an organophosphorus compound comprising a phosphorus-halide, a phosphorus-cyanide, or a combination thereof.

Examples of phosphoric triester hydrolases and cleaved OP compounds and bond types are shown at Table 8.

<td colspan="6"> Table 8: Phosphoric Triester Hydrolases</td>

<td> Enzyme</td><td colspan="5"> OP Compound Phosphoryl Bond-Type and Phosphoryl Bond Types Cleaved by Enzyme</td>

<td></td><td></td><td> Various OP Pesticides</td><td> Sarin, Soman</td><td> vx, R-VX</td><td> Tabun</td>

<td></td><td> P-C</td><td> P-0</td><td> P-F</td><td> P-S</td><td> P-CN</td>

<td> Qp}p|BrD,C,O,e J.g</td><td> -</td><td> +</td><td> +</td><td> +</td><td> +</td>

<td> Human Paraoxonase^h׳'^J</td><td> +</td><td> +</td><td> +</td><td> ־</td><td> +</td>

<td> OPAA-2^K’'</td><td></td><td> +</td><td> 4*</td><td> -</td><td> +</td>

<td> Squid DFPase™</td><td> -</td><td> -</td><td> +</td><td> -</td><td> -</td>

®Dumas, D. P. et al., 1989a; 'Dumas, D. P. et al., 1989b; 'Dumas, D. P. et ai ΓΪ990; ^dDave, K. I. et al., 1993; ®Chae, Μ. Y. et al., 1994; ^fLai, K. et al., 1995; ”Kolakowski, J.

E. et al., 1997; ^hHassett, C. et al., 1991; ׳Josse, D. et al., 2001; ¹Josse, D. et al., 1999; ^kDeFrank,

J. J. et al. 1993; ׳Cheng, T.-C. et al1996 ״; ™Hoskin, F. C. G. and Roush, A. H., 1982.

A preferred substrate for a composition of the present invention comprises an organophosphorus compound. As used herein, an organophosphorus compound is a compound comprising a phosphoryl center, and further comprises two or three ester linkages. In some aspects, the type of phosphoester bond and/or additional covalent bond at the phosphoryl center classifies an organophosphorus compound. In embodiments wherein the phosphorus is linked to an oxygen by a double bond (P=O), the OP compound is known as an “oxon OP compound or “oxon organophosphorus compound. In embodiments wherein the phosphorus is linked to a sulfur by a double bond (P=S), the OP compound Is known as a “thion OP compound or thion organophosphorus compound. Additional examples of bond-type classified OP compounds include a phosphonocyanate, which comprises a P-CN bond; a ph os pho roa mid ate, which comprises a P-N bond; a phosphotriester, which comprises a P-0 bond; a phosphodiester, which comprises a P-0 bond; a phosphonofluoridate, which comprises a P-F bond; and a

48q phosphonothiolate, which comprises a P־S bond. A dimethyl OP compound comprises two methyl moieties covalently bonded to the phosphorus atom, such as, for example, malathion. A diethyl OP compound comprises two ethoxy moieties covalently bonded to the phosphorus atom, such as, for example, diazinon.

In general embodiments, an OP compound comprises an organophosphorus nerve agent or an organophosphorus pesticide. As used herein, a “nerve agent is an inhibitor of a cholinesterase, including but not limited to, an acetyl cholinesterase, a butyl cholinesterase, or a combination thereof. The toxicity of an OP compound depends on the rate of release of its phosphoryl center (e.g., P-C, P-O, P-F, P-S, P-CN) from the target enzyme (Millard, C. B. etal., 1999). Preferred nerve agents are inhibitors of a cholinesterase (e.g., acetyl cholinesterase) whose catalytic activity is often critical for health and survival in animals, including humans.

Certain OP compounds are so toxic to humans that they have been adapted for use as chemical warfare agents, such as tabun, soman, sarin, cyclosarin, VX, and R-VX. A CWA may be in airborne form and such a formulation is known herein as an OP-nerve gas.” Examples of airborne forms include a gas, a vapor, an aerosol, a dust, or a combination thereof. Examples of an OP compounds that may be formulated as an OP nerve gas include tabun, sarin, soman, VX, GX, or a combination thereof.

In addition to the initial inhalation route of exposure common to such agents, CWAs, especially persistent agents such as VX and thickened soman, pose threats through dermal absorption [In “Chemical Warfare Agents: Toxicity at Low Levels, (Satu M. Somani and James A. Romano, Jr., Eds.) p. 414,2001]. As used herein, a persistent agent is a CWA formulated to be non-volatile and thus remain as a solid or liquid while exposed to the open air for more than three hours. Often after release, a persistent agent may convert from an airborne dispersal form to a solid or liquid residue on a surface, thus providing the opportunity to contact the skin of a human. The toxicities for common OP chemical warfare agents after contact with skin are shown at Table 9.

<td colspan="2"> Table 9: LD_SO Values* of Common Organophosphorus Chemical Warfare Agents</td>

<td> Common OP CWA</td><td> Estimated human LD_SO - percutaneous (skin) administration</td>

<td> Tabun</td><td> 1000 milligrams (mg)</td>

<td> Sarin</td><td> 1700 mg</td>

48r

<td colspan="2"> Table 9: LD_SO Values* of Common Organophosphorus Chemical Warfare Agents</td>

<td> Common OP CWA</td><td> Estimated human LD₅₀ - percutaneous (skin) administration</td>

<td> Soman</td><td> 100 mg</td>

<td> VX</td><td> 10 mg</td>

*LD_s0-the dose need to kill 50% of individuals in a population after administration, wherein the individuals weigh approximately 70 kg.

In some embodiments, an OP compound may be a particularly poisonous organophosphorus nerve agent. As used herein, a particularly poisonous agent is a composition with a LD_M of 35 mg/kg or less for an organism after percutaneous (skin) administration of the agent. Examples of a particularly poisonous OP nerve agent include tabun, sarin, cyclosarin, soman, VX, R-VX, or a combination thereof.

As used herein, “detoxification, “detoxify, detoxified, degradation, degrade, and “degraded refers to a chemical reaction of a compound that produces a chemical byproduct that is less harmful to the health or survival of a target organism contacted with the chemical product relative to contact with the parent compound. OP compounds may be detoxified using chemical hydrolysis or through enzymatic hydrolysis (Yang, Y.-C. et al., 1992; Yang, Y.-C. et al., 1996; Yang, Y.-C. etal., 1990; LeJeune, K. E. etal., 1998a). In general embodiments, the enzymatic hydrolysis is a specifically targeted reaction wherein the OP compound is cleaved at the phosphoryl center's chemical bond resulting in predictable byproducts that are acidic in nature but benign from a neurotoxicity perspective (Kolakowski, J. E. et al., 1997; Rastogi, V, K. et al., 1997; Dumas, D. P. etal., 1990; Raveh, L. et al., 1992). By comparison, chemical hydrolysis can be much less specific, and in the case of VX may produce some quantity of byproducts that approach the toxicity of the intact agent (Yang, Y.-C. et ai., 1996; Yang, Y.-C. et al., 1990). In preferred facets, an enzyme composition of the present invention degrades a CWA, a particularly poisonous organophosphorus nerve agent, or a combination thereof into byproduct that is not particularly poisonous.

Many OP compounds are pesticides that are not particularly poisonous to humans, though they do possess varying degrees of toxicity to humans and other animals. Examples of an OP pesticide include bromophos-ethyl, chiorpyrifos, chlorfenvinphos, chlorothiophos, chlorpyrifos-methyt, coumaphos, crotoxyphos, crufomate, cyanophos, diazinon, dichlofenthion, dichlorvos, dursban, EPN, ethoprop, ethyl-parathion, etrimifos, famphur, fensulfothion, fenthion, fenthrothion, isofenphos, jodfenphos, leptophos-oxon, malathion, methyl-parathion, mevinphos,

48s paraoxon, parathion, parathion-methyl, pirimiphos-ethyl, plrimiphos-methyl, pyrazophos, quinalphos, ronnel, sulfopros, sulfotepp, trichloronate, or a combination thereof. In some embodiments, a composition of the present invention degrades a pesticide into a byproduct that is less toxic to an organism. In specific aspects, the organism is an animal, such as a human.

a. OPH

Organophosphorus hydrolase (E.C.3.1.8.1) has been also refered to in that art as organophosphate-hydrolyzing enzyme, “phosphotriesterase,” “PTE,” organophosphatedegrading enzyme, OP anhydrolase, “OP hydrolase, “OP thiolesterase,” “organophosphorus triesterase, parathion hydrolase, paraoxonase, “DFPase, “somanase, VXase, and “sarinase. As used herein, this type of enzyme will be referred to herein as organophosphorus hydrolase” or “OPH.”

The initial discovery of OPH was from two bacterial strains from the closely related genera: Pseudomonas dlminuta and Flavobacterium spp. (McDaniel, S. et al., 1988; Harper, L. et al., 1988), which encoded identical organophosphorus degrading opd genes on large plasmids (Genbank accession no. M20392 and Genbank accession no. M22863) (copending

U.S. Patent Application Serial No. 07/898,973, incorporated herein in its entirety by reference), ft is likely that Pseudomonas diminuta was derived from the Flavobacterium spp. Subsequently, other such OPH encoding genes have been discovered. The use of any opd gene or their gene product in the described compositions and methods is contemplated. Examples of opd genes and gene products that may be used include the Agrobacterium radiobacter P230 organophosphate hydrolase gene, opdA (Genbank accession no. AY043245; Entrez databank no. AAK85308); the Flavobacterium balustinum opd gene for parathion hydrolase (Genbank accession no. AJ426431; Entrez databank no. CAD19996); the Pseudomonas diminuta phosphodiesterase opd gene (Genbank accession no. M20392; Entrez databank no. AAA98299; Protein Data Bank entries 1JGM, 1DPM, 1EYW, 1EZ2, 1HZY, 1IOB, 1IOD, IPSCand 1PTA); the Flavobacterium sp opd gene (Genbank accession no. M22863; Entrez databank no. AAA24931; ATCC 27551); the Flavobacterium sp. parathion hydrolase opd gene (Genbank accession no. M29593; Entrez databank no. AAA24930: ATCC 27551); or a combination thereof (Home, I. et al., 2002; Somara, S. et al., 2002; McDaniel, C. S. et al1988 ״a; Harper, L. L. et al., 1988; Mulbry, W. W. and Kams, J. S., 1989).

Because OPH possesses the desirable property of cleaving a broad range of OP compounds (Table 8), it is the OP detoxifying enzyme that has been most studied and characterized, with the enzyme obtained from Pseudomonas being the target of focus for most studies. This OPH was initially purified following expression from a recombinant bacutoviral

48t vector in insect tissue culture of the Fall Armyworm, Spodoptera frugiperda (Dumas, D. P. et al., 1989b). Purified enzyme preparations have been shown to be able to detoxify via hydrolysis a wide spectrum of structurally related insect and mammalian neurotoxins that function as acetylcholinesterase inhibitors. Of great interest, this detoxification ability included a number of organophosphorofluoridate nerve agents such as sarin and soman. This was the first recombinant DNA construction encoding an enzyme capable of degrading these potent nerve gases. This enzyme was capable of degrading the common organophosphorus insecticide analog (paraoxon) at rates exceeding 2 x 10⁷ M‘¹ (mole enzyme)‘¹, which is equivalent to the most catalytically efficient enzymes observed in nature. The purified enzyme preparations are capable of detoxifying sarin and the less toxic model mammalian neurotoxin O, O-diisopropyl phosphorofluoridate (DFP”) at the equivalent rates of 50-60 molecules per molecule of enzymedimer per second. In addition, the enzyme can hydrolyze soman and VX at approximately 10% and 1% of the rate of sarin, respectively. The breadth of substrate utility (e.g., V agents, sarin, soman, tabun, cycosarin, OP pesticides) and the efficiency for the hydrolysis exceeds the known abilities of other prokaryotic and eukaryotic organophosphorus acid anydrases, and it is clear that this detoxification is due to a single enzyme rather than a family of related, substrate-limited proteins.

The X-ray crystal structure of Pseudomonas OPH has been determined (Benning, M.

M. et al., 1994; Benning, Μ. M. et al., 1995; Vanhooke, J. L. et al., 1996). Each OPH monomer's active site binds two atoms of Zn²⁺; however, OPH is usually prepared wherein Co²* replaces Zn²*, which enhances catalytic rates. Examples of the catalytic rates (k^!) and specificities (kca/Κπ,) for Co²* substituted OPH against various OP compounds are shown at Table 10 below.

<td colspan="3"> Table 10: Catalytic Activity of Wild-Type OPH binding Co'’</td>

<td> OP Pesticide Substrate</td><td> kcat )</td><td> (Μ¹־ s־')</td>

<td> Paraoxon</td><td> 15000^a</td><td> 1.3 X 10^B</td>

<td></td><td></td><td></td>

<td> OP CWA Substrates</td><td></td><td></td>

<td> Sarin</td><td> 56“</td><td> 8x10’</td>

<td> Soman</td><td> 5^d</td><td> 1 x10⁴</td>

<td> VX</td><td> 0.3^b</td><td> 7.5 x 10²</td>

<td> R-VX</td><td> 0.5^c</td><td> 105</td>

<td> Tabun*</td><td> 77^d</td><td> 7.6x10“</td>

׳Wild-type Zn²* OPH was used in obtaining these kinetic parameters; ^adiSioudi,

B. et al., 1999a; ^bKolakoski, J. E. et al., 1997; ^cRastogi, V. K. et al., 1997; ^dRaveh, L. et al., 1992.

48u

The phosphoryl center of OP compounds is chiral, and Pseudomonas OPH preferentially binds and/or cleaves S_p enantiomers over R_p enantiomers of the chiral phosphorus in various substrates by a ratio of about 10:1 to about 90:1 (Chen-Goodspeed, M, etal., 2001a; Hong, S.-B. and Raushel, F. M., 1999a; Hong, S.-B. and Raushel, F. M., 1999b). CWAs such as VX, sarin, and soman are usually prepared and used as a mixture of sterioisomers of varying toxicity, with VX and sarin having two enantomers each, with the chiral center around the phosphorus of the cleavable bond. Soman possesses four enantomers, with one chiral center based on the phosphorus and an additional chiral center based on a pinacolyl moeity [In Chemical Warfare Agents: Toxicity at Low Levels (Satu M. Somani and James A. Romano, Jr., Eds.) pp 26-29, 2001; Li, W.-S. et aL, 2001; Yang, Y.-C. et aL, 1992; Benshop, Η. P. et al., 1988]. The Sp enantiomer of sarin is about 10⁴ times faster in inactivating acetylcholinesterase than the RP enantiomer (Benschop, Η. P. and De Jong, L. P. A. 1988), while the two Sp enantiomers of soman is about 10⁵ times faster in inactivating acetylcholinesterase than the RP enantiomers (Li, W.-S. et al., 2001; Benschop, Η. P. et al., 1984). Wild-type organophosphorus hydrolase seems to have greater specificity for the less toxic enantiomers of sarin and soman. OPH is about 9-fold faster cleaving an analog of the R_P enantiomer of sarin relative to an analog of the S_p enantiomer, and about 10-fold faster in cleaving analogs of the R_o enantiomers of soman relative to analogs of the S_c enantiomers (Li, W.-S. et al., 2001).

b. Paraoxonase

Human paraoxonase (EC 3.1.8.1), is a calcium dependent protein, and is also known as an “arylesterase or aryl-ester hydrolase (Josse, D. etal., 1999; Vitarius, J. A. and Sultanos, L.

G., 1995). Examples of the human paraoxonase (ΉΡΟΝΓ) gene and gene products can be accessed at (Genbank accession no. M63012; Entrez databank no. AAB59538) (Hassett,

C. etal., 1991).

c. Carboxylases

It is contemplated that a carboxylase gene isolated from an animal may be used as an organophosphate hydrolase in the present invention. As used herein, a carboxylase or “allesterase (EC 3.1.1.1) is an enzyme that hydrolytically cleaves carboxylic esters (e.g., C-0 bonds). As is well known to those of ordinary skill in the art, most genes in eukaryatic organisms have multiple alleles which comprise varient nucleotide and/or expressed protein sequences for a particular gene. Certain insect species have been identified with reduced carboxylase activity and enhanced resistance to OP compounds such as malathion or diazinon. Examples of insect species include Plodia interpunctella, Chrysomya putoria, Lucilia cuprina, and Musca domestica. In particular, an allele of a carboxylase gene possessing organophosphate hydrolase (EC 3.1.8.1) activity is thought to be responsible for OP compound resistance. Examples of such carboxylase

48v genes include alleles isolated from Lucilia cuprina (Genbank accession no. U56636; Entrez databank no. AAB67728), Musca domestica (Genbank accession no. AF133341; Entrez databank no. AAD29685), or a combination thereof (Claudianos, C. etal., 1999; Campbell, P.

M. et al., 1998; Newcomb, R. D. et al., 1997). Additionally, carboxylases or carbamoyl lyases are useful against the carbamate nerve agents, and are specifically contemplated for use in biomolecufe composition of the present invention for use against such agents.

d. OPAAs, Prolidases, Aminopeptideases and PepQ

Organophosphorus acid anhydrolases (E.C.3.1.8.2), known as OPAAs,” have been isolated from microorganisms and identified as enzymes that detoxify OP compounds (Serdar, C. M. and Gibson, D. T., 1985; Mulbry, W. W. et aL, 1986; DeFrank, J. J. and Cheng, T.-C,, 1991). The better-characterized OPAAs have been isolated from Altermonas species, such as Alteromonas sp JD6.5, Alteromonas haloplanktis and Altermonas undina (ATCC 29660) (Cheng,

T.-C. et al., 1996; Cheng, T.-C. et al., 1997; Cheng, T. C. etal., 1999; Cheng, T.-C. etal., 1993). Examples of OPAA genes and gene products that may be used include the Alteromonas sp JD6.5 opaA gene, (GeneBank accession no. U29240; Entrez databank no. AAB05590); the Alteromonas haloplanktis prolidase gene (GeneBank accession no. U56398; Entrez databank AAA99824; ATCC 23821); or a combination thereof (Cheng.T.C. et al., 1996; Cheng, T.-

C. et al, 1997). The wild-type encoded OPAA from Alteromonas sp JD6.5 is 517 amino acids, while the wild-type encoded OPAA from Alteromonas haloplanktis is 440 amino acids (Cheng, T.

C. et al., 1996; Cheng, T.-C. et al., 1997). The Alteromonas OPAAs accelerates the hydrolysis of phosphotrlesters and phosphofiuoridates, including cyclosarin, sarin and soman (Table 11).

<td colspan="4"> Table 11. Catalytic Activity of Wild-Type OPAAS</td>

<td></td><td colspan="3"> heat (s~ ) per species OPAA per OP Substrate</td>

<td> OP Compound Substrate</td><td> A. sp JD6.5</td><td> A. haloplanktis</td><td> A. undina</td>

<td> DFP</td><td> 1650^a</td><td> 575^a</td><td> 1239^a</td>

<td> OP CWA Substrates</td><td></td><td></td><td></td>

<td> Sarin</td><td> 611“</td><td> 257⁰</td><td> 376“</td>

<td> Cyclosarin</td><td> *׳1650</td><td> 269^a</td><td> 1586“</td>

<td> Soman</td><td> 3145^a</td><td> 1389°</td><td> 2496^a</td>

<td> Tabun</td><td> 85^a</td><td> 113“</td><td> 292^s</td>

^aCheng, T. C. etal., 1999

Similar to OPH, OPAA from Alteromonas sp JD6.5 (“OPAA-2) has a general binding and cleavage preference up to 112:1 for the S_p enantiomers of various p-nitrophenyl phosphotriesters (Hill, C. M. et al., 2000). Additionally, OPAA from Alteromonas sp JD6.5 is over 2 fold faster at

48w cleaving an S_p enantiomer of a sarin analog, and over 15-fold faster in cleaving analogs of the

Ra enantiomers of soman relative to analogs of the S_c enantiomers (Hill, C. M. et al., 2001).

Additionally, a prolidase (“imidodipeptidase, “proline dipeptidase, “peptidase D, gpeptidase), PepQ and/or aminopeptidase P gene or gene product with OPAA activity, or a functional equivalent thereof may be used in the present invention. OPAAs possess sequence and structural similarity to human prolidase, Escherichia coli aminopeptidase P and Escherichia coli PepQ (Cheng, T.-C. etal., 1997; Cheng, T.-C. etal., 1996). A prolidase or a PepQ protein (E.C. 3.4.13.9) hydrolyzes a C-N bond of a dipeptide with a prolyl residue at the carboxylterminus, and OPAAs are also classified as prolidases. An aminopeptidase P (EC 3.4.11,9) hydrolyzes the C-N amino bond of a proline at the penultimate position from the amino terminus of an amino acid sequence. Partly purified human and porcine prolidase demonstrated the ability to cleave DFP and G-type nerve agents (Cheng, T.-C. et. al., 1997). Examples of prolidase genes and gene products include the Mus musculus prolidase gene (GeneBank accession no. D82983; Entrez databank no. BAB11685); the Homo sapien prolidase gene (GeneBank accession no. J04605; Entrez databank AAA60064); the Lactobacillus helveticus prolidase (PepQ) gene (GeneBank accession no. AF012084; Entrez databank AAC24966); the Escherichia coli prolidase (pepQ”) gene (GeneBank accession no. X54687; Entrez databank CAA38501); the Escherichia coli aminopeptidase P (pepP”) gene (GeneBank accession no. D00398; Entrez databank BAA00299; Protein Data Bank entries 1A16, 1AZ9, 1JAW and 1M35); or a combination thereof (Ishii, T. et al., 1996; Endo, F. et at., 1989; Nakahigashi, K. and Inokuchi,

H., 1990; Yoshimoto, T. et a!1989 ״).

e. Squid-Type DFPases

As used herein, a “squid-type DFPase (EC 3.1.8.2) refers to an enzyme that catalyzes the cleavage of both DFP and soman, and is isolated from organisms of the Loligo genus. Generally, a squid-type DFPase cleaves DFP at a faster rate than soman. Squid-type DFPases include, for example, a DFPase from Loligo vulgaris, Loligo pealei, Loligo opalescens, or a combination thereof (Hoskin, F. C. G. et al., 1984; Hoskin, F. C. G. et al., 1993; Garden, J. M. et al., 1975).

A well-characterized example of a squid-type DFPase includes the DFPase that has been isolated from the optical ganglion of Loligo vulgaris (Hoskin, F. C. G. etai., 1984). This squid-type DFPase cleaves a variety of OP compounds, including DFP, sarin, cyclosarin, soman, and tabun (Hartleib, J. and Ruterjans, H2001 ״a). The gene encoding this squid-type DFP has been isolated, and can be accessed at GeneBank accession no. AX018860 (international patent publication: WO 9943791-A). Further, this enzyme's X-ray crystal structure has been determined

48x (Protein Data Bank entry 1E1 A) (Koepke, J. et al., 2002: Scharff, E. I. et al., 2001). This squidtype DFPase binds two Ca²* ions, which are important in catalytic activity and enzyme stability (Hartleib, J. et al., 2001). Both the DFPase from Loligo vulgaris and Loligo pealei are susceptible to proteolytic cleavage into a 26-kDa and 16 kDa fragments, and the fragments from Loligo vulgaris are capable of forming active enzyme when associated together (Hartleib, J. and Ruterjans, H., 2001a).

f. Mazur-Type DFPases

As used herein, a Mazur-type DFPase (EC 3.1.8.2) refers to an enzyme that catalyzes the cleavage of both DFP and soman. Generally, Mazur-type DFPases cleaves soman at a faster rate than DFP. Examples of a Mazur-type DFPases include the DFPase isolated from mouse liver (Billecke, S. S. et al., 1999), which may be the same as the DFPase known as SMP30 (Fujita,T. et al., 1996; Billecke, S. S. et al., 1999; Genebank accession no. U28937; Entrez databank AAC52721); a DFPase isolated from rat liver (Little, J. S. et al., 1989); a DFPase isolated from hog kidney; a DFPase isolated from Bacillus stearothermophiius strain OT, a DFPase isolated from Escherichia coli (ATCC25922) (Hoskin, F. C. G. et al., 1993; Hoskin, F. C. G, 1985); or a combination thereof,

g. Other Phosphoric Triester Hydrolases

It is contemplated that any phosphoric triester hydrolase that is known in the art may be used in preferred embodiments of the present invention. An example of an additional phosphoric triester hydrolase includes the product of the gene, mpd, (GenBank accession number AF338729; Entrez databank AAK14390) isolated from Plesiomonas sp. strain M6 (Zhongli,

C. et al., 2001). Other examples include the phosphoric triester hydrolase identified in a Xanthomonas sp. (Tchelet, R. et al., 1993); Tetrahymena (Landis, W. G. et al., 1987); certain plants such as Myriophyilum aquaticum, Spirodela origorrhiza L. Elodea Canadensis and Zea mays (Gao, J. et al. 2000; Edwards, R. and Owen, W. J., 1988); and in hen liver and brain (DiazAlejo, N. et al., 1998). Additional, cholinesterases (e.g., an acetyl cholinesterase) with OP degrading activity have been identified in insects resistant OP pesticides (see, for example, Baxter, G. D. et al., 1998; Baxter, G. D. et aL, 2002; Rodrigo. L. et al., 1997, Vontas, J. G., et al., 2002; Walsh, S. B., et al., 2001; Zhu, K. Y., et al., 1995), and are contemplate for use a bimolecular composition of the present invention.

2. Functional Equivalents of Wild-Type Enzymes

It is possible to optimize a proteinaceous molecule with a defined amino acid sequence and/or length for one or more properties. An alteration in a desirable property is possible because such molecules can be manipulated, for example, by chemical modification, as

48y described herein or 3s would be known to one of ordinary skill in the art, in light of the present disclosures. As used herein “alter” or “alteration” may result in an increase or a decrease in the measured value for a particular property. As used herein a “property, in the context of an proteinaceous molecule, includes, but is not limited to, a ligand binding property, a catalytic property, a stability property, a property related to environmental safety, or a combination thereof. Examples of a catalytic property that may be altered include a kinetic parameter, such as K_m, a catalytic rate (k_cat) for a substrate, an enzyme’s specificity for a substrate (k_cat/K_m), or a combination thereof. Examples of a stability property that may be altered include thermal stability, half-life of activity, stability after exposure to a weathering condition, or a combination thereof. Examples of a property related to environmental safety include an alteration in toxicity, antigenicity, bio-degradability, or a combination thereof. However, as would be readily apparent to one of ordinary skill in the art, an alteration to increase an enzyme's catalytic rate for a substrate, an enzyme's specificity for a substrate, a proteinaceous molecule's thermal stability, a proteinaceous molecule's half-life of activity, or a proteinaceous molecule’s stability after exposure to a weathering condition may be preferred for some applications, while a decrease in toxicity and/or antigenicity for a proteinaceous molecule may be preferred in additional applications. An enzyme comprising a chemical modification that function as an enzyme of the present invention is a functional equivalent to, and in accordance with, an un-modified enzyme.

It is also understood by those of skill in the art that there is a limit to the number of chemical modifications that can be made to an enzyme of the present invention before a preferred property is undesirably altered. However, in light of the disclosures herein of assays for determining whether a composition possesses one or more desirable properties, including, for example, a preferred enzymatic activity, a stability property, etc., and that which is known in the art regarding such assays, it is well within the ability of one of ordinary skill in the art to determine whether a given chemical modification to an enzyme of the present invention produces a molecule that still possesses a suitable set of properties for use in a particular application. In certain aspects, a functional equivalent enzyme comprising a plurality of different chemical modifications can be produced in accordance with the present invention.

It is particularly contemplated that a functional equivalent enzyme comprising a structural analog and/or sequence analog may possess an enhanced desirable property and/or a reduced undesirable property, in comparison to the enzyme upon which it is based. All such functional equivalent enzymes described herein, or as would be known to one of ordinary skill in the art in light of the present disclosures, are considered part of the present invention. As used herein, a “structural analog refers to one or more chemical modifications to the peptide backbone or

48z non-side chain chemical moieties of a proteinaceous molecule. In certain aspects, a subcomponent of an enzyme such as an apo-enzyme, a prosthetic group, a co-factor, or a combination thereof, may be modified to produce a functional equivalent structural analog. In particular facets, such an enzyme sub-component that does not comprise a proteinaceous molecule may be altered to produce a functional equivalent structural analog of an enzyme when combined with the other sub-components. As used herein, a '¹sequence analog refers to one or more chemical modifications to the side chain chemical moieties, also known herein as a residue of one or more amino acids that define a proteinaceous molecule's sequence. Often such a sequence analog” comprises an amino acid substitution, which is generally produced by recombinant expression of a nucleic acid comprising a genetic mutation to produce a mutation in the expressed amino acid sequence.

As used herein, an amino acid' may be a common or uncommon amino acid. The common amino acids include: alanine (Ala, A): arginine (Arg, R); aspartic acid (a.k.a. aspartate; Asp, D); asparagine (Asn, N); cysteine (Cys, C); glutamic acid (a.k.a. glutamate; Glu, E); glutamine (Gin, Q); glycine (Gly, G); histidine (His, H); isoleucine (lie, I); leucine (Leu, L); lysine (Lys, K); methionine (Met, M); phenylalanine (Phe, F); proline (Pro, P); serine (Ser, S); threonine (Thr, T); tryptophan (Trp, W); tyrosine (Tyr, Y); and valine (Val, V). Common amino acids are often biologically produced in the biological synthesis of a peptide or a polypeptide. An uncommon amino acid refers to an analog of a common amino acid, as well as a synthetic amino acid whose side chain is chemically unrelated to the side chains of the common amino acids. Various uncommon amino acids are well known to those of ordinary skill in the art though it is contemplated that in general embodiments, an enzyme of the present invention will be biologically produced, and thus lack or possess relatively few uncommon amino acids prior to any subsequent non-mutation based chemical modifications.

As is well known in the art, the side chains of amino acids comprise moieties with specific chemical and physical properties. Certain side chains contribute to a ligand binding property, a catalytic property, a stability property, a property related to environmental safety, or a combination thereof. For example, cysteines can form covalent bonds between different parts of a contiguous amino acid sequence, or between non-contlguous amino acid sequences to confer enhanced stability to a secondary, tertiary or quaternary structure. In an additional example, the presence of hydrophobic or hydrophilic side chains exposed to the outer environment can alter the hydrophobicity or hydrophilicity of part of a proteinaceous sequence such as in the case of a transmembrane domain that is embedded in a lipid layer of a membrane. In another example, hydrophilic side chains may be exposed to the environment surrounding a proteinaceous molecule, which can enhance the overall solubility of a proteinaceous molecule in a polar liquid,

48aa such as water or a liquid component of a coating. In a further example, various acidic, basic, hydrophobic, hydrophilic, and/or aromatic side chains present at or near a binding site of a proteinaceous structure can affect the affinity for a proteinaceous sequence for binding a ligand and/or a substrate, based on the covalent, ionic, Van der Waal forces, hydrogen bond, hydrophilic, hydrophobic, and/or aromatic interactions at a binding site. Such interactions by residues at or near an active site also contribute to a chemical reaction that occurs at the active site of an enzyme to produce enzymatic activity upon a substrate. As used herein, a residue is at or near another residue or group of residues when it is within 15A, 14A, 13A, 12A, 11A, 10A, 9A, 8A, 7A, 6A, 5A, 4A, 3A, 2A, or 1A the residue or group of residues such as residues identified as contributing to the active site and/or binding site.

Identification of an amino acid whose chemical modification would likely change a desirable property of a proteinaceous molecule can be accomplished using such methods as a chemical reaction, mutation, X-ray crystallography, nuclear magnetic resonance (NMR”), computer based modeling or a combination thereof. Selection of an amino acid on the basis of such information can then be used in the rational design of a mutant proteinaceous sequence that would possess an altered desired property. Preferred alterations include those that alter enzymatic activity to produce a functional equivalent of an enzyme.

For example, many residues of a proteinaceous molecule that contribute to the properties of a proteinaceous molecule comprise chemically reactive moieties. These residues are often susceptible to chemical reactions that can inhibit their ability to contribute to a desirable property of the proteinaceous molecule. Thus, a chemical reaction can be used to identify one or more amino acids comprised within the proteinaceous molecule that may contribute to a desirable property. The identified amino acids then can be subject to modifications such as amino acid substitutions to produce a functional equivalent. Examples of amino acids that can be so chemically reacted include Arg, which can be reacted with butanedione; Arg and/or Lys, which can be reacted with phenylglyoxai; Asp and/or Glu, which can be reacted with carbodiimide and HCI; Asp and/or Glu, which can be reacted with N-ethyl-5-phenylisoxazolium-3'-sulfonate (“Woodward's reagent K”); Asp and/or Glu, which can be reacted with 1,3-dicyclohexyl carbodiimide; Asp and/or Glu, which can be reacted with 1-ethy 1-3-(3dimethylaminopropyljcarbodiimide (“EDC“); Cys, which can be reacted with p-hydroxy mercuribenzoate; Cys, which can be reacted with dithiobisnitrobenzoate (DTNB”); Cys, which can be reacted with iodoacetamide; His, which can be reacted with diethylpyrocarbonate (“DEPC); His, which can be reacted with diazobenzenesulfonic acid (“DBS); His, which can be reacted with 3,7-bis(dimethylamino)phenothiazin-5-ium chloride (methylene blue); Lys, which can be reacted with dimethylsuberimidate; Lys and/or Arg, which can be reacted with 2,448bb dinitrofluorobenzene; Lys and/or Arg, which can be reacted with trinitrobenzene sulfonic acid (“TNBS”); Trp, which can be reacted with 2-hydroxy-5-nitrobenzyl bromide 1-ethyl-3(3dimethylaminopropyl); Trp, which can be reacted with 2-acetoxy-5-nitrobenzyl chloride; Trp, which can be reacted with N-bromosucinimide; Tyr, which can be reacted with N-acetylimidazole (״ΝΑΓ); or a combination thereof (Hartleib, J. and Ruterjans, H., 2001b; Josse, D. et al., 1999; Josse, D. et al., 2001).

In an additional example, the secondary, tertiary and/or quaternary structure of a proteinaceous molecule may be modeled using techniques known in the art, including X-ray crystallography, nuclear magnetic resonance, computer based modeling, or a combination thereof to aid in the identification of active-site, binding site, and other residues for the design and production of a mutant form of an enzyme (Bugg, C. E. et al., 1993; Cohen, A. A. and Shatzmiller,

S. E., 1993; Hruby, V. J., 1993; Moore, G. J., 1994; Dean, P. M., 1994; Wiley, R. A. and Rich, D. H., 1993). The secondary, tertiary and/or quaternary structures of a proteinaceous molecule may be directly determined by techniques such as X-ray crystallography and/or nuclear magnetic resonance to identify amino acids most likely affect one or more desirable properties. Additionally, many primary, secondary, tertiary, and/or quaternary structures of proteinaceous molecules can be obtained using a public computerized database. An example of such a databank that may be used for this purpose is the Protein Data Bank (PDB), which is an international repository of the 3-dimensional structures of many biological macromolecules, and can be accessed at http://www.rcsb.org/pdb/index.html. Additional examples of such databases are listed at: http://www.rcsb.0rg/pdb/links.html#Databases.

Computer modeling can be used to identify amino acids most likely to affect one or more desirable properties. Often, a structurally related proteinaceous molecule comprises primary, secondary, tertiary and/or quaternary structures that are evolutionary conserved In the wild-type protein sequences of various organisms. As would be known to those of ordinary skill in the art, the secondary, tertiary and/or quaternary structure of a proteinaceous molecule can be modeled using a computer to overlay the proteinaceous molecule’s amino acid sequence, which is also known as the “primary structure, onto the computer model of a described primary, secondary, tertiary, and/or quaternary structure of another, structurally related proteinaceous molecule. Often the amino acids that may participate in an active site, a binding site, a transmembrane domain, the general hydrophobicity and/or hydrophilicity of a proteinaceous molecule, the general positive and/or negative charge of a proteinaceous molecule, etc, may be identified by such comparative computer modeling.

48cc

In embodiments wherein an amino acid of particular interest have been identified using such techniques, functional equivalents may be created using mutations that substitute a different amino acid for the identified amino acid of interest. Examples of substitutions of an amino acid side chain to produce a functional equivalent” proteinaceous molecule are also known in the art, and may involve a conservative side chain substitution a non-conservative side chain substitution, or a combination thereof, to rationally alter a property of a proteinaceous molecule. Examples of conservative side chain substitutions include, when applicable, replacing an amino acid side chain with one similar in charge (e.g., an arginine, a histidine, a lysine); similar in hydropathic index; similar in hydrophilicity; similar in hydrophobicity; similar in shape (e.g., a phenylalanine, a tryptophan, a tyrosine); similar in size (e.g., an alanine, a glycine, a serine); similar in chemical type (e.g., acidic side chains, aromatic side chains, basic side chains); or a combination thereof. Conversely, when a change to produce a non-conservative substitution is contemplated to alter a property of proteinaceous molecule, and still produce a functional equivalent proteinaceous molecule, these guidelines can be used to select an amino acid whose side-chains relatively nonsimilar in charge, hydropathic index, hydrophilicity, hydrophobicity, shape, size, chemical type, or a combination thereof. Various amino acids have been given a numeric quantity based on the characteristics of charge and hydrophobicity, called the hydropathic index (Kyte, J. and Doolittle, R. F. 1982), which can be used as a criterion for a substitution. The hydropathic index of the common amino acids are: Arg (-4.5); Lys (-3.9); Asn (-3.5); Asp (-3.5); Gin (-3.5); Glu (-3.5); His (-3.2); Pro (-1.6); Tyr (-1.3); Trp (-0.9); Ser (-0.8); Thr (-0.7); Gly (-0.4); Ala (+1.8); Met (+1.9); Cys (+2.5); Phe (+2.8); Leu (+3.8); Val (+4.2); and lie (+4.5). Additionally, a value has also been given to various amino acids based on hydrophilicity, which can also be used as a criterion for substitution (U.S. Pat. No. 4,554,101). The hydrophilicity values for the common amino acids are: Trp (-3.4); Phe (-2.5); Tyr (-2.3); He (-1.8); Leu (-1.8); Val (-1.5); Met (-1.3); Cys (-1.0); Ala (-0.5); His (-0.5); Pro (-0.5+/-0.1); Thr (-0.4); Gly (0); Asn (+0.2); Gin (+0.2); Ser (+0.3); Asp (+3.0+/-0.1); Glu (+3.0+/-0.1); Arg (+3.0); and Lys (+3.0). In aspects wherein an amino acid is being conservatively substituted for an amino acid whose hydropathic index or hydrophilic value is similar, the difference between the respective index and/or value is preferably within +/- 2, more preferably within +/- 1, and most preferably within +/-0.5. In aspects wherein an amino acid is being non-conservatively substituted for an amino acid whose hydropathic index or hydrophilic value is similar, the difference between the respective index and/or value is preferably greater than +/-0.5, more preferably greater than +/-1, and most preferably greater than +/- 2.

In certain embodiments, a functional equivalent may be produced by a non-mutation based chemical modification to an amino acid, a peptide or a polypeptide. Examples of chemical modifications include, when applicable, a hydroxylation of a proline or a lysine; a phosphorylation of a hydroxyl group of a serine and/or a threonine; a methylation of an alpha-amino group of a

48dd lysine, an arginine and/or a histidine (Creighton, T. E., 1983); adding a detectable label such as a fluorescein isothiocyanate compound (FITC) to a lysine side chain and/or a terminal amine (Rogers, K. R, etal., 1999); covalent attachment of a poly ethylene glycol (Yang, Z. et al., 1995; Kim, C. et al., 1999; Yang, Z. et al., 1996; Mijs, M. et al., 1994); an acylatylation of an amino acid, particularly at the N-terminus; an amination of an amino acid, particularly at the C-terminus (Greene, T. W. and Wuts, P. G. M. Productive Groups in Organic Synthesis, Second Edition, pp. 309-315, John Wiley &amp; Sons, Inc., USA, 1991); a deamidation of an asparagine or a glutamine to an aspartic acid or glutamic acid, respectively; a derivation of an amino acid by a sugar moiety, a lipid, a phosphate, or a farnysyl group; an aggregation (e.g., a dimerization) of a plurality of proteinaceous molecules, whether of identical sequence or varying sequences; a cross-linking of a plurality of proteinaceous molecules of the present invention using a cross-linking agent [e.g., a 1,1-bis(diazoacety!)-2-phenylethane; a glutaraldehyde; a N-hydroxysuccinimide ester; a 3,3’-dithiobis (succinimidyl-propionate); a bis-N-maleimido-1,8-octane]; an ionization of an amino acid into an acidic, basic or neutral salt form; an oxidation of an amino acid; or a combination thereof of any of the forgoing. Such modifications may produce a desirable alteration in a property of a proteinaceous molecule, as would be known to those of ordinary skill in the art. For example, it is contemplated that a N-terminal glycosylation may enhance a proteinaceous molecule's stability (Powell, M.

F. et al., 1993). In an additional example, it is contemplated that substitution of a beta-amino acid isoserine for a serine may enhance the aminopeptidase resistance a proteinaceous molecule (Caller, B. S. etal., 1993).

A proteinaceous molecule for use in the present invention may comprise a proteinaceous molecule longer or shorter than the wild-type amino acid sequences specifically disclosed herein, or that would be known to those of ordinary skill in the art in light of the present disclosure. For example, an enzyme comprising longer or shorter sequences is encompassed as part of the present invention, insofar as it retains enzymatic activity. In some embodiments, a proteinaceous molecule for use in the present invention may comprise one or more peptide and/or polypeptide sequences. In certain embodiments, a modification to a proteinaceous molecule may add and/or subtract one or two amino acids from a peptide and/or polypeptide sequence. In other embodiments, a change to a proteinaceous molecule may add and/or remove one or more peptide and/or polypeptide sequences. Often a peptide or a polypeptide sequence may be added or removed to confer or remove a specific property from the proteinaceous molecule, and numerous examples of such modifications to a proteinaceous molecule are described herein, particularly in reference to fusion proteins. In particular, the native OPH of Pseudomonas diminuta is produced with a short amino acide sequence at its N-terminas that promotes the exportation of the protein through the cell membrane and is later cleaned. Thus, in certain

48ee embodiment, this signal sequence amino acide sequence is deleted by genetic modification in the DNA construction placed into Escherichia coli host cells in order to enhance its production.

As used herein, a peptide comprises a contiguous molecular sequence from 3 to 100 amino acids in length, including all intermediate ranges and combinations thereof. A sequence of a peptide may be 3 to 100 amino acids in length, including all intermediate ranges and combinations thereof. As used herein a polypeptide comprises a contiguous molecular sequence 101 amino acids or greater. Examples of a sequence length of a polypeptide include 101 to 10,000 amino acids, including all intermediate ranges and combinations thereof. As used herein a protein is a proteinaceous molecule comprising a contiguous molecular sequence three amino acids or greater in length, matching the length of a biologically produced proteinaceous molecule encoded by the genome of an organism.

It is recognized that removal of one or more amino acids from an enzyme s sequence may reduce or eliminate a detectable, desirable property such asenzymatic activity, and therefore would not be preferred. However, it is further contemplated that a longer sequence, particularly a proteinaceous molecule that consecutively or non-consecutively comprises or even repeats one or more enzymatic sequences disclosed herein, or as would be known to those of ordinary skill in the art in light of the present disclosure, would be encompassed within the present invention. Additionally, fusion proteins may be bioengineered to comprise a wild-type sequence and/or a functional equivalent of an enzyme sequence and an additional peptide or polypeptide sequence that confers a desirable property and/or function.

a. OPH Functional Equivalents

Using recombinant DNA technology, wild-type and mutant forms of the opd gene have been expressed, predominantly in Escherichia coli, for further characterization and analysis. Unless otherwise noted, the various OPH enzymes, whether wild-type or mutants, that act as functional equivalents were prepared using the OPH genes and encoded enzymes first isolated from Pseudomonas diminuta and Flavobacterium spp.

OPH normally binds two atoms of Zn²⁺ per monomer when endogenously expressed. While binding Zn²⁺, this enzyme is one of the most stable dimeric enzymes known, with a thermal temperature of melting {^,7/) of approximately 75°C and a conformational stability of approximately 40 killocalorie per mole (“kcal/mol) (Grimsley, J. K. et al., 1997). However, structural analogs have been made wherein Co^z+, Fe² , Cu^z , Mn² , Cd , or Ni are bound instead to produce enzymes with altered stability and rates of activity (Omburo, G. A. etal., 1992). For example, Co^z+ substituted OPH does possess a reduced conformational stability (~22 kcal/mol). But this reduction in thermal stability is offset by the superior catalytic

48ff activity of Co²⁺ substituted OPH in degrading various OP compounds. For example, five-fold or greater rates of detoxification of sarin, soman, and VX were measured for Co²* substituted OPH relative to OPH binding Zn²* (Kolakoski, J. E.etal., 1997). It is contemplated that structural analogs of an OPH sequence may be prepared comprising a Zn²⁺, Co²⁺, Fe²*, Cu^z+, Mn²⁺, Cd²⁺, Ni²*, or a combination thereof. Generally, changes in the bound metal can be achieved by using ceil growth media during cell expression of the enzyme wherein the concentration of a metal present is defined, and/or removing the bound metal with a chelator (e.g., 1,10-phenanthroline; 8hydroxyquinoline-5-sulfphonic acid; ethylenediamlnetetraacetic acid) to produce an apo-enzyme, followed by reconstitution of a catalytically active enzyme by contact with a selected metal (Omburo, G. A. et al., 1992; Watkins, L. M. et al., 1997a; Watkins, L M. et al., 1997b). It is further contemplated that structural analogs of an OPH sequence may be prepared to comprise only one metal atom per monomer.

In an additional example, OPH structure analysis has been conducted using NMR (Omburo, G. A. et al1993 ״). in a further example, the X-ray crystal structure for OPH has been determined (Benning, Μ. M. et al., 1994; Benning, Μ. M. et al., 1995; Vanhooke, J.

L. et al., 1996), including the structure of the enzyme while binding a substrate, further identifying residues involved in substrate binding and catalytic activity (Benning, Μ. M. et al., 2000). From these structure evaluations, the amino acids His55, His57, His201, His230, Asp301, and the carbamylated lysine, Lys169, have been identified as coordinating the binding of the active site metal. Additionally, the positively charged amino acids His55, His57, His201, His230, His254, and His257 are counter-balanced by the negatively charged amino acids Asp232, Asp233, Asp235, Asp 253, Asp301, and the carbamylated lysine Lys169 at the active site area. A water molecule and amino acids His55, His57, Lys169, His201, His230, and Asp301 are thought to be involved in direct metal binding. The amino acid Asp301 is thought to aid a nucleophilic attack by a bound hydroxide upon the phosphorus to promote cleavage of an OP compound, while the amino acid His354 may aid the transfer of a proton from the active site to the surrounding liquid in the latter stages of the reaction (Raushel, F. M., 2002). The amino acids His254 and His257 are not thought to be direct metal binding amino acids, but may be residues that interact (e.g., a hydrogen bond, a Van der Waal interaction) with each other and other active site residues, such as residues that directly contact a substrate or bind a metal atom. In particular, amino acid His254 is thought to interact with the amino acids His230, Asp232, Asp233, and Asp301. Amino acid His257 is thought to be a participant in a hydrophobic substrate-binding pocket. The active site pocket comprises various hydrophobic amino acids, Trp131, Phe132, Leu271, Phe306, and Tyr309. These amino acids may aid the binding of hydrophobic OP compounds (Benning, M.

M. etal., 1994; Benning, Μ. M. etal., 1995; Vanhooke, J. L. et al., 1996). Electrostatic interactions may occur between phosphoryl oxygen, when present, and the side chains of Trp131

48gg and His201. Additionally, the side chains of amino acids Trp131, Phe132, and Phe306 are thought to be orientated toward the atom of the cleaved substrate’s leaving group that was previously bonded to the phosphorus atom (Watkins, L. M. et al., 1997a).

Substrate binding subsites known as the small subsite, the large subsite, and the leaving group subsite have been identified (Benning, Μ. M. et al., 2000; Benning, Μ. M. et al., 1994; Benning, Μ. M. et al., 1995; Vanhooke, J. L. et al., 1996). The amino acids Gly60, lle106, Leu303, and Ser308 are thought to comprise the small subsite. The amino acids Cys59 and Ser61 are near the small subsite, but with the side chains thought to be orientated away from the subsite. The amino acids His254, His257, Leu271, and Met317 are thought to comprise the large subsite. The amino acids Trp131, Phe132, Phe306, and Tyr309 are thought to comprise the leaving group subsite, though Leu271 is sometimes considered part of this subsite as well (Watkins, L. M. et al., 1997a). Comparison of this opd product with the encoded sequence of the opdA gene from Agrobacterium radiobacter P230 revealed that the large subsite possessed generally larger residues that affected activity, specifically the amino acids Arg254, Tyr257, and Phe271 (Horne, I. et al., 2002). Few electrostatic interactions are apparent from the X-ray crystal structure of the inhibitor bound by OPH, and it is thought that hydrophobic interactions and the size of the subsites affect substrate specificity, including steriospecificity for a stereoisomer, such as a specific enantiomer of an OP compound's chiral chemical moiety (Chen-Goodspeed, M. et al., 2001b).

Using the sequence and structural knowledge of OPH, numerous mutants of OPH comprising a sequence analog have been specifically produced to alter one or more properties relative to a substrate's cleavage rate (k_cat) and/or specificity (k_cat/K_m). Examples of OPH sequence analog mutants include H55C, H57C, C59A, G60A, S61A, I106A, I106G, W131A, W131F, W131K, F132A, F132H, F132Y, L136Y, L140Y, H201C, H230C, H254A, H254R, H254S, H257A, H257L, H257Y, L271A, L271Y, L303A, F306A, F306E, F306H, F306K, F306Y, S308A, S308G, Y309A, M317A, M317H, M317K, M317R, H55C/H57C, H55C/H201C, H55C/H230C, H57C/H201C, H57C/H230C, A80V/S365P, I106A/F132A, I106A/S308A, I106G/F132G, 1106G/S308G, F132Y/F306H, F132H/F306H, F132H/F306Y, F132Y/F306Y, F132A/S308A, F132G/S308G, L182S/V310A, H201C/H230C, H254R/H257L, H55C/H57C/H201C,

H55C/H57C/H230C, H55C/H201C/H230C, I106A/F132A/H257Y, I106A/F132A/H257W,

I106G/F132G/S308G, L130M/H257Y/I274N, H257Y/I274N/S365P, H55C/H57C/H201C/H230C, I106G/F132G/H257Y/S308G, or A14T/A80V/L185R/H257Y/I274N (Li, W.-S. et al., 2001; Gopal, S. et al., 2000; Chen-Goodspeed, M. et al., 2001a; Chen-Goodspeed, M. et al., 2001b; Watkins, L. M. et al., 1997a; Watkins, L. M. et al., 1997b; diSioudi, B. et al., 1999; Cho, C. M.48hh

H. et al., 2002; Shim, H. et al., 1996; Raushel, F. M., 2002; Wu, F. et a!., 2000a; diSioudi, B.

D. etal., 1999).

For example, the sequence and structural information has been used in production of mutants of OPH possessing cysteine substitutions at the metal binding histidines His55, His57, His201, and His230. OPH mutants H55C, H57C, H201C, H230C, H55C/H57C, H55C/H201C, H55C/H230C, H57C/H201C, H57C/H230C, H201C/H230C, H55C/H57C/H201C,

H55C/H57C/H230C, H55C/H201C/H230C, H57C/H201C/H230C, and

H55C/H57C/H201C/H230C were produced binding either Zn²*; Co²* or Cd²*. The H57C mutant had between 50% (i.e., binding Cd²⁺, Zn²*) and 200% (i.e., binding Co²*) wild-type OPH activity for paraoxon cleavage. The H201C mutant had about 10% activity, the H230C mutant had less than 1% activity, and the H55C mutant bound only one atom of Co²* and possessed little detectable activity, but may still be useful if possessing a desirable property (e.g., enhanced stability) (Watkins, L. M., 1997b).

In an additional example, the sequence and structural information has been used in production of mutants of OPH possessing altered metal binding and/or bond-type cleavage properties. OPH mutants H254R, H257L, and H254R/H257L have been made to alter amino acids that are thought to interact with nearby metal-binding amino acids. These mutants also reduced the number of metal ions (i.e., Co^z+, Zn²⁺) binding the enzyme dimer from four to two, while still retaining 5% to greater than 100% catalytic rates for the various substrates. These reduced metal mutants possess enhanced specificity for larger substrates such as NPPMP and demeton-S, and reduced specificity for the smaller substrate diisopropyl fluorophosphonate (diSioudi, B. et al., 1999). In a further example, the H254R mutant and the H257L mutant each demonstrated a greater than four-fold increase in catalytic activity and specificity against VX and its analog demeton S. The H257L mutant also demonstrated a five-fold enhanced specificity against soman and its analog NPPMP (diSioudi, B. D. et al., 1999).

In an example, specific mutants of OPH (a phosphotriesterase), were designed and produced to aid phosphodiester substrates to bind and be cleaved by OPH. These substrates either comprised a negative charge and/or a large amide moiety. A M317A mutant was created to enlarge the size of the large subsite, and M317H, M317K, and M317R mutants were created to incorporate a cationic group in the active site. The M317A mutant demonstrated a 200-fold cleavage rate enhancement in the presence of aikyiamines, which were added to reduce the substrate’s negative charge. The M317H, M317K, and M317R mutants demonstrated modest improvements in rate and/or specificity, including a 7-fold kcat/Km improvement for the M317K mutant (Shim, H. et al., 1998).

48ii

In a further example, the W131K, F132Y, F132H, F306Y, F306H, F306K, F306E, F132H/F306H, F132Y/F306Y, F132Y/F306H, and F132H/F306Y mutants were made to add or change the side chain of active site residues to form a hydrogen bond and/or donate a hydrogen to a cleaved substrate's leaving group, to enhance the rate of cleavage for certain substrates, such as phosphofluoridates. The F132Y, F132H, F306Y, F306H, F132H/F306H, F132Y/F306Y, F132Y/F306H, and F132H/F306Y mutants all demonstrated enhanced enzymatic cleavage rates, of about three- to ten-fold improvement, against the phosphonofluoridate, diisopropyl fluorophosphonate (Watkins, L. M. et al., 1997a).

In an additional example, OPH mutants W131F, F132Y, L136Y, L140Y, L271Y and H257L were designed to modify the active site size and placement of amino acid side chains to refine the structure of binding subsites to specifically fit the binding of a VX substrate. The refinement of the active site structure produced a 33% increase in cleavage activity against VX in the L136Y mutant (Gopal, S. et al., 2000).

Various mutants of OPH have been made to alter the steriospecificity, and in some cases, the rate of reaction, by substitutions in substrate binding subsites. For example, the C59A, G60A, S61A, I106A, W131A, F132A, H254A, H257A, L271A, L303A, F306A, S308A, Y309A, and M317A mutants of OPH have been produced to alter the size of various amino acids associated with the small subsite, the large subsite and the leaving group subsite, in order to alter enzyme activity and selectivity, including sterioselectivity, for various OP compounds. The G60A mutant reduced the size of the small subsite, and decreased both rate (k״t) and specificity (k_cat/K_a) for Rp-enantiomers, thereby enhancing the overall specificity for some S_p-enantiomers to over 11,000:1. Mutants I106A and S308A, which enlarged the size of the small subsite, as well as mutant F132A, which enlarged the leaving group subsite, all increased the reaction rates for Rp-enantiomers and reduced the specificity for S_p-enantiomers (Chen-Goodspeed, M. etal., 2001a).

Additional mutants I106A/F132A, I106A/S308A, F132A/S308A, I106G, F132G, S308G, I106G/F132G, I106G/S308G, F132G/S308G, and I106G/F132G/S308G were produced to further enlarge the small subsite and leaving group subsite. These OPH mutants demonstrated enhanced selectivity for R_p-enantiomers. Mutants H254Y, H254F, H257Y, H257F, H257W, H257L, L271Y, L271F, L271W, M317Y, M317F, and M317W were produced to shrink the large subsite, with the H257Y mutant, for example, demonstrating a reduced selectivity for S_penantiomers (Chen-Goodspeed, M. et al., 2001b). Further mutants I106A/H257Y, F132A/H257Y, I106A/F132A/H257Y, I106A/H257Y/S308A, I106A/F132A/H257W, F132A/H257Y/S308A,

48jj

I106G/H257Y, F132G/H257Y, 1106G/F132G/H257Y, 1106G/H257Y/S308G, and

I106G/F132G/H257Y/S308G were made to simultaneously enlarge the small subsite and shrink the large subsite. Mutants such as H257Y, I106A/H257Y, I106G, I106A/F132A, and I106G/F132G/S308G were effective in altering steriospecificity for S_P:R_P enantiomer ratios of some substrates to less than 3:1 ratios. Mutants including F132A/H257Y, I106A/F132A/H257W, I106G/F132G/H257Y, and I106G/F132G/H257Y/S308G demonstrated a reversal of selectivity for Sp.Rp enantiomer ratios of some substrates to ratios from 3.6:1 to 460:1. In some cases, such a change in steriospecificity was produced by enhancing the rate of catalysis of a less preferred R_penantiomer with little change on the rate of S_p enantiomer cleavage (Chen-Goodspeed, M. et al., 2001b; Wu, F. et al., 2000a).

Such alterations in sterioselectivity can enhance OPH performance against a specific OP compound that is a preferred target of detoxification, including a CWA. Enlargement of the small subsite by mutations that substitute the Ile106 and Phe132 residues with the less bulky amino acid alanine and/or reduction of the large subsite by a mutation that substitutes His257 with the bulkier amino acid phenylalanine increased catalytic rates for the S_p-isomer; anddecreased the catalytic rates for the R_p-isomers of a sarin analog, thus resulting in a triple mutant, I106A/F132A/H257Y, with a reversed sterioselectivity such as a S_P:R_P preference of 30:1 for the isomers of the sarin analog. A mutant of OPH designated G60A has also been created with enhanced steriospecificity relative to specific analogs of enantiomers of sarin and soman (Li, W.S. et al, 2001; Raushel, F. M, 2002). Of greater interest, these mutant forms of OPH have been directly assayed against sarin and soman nerve agents, and demonstrated enhanced detoxification rates for racemic mixtures of sarin or soman enantiomers. Wild-type OPH has a kct for sarin of 56 s¹־, while the I106A/F132A/H257Y mutant has kCBt for sarin of 1000 s“¹. Additionally, wild-type OPH has a kcat for soman of 5 s'¹, while the G60A Mutant has k_cal for soman of 10 s“¹ (Kolakoski, Jan E. et al. 1997; Li, W.-S. et al, 2001).

It is also possible to produce a mutant enzyme with an enhanced enzymatic property against a specific substrate by evolutionary selection rather than rational design. Such techniques can screen hundreds or thousands of mutants for enhanced cleavage rates against a specific substrate. The mutants identified may possess substitutions at amino acids that have not been identified as directly comprising the active site, or its binding subsites, using techniques such as NMR, X-ray crystallography and computer structure analysis, but still contribute to activity for one or more substrates. For example, selection of OPH mutants based upon enhanced cleavage of methyl parathion identified the A80V/S365P, L182S/V310A, I274N, H257Y, H257Y/I274N/S365P, L130M/H257Y/I274N, and A14T/A80V/L185R/H257Y/I274N mutants as having enhanced activity. Amino acids Ile274 and Val310 are within 10A of the active site,

48kk though not originally identified as part of the active site from X-ray and computer structure analysis. However, mutants with substitutions at these amino acids demonstrated improved activity, with mutants comprising the I274N and H257Y substitutions particularly active against methyl parathion. Additionally, the mutant, A14T/A80V/L185R/H257Y/I274N, further comprising a L185R substitution, was most active having a 25-fold improvement against methyl parathion (Cho, C. M.-H. et al., 2002).

In an example, a functional equivalent of OPH may be prepared that lacks the first 29-31 amino acids of the wild-type enzyme. The wild-type form of OPH endogenously or recombinantly expressed in Pseudomonas or Flavobacterium removes the first N-terminal 29 amino acids from the precursor protein to produce the mature, enzymatically active.protein (Mulbry, W. and Karns, J1989 ״; Serdar, C. M. et al., 1989). Recombinant expressed OPH in Gliocladium virens apparently removes part or all of this sequence (Dave, K. I. et al., 1994b). Recombinant expressed OPH in Streptomyces lividans primarily has the first 29 or 30 amino acids removed during processing, with a few percent of the functional equivalents having the first 31 amino acids removed (Rowland, S. S. et al., 1992). Recombinant expressed OPH in Spodoptera frugiperda cells has the first 30 amino acids removed during processing (Dave, K. I. et al., 1994a).

The 29 amino acid leader peptide sequence targets OPH enzyme to the cell membrane in Escherichia coli, and this sequence is partly or fully removed during cellular processing (Dave,

K. I. et al., 1994a; Miller, C. E., 1992; Serdar, C. M. et al., 1989; Mulbry, W. and Karns, J., 1989). The association of OPH comprising the leader peptide sequence with the cell membrane in Escherichia coii expression systems seems to be relatively weak, as brief 15 second sonication releases most of the activity into the extracellular environment (Dave, K. I. etal., 1994a). For example, recombinant OPH often is expressed without this leader peptide sequence to enhance enzyme stability and expression efficiency in Escherichia coli (Serdar, C. M., et al. 1989). In another example, recombinant expression efficiency in Pseudomonas putida for OPH was improved by retaining this sequence, indicating that different species of bacteria may have varying preferences for a signal sequence (Walker, A. W. and Keasling, J. D., 2002). However, it is contemplated that one of ordinary skill in the art can easily modify the length of an enzymatic sequence to optimize expression or other properties in a particular organism, or select a cell with a relatively good ability to express a biomotecule, in light of the present disclosures and methods known in the art (see U.S. Patent Nos. 6,469,145, 5,589,386 and 5,484,728)

In an example, recombinant OPH sequence-fength mutants have been expressed wherein the first 33 amino acids of OPH have been removed, and a peptide sequence M-l-T-N-S added at the N-terminus (Omburo, G. A. et al., 1992; Mulbry, W. and Karns, J., 1989). Often

4811 removal of the 29 amino acid sequence is used when expressing mutants of OPH comprising one or more amino acid substitutions such as the C59A, G60A, S61A, I106A, W131A, F132A, H254A, H257A, L271A, L303A, F306A, S308A, Y309A, M317A, I106A/F132A, I106A/S308A, F132A/S308A, I106G, F132G, S308G, I106G/F132G, I106G/S308G, F132G/S308G, I106G/F132G/S308G, H254Y, H254F, H257Y, H257F, H257W, H257L, L271Y, L271W, M317Y, M317F, M317W, I106A/H257Y, F132A/H257Y, I106A/F132A/H257Y, I106A/H257Y/S308A, I106A/F132A/H257W, F132A/H257Y/S308A, I106G/H257Y, F132G/H257Y,

I106G/F132G/H257Y, I106G/H257Y/S308G, and I106G/F132G/H257Y/S308G mutants (ChenGoodspeed, M. et al., 2001a). In a further example, LacZ-OPH fusion protein mutants lacking the 29 amino acid leader peptide sequence and comprising an amino acid substitution mutant such as W131F, F132Y, L.136Y, L140Y, H257L, L271L, L271Y, F306A, or F3O6Y have been recombinantly expressed (Gopal, S. et al., 2000).

In an additional example, OPH mutants that comprise additional amino acid sequences are also known in the art. An OPH fusion protein lacking the 29 amino acid leader sequence and possessing an additional C-terminal flag octapeptide sequence was expressed and localized in the cytoplasm of Escherichia coli (Wang, J. et al., 2001). In another example, nucleic acids encoding truncated versions of the ice nucleation protein (“InaV) from Pseudomonas syringae have been used to construct vectors that express OPH-lnaV fusion proteins in Escherichia coli. The InaV sequences targeted and anchored the OPH-lnaV fusion proteins to the cells' outer membrane (Shimazu, M. et al., 2001a; Wang, A. A. et al., 2002). In a further example, a vector encoding a similar fusion protein was expressed in Moraxella sp., and demonstrated a 70-fold improved OPH activity on the cell surface compared to Escherichia coli expression (Shimazu, M. et al2001 ״b). In a further example, fusion proteins comprising the signal sequence and first nine amino acids of lipoprotein, a transmembrane domain of outer membrane protein A (LppOmpA), and either a wild-type OPH sequence or an OPH truncation mutant lacking the first 29 amino acids has been expressed in Escherichia coli. These OPH-Lpp-OmpA fusion proteins were targeted and anchored to the Escherichia coli ceil membrane, though the OPH truncation mutant had only 5% to 10% the activity of the wild-type OPH sequence (Richins, R.

D. etal,, 1997; Kaneva, I. etal., 1998). In one example, a fusion protein comprising N-terminus to C-terminus, a (His)6 polyhistidine tag, a green fluorescent protein (GFP), an enterokinase recognition site, and an OPH sequence lacking the 29 amino acid leader sequence has been expressed within Escherichia coli cells (Wu, C.-F. et al., 2000b, Wu, C.-F. et af., 2002), A similar fusion protein a (His)6 polyhistidine tag, an enterokinase recognition site, and an OPH sequence lacking the 29 amino acid leader sequence has also been expressed within Escherichia coli cells (Wu, C.-F. et al., 2002). Additionally, variations of these GFP-OPH fusion proteins have been expressed within Escherichia coli cells where an second enterokinase recognition site was placed

48mm at the C-terminus of the OPH gene fragment sequence, followed by a second OPH gene fragment sequence (Wu, C.-F. et al., 2001b). The GFP sequence produced fluorescence that was proportional to both the quantity of the fusion protein, and the activity of the OPH sequence, providing a fluorescent assay of enzyme activity and stability in GFP-OPH fusion proteins (Wu,

C.-F. et at., 2000b, Wu, C.-F. et al., 2002).

In a further example, a fusion protein comprising an elastin-like polypeptide (“ELP”) sequence, a polyglycine linker sequence, and an OPH sequence was expressed in Escherichia coli (Shimazu, M. et al., 2002). In an additional example, a cellulose-binding domain at the Nterminus of an OPH fusion protein lacking the 29 amino acid leader sequence, and a similar fusion protein wherein OPH possessed the leader sequence, where both predominantly excreted into the external medium as soluble proteins by recombinant expression in Escherichia coli (Richins, R. D. et al., 2000).

b. Paraoxonase Functional Equivalents

Various chemical modifications to the amino acid residues of the recombinantly expressed human paraoxonase have been used to identify specific residues including tryptophans, histidines, aspartic acids, and glutamic acids as of importance to enzymatic activity for the cleavage of phenylacetate, paraoxon, chlorpyrifosoxon. and diazoxon. Additionally, comparison to conserved residues in human, mouse, rabbit, rat dog, chicken, and turkey paraoxonase enzymes was used to further identify amino acids for the production of specific mutants. Site-directed mutagenesis was used to alter the enzymatic activity of human paraoxonase through conservative and non-conservative substitutions, and thus clarify the specific amino acids of particular importance for enzymatic activity. Specific paraoxonase mutants include the sequence analogs E32A, E48A, E52A, D53A, D88A, D107A, H114N, D121A, H133N, H154N, H160N, W193A, W193F, W201A, W201F, H242N, H245N, H250N, W253A, W253F, D273A, W280A, W280F, H284N, or H347N.

The various paraoxonase mutants generally had different enzymatic properties. For example, W253A had a 2-fold greater k_c0t; and W201F, W253A and W253F each had a 2 to 4 fold increase in k_cal, though W201F also had a lower substrate affinity. A non-conservative substitution mutant W280A had 1% wild-type paraoxonase activity, but the conservative substitution mutant W280F had similar activity as the wild-type paraoxonase (Josse,

D. et al., 1999; Josse, D. et al., 2001).

48nn

c. Squid-type DFPase Functional Equivalents

Various chemical modifications to the amino acid residues of the recombinantly expressed squid-type DFPase from Loligo vulgaris has been used to identify which specific types of residues of modified arginines, aspartates, cysteines, glutamates, histidines, lysines, and tyrosines, are important to enzymatic activity for the cleavage of DFP. Modification of histidines generally reduced enzyme activity, and site-directed mutagenesis was used to clarify which specific histidines are of importance for enzymatic activity. Specific squid-type DFPase mutants include the sequence analogs H181N, H224N, H274N, H219N, H248N, or H287N.

The H287N mutant lost about 96% activity, and is thought to act as a hydrogen acceptor in active site reactions. The H181N and H274N mutants lost between 15% and 19% activity, and are thought to help stabilize the enzyme. The H224N mutant gained about 14% activity, indicating that alterations to this residue may also affect activity (Hartleib, J. and Ruterjans, H״ 2001b).

In a further example of squid-type DFPase functional equivalents, recombinant squidtype DFPase sequence-length mutants have been expressed wherein a (His)6 tag sequence and a thrombin cleavage site has been added to the squid-type DFPase (Hartleib, J. and Ruterjans, H2001 ״a). In an additional example, a polypeptide comprising amino acids 1-148 of squid-type DFPase has been admixed with a polypeptide comprising amino acids 149-314 of squid-type DFPase to produce an active enzyme (Hartleib, J. and Ruterjans, H., 2001a).

3, Combinations of Blomolecules

It is contemplated that in various embodiments, a composition of the present invention may comprise one or more selected biomolecules, with an enzyme being a preferred biomolecule. It is contemplated that in specific embodiments, a composition of the present invention may comprise an endogenously expressed wild-type enzyme, a recombinant enzyme, or a combination thereof. In specific aspects, a recombinant enzyme comprises a wild-type enzyme, a functional equivalent enzyme, or a combination thereof. Numerous examples of enzymes with different properties are described herein, and any such enzyme as would be known to one of ordinary skill in the art is contemplated for inclusion in a composition of the present invention.

It is contemplated that a combination of biomolecules may be selected for inclusion in the biomolecuie composition, coating and/or paint, to optimize one or more properties of such a composition of the present invention. Thus, a composition of the present invention may comprise 1 to 100 or more different selected biomolecules of interest, including all intermediate ranges and

4800 combinations thereof. For example, as various enzymes have differing binding properties, catalytic properties, stability properties, properties related to environmental safety, etc, one may select a combination of enzymes to confer the a more desirable range of properties to a composition of the present invention. In a specific example, it is contemplated that phosphoric triester hydrolases, with differing but desirable abilities to cleave the chiral centers of OP compounds, may be admixed to confer a more desirable range of catalytic properties to a composition of the present invention than would be achieved by the selection of a single phosphoric triester hydrolase.

C. Recombinantly Produced Enzymes

In certain aspects, an enzyme of the present invention may be biologically produced in cell, tissue and/or organism transformed with a genetic expression vector. As used herein, an “expression vector refers to a carrier nucleic acid molecule, into which a nucleic acid sequence can be inserted, wherein the nucleic acid sequence is capable of being transcribed into a ribonucleic acid (RNA) molecule after introduction into a cell. Usually an expression vector comprises deoxyribonucleic acid (“DNA). As used herein, an expression system refers to an expression vector, and may further comprise additional reagents needed to promote insertion of a nucleic acid sequence, introduction into a cell, transcription and/or translation. As used herein, a “vector, refers to a carrier nucleic acid molecule into which a nucleic acid sequence can be inserted for introduction into a cell. Certain vectors are capable of replication of the vector and/or any inserted nucleic acid sequence in a cell. For example, a viral vector may be used in conjunction with either an eukaryotic or prokaryotic host cell, particularly one that is permissive for replication or expression of the vector. A cell that is capable of being transformed with a vector is known herein as a host cell.

In general embodiments, the inserted nucleic acid sequence encodes for at least part of a gene product. In some embodiments wherein the nucleic acid sequence is transcribed into an RNA molecule, the RNA molecule is then translated into a proteinaceous molecule. As used herein, a “gene refers to a nucleic acid sequence isolated from an organism, and/or man-made copies or mutants thereof, that comprises a nucleic acid sequence capable of being transcribed and/or translated in an organism. A “gene product” is the transcribed RNA and/or translated proteinaceous molecule from a gene. Often, only partial nucleic acid sequences of a gene, known herein as a gene fragment,” are used COME BACK to produce a part of the gene product. Many gene and gene fragment sequences are known in the art, and are both commercially available and/or publicly disclosed at a database such as Genbank, it is contemplated that a gene and/or a gene fragment can be used to recombinantly produce an enzyme for use in the present invention. It is further contemplated that a gene and/or a gene

48pp fragment can be use in construction of a fusion protein comprising an enzyme, for use in the present invention.

In certain embodiments, a nucleic acid sequence such as a nucleic acid sequence encoding an enzyme, or any other desired RNA or proteinaceous molecule (as well as a nucleic acid sequence comprising a promoter, a ribosome binding site, an enhancer, a transcription terminator, an origin of replication, or other nucleic acid sequences described herein or would be known by one of ordinary skill in the art in light of the present disclosures) may be recombinantly produced or synthesized using any method or technique known to those of ordinary skill in the art in various combinations, [in “Molecular Cloning (Sambrook, J., and Russell, D.W., Eds.) 3rd Edition, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press, 2001; in Current Protocols in Molecular Biology (Chanda, V. B. Ed.) John Wiley &amp; Sons, 2002; In Current Protocols in Cell Biology (Morgan, K. Ed.) John Wiley &amp; Sons, 2002; In Current Protocols in Nucleic Acid Chemistry׳ (Harkins, E. W. Ed.) John Wiley &amp; Sons, 2002; in Current Protocols in Protein Science” (Taylor, G. Ed.) John Wiley &amp; Sons, 2002; In Current Protocols in Pharmacology (Taylor, G. Ed.) John Wiley &amp; Sons, 2002; In “Current Protocols in Cytometry״ (Robinson, J. P. Ed.) John Wiley &amp; Sons, 2002; In “Current Protocols in immunology (Coico, R. Ed.) John Wiley &amp; Sons, 2002]. For example, a gene and/or a gene fragment encoding the enzyme of interest may be isolated and/or amplified through polymerase chain reaction (“PCR™“) technology. Often such nucleic acid sequence is readily available from a public database and/or a commercial vendor, as previously described.

Nucleic acid sequences, called codons, encoding for each amino acid are well known in the art, and used to copy and/or mutate a nucleic acid sequence to produce a desired mutant in an expressed amino acid sequence. Codons comprise nucleotides such as adenine ( A ), cytosine (C), guanine (“G), thymine (“T) and uracil (U). The common amino acids are generally encoded by the following codons: alanine is encoded by GCU, GCC, GCA, or GCG; arginine is encoded by CGU, CGC, CGA, CGG, AGA, or AGG; aspartic acid is encoded by GAU or GAC; asparagine is encoded by AAU or AAC; cysteine is encoded by UGU or UGC; glutamic acid is encoded by GM or GAG; glutamine is encoded by CM or CAG; glycine is encoded by GGU, GGC, GGA, or GGG; histidine is encoded by CAU or CAC; isoleucine is encoded by AUU, AUC, or AUA; leucine is encoded by UUA, UUG, CUU, CUC, CUA ,or CUG; lysine is encoded by AM or MG; methionine is encoded by AUG; phenylalanine is encoded by UUU or UUC; proline is encoded by CCU, CCC, CCA, or CCG; serine is encoded by AGL), AGC, UCU, UCC, UCA, or UCG; threonine is encoded by ACU, ACC, ACA, or ACG; tryptophan is encoded by UGG, tyrosine is encoded by UAU or UAC; and valine is encoded by GUU, GUC, GUA, or GUG.

qq

A mutation in a nucleic acid encoding a proteinaceous molecule may be introduced into the nucleic acid sequence through any technique known to one of ordinary skill in the art. As would be well understood by those of ordinary skill in the art, such a mutation may be bioengineered to a specific region of a nucleic acid comprising one or more codons using a technique such as site-directed mutagenesis or cassette mutagenesis. Numerous examples of phosphoric triester hydrolase mutants have been produced using site-directed mutagenesis or cassette mutagenesis, and are described herein.

It is contemplated that for recombinant expression, the choice of codons may be made to mimic the host cell's molecular biological activity, in order to optimize the efficiency of expression from an expression vector. For example, codons may be selected to match the preferred codons used by a host cell in expressing endogenous proteins. In some aspects, the codons selected may be chosen to approximate the G-C content of an expressed gene and/or a gene fragment in a host cell’s genome, or the G-C content of the genome itself. In other aspects, a host ceil may be genetically altered to recognize more efficiently use a variety of codons, such as Escherichia coli host cells that are dnaYgene positive (Brinkmann, U. et al., 1989).

1. General Expression Vector Components and Use

An expression vector may comprise specific nucleic acid sequences such as a promoter, a ribosome binding site, an enhancer, a transcription terminator, an origin □f replication, or other nucleic acid sequence described herein or would be known by one of ordinary skill in the art in light of the present disclosures, in various combinations. A nucleic acid sequence can be exogenous, which means that it is foreign to the cell into which the vector is being introduced or that the sequence is homologous to a sequence in the cell, but in a position within the host cell nucleic acid in which the sequence is ordinarily not found. An expression vector may have one or more nucleic acid sequences removed by restriction enzyme digestion, modified by mutagenesis, and/or replaced with another more appropriate nucleic acid sequence, for transcription and/or translation in a host cell suitable for the expression vector selected.

One of skill in the art can construct a vector through standard recombinant techniques, which are well known and routine in the art. Further, one of skill in the art would know how to express a vector to transcribe a nucleic acid sequence and/or translate its cognate proteinaceous molecule. One of skill in the art would further understand the conditions under which to incubate all of the above described host cells to maintain them and to permit replication of a vector. Also understood and known are techniques and conditions that would allow large-scale production of a vector, as well as production of a nucleic acid sequence encoded by a vector into an RNA molecule and/or translation of the RNA molecule into a cognate proteinaceous molecule.

48rr

In certain embodiments, a cell may express multiple gene and/or gene fragment products from the same vector, and/or express more than one vector. Often this occurs simply as part of the normal function of a multi-vector expression system. For example, one gene or gene fragment is often used to produce a repressor that suppresses the activity of a promoter that controls the expression of a gene or a gene fragment of interest. The repressor gene and the desired gene may be on different vectors. However, multiple gene, gene fragment and/or expression systems may be used to express an enzymatic sequence of interest and another gene or gene fragment that is desired for a particular function. In an example, recombinant Pseudomonas putida has co-expressed OPH from one vector, and the multigenes encoding the enzymes for converting p-nitrophenol to β-ketoadipate from a different vector. The expressed OPH catalyzed the cleavage of parathion to p-nitrophenol. The additionally expressed recombinant enzymes converted the p-nitrophenol, which is a moderately toxic compound, to βketoadipate, thereby detoxifying both an OP compound and the byproducts of its hydrolysis (Walker, A. W. and Keasling, J. D., 2002). In a further example, Escherichia coli cells expressed a cell surface targeted INPNC-OPH fusion protein from one vector to detoxify OP compounds, and co-expressed from a different vector a cell surface targeted Lpp-OmpA-cellulose binding domain fusion protein to immobilize the cell to a cellulose support (Wang, A. A. et al., 2002). In an additional example, a vector co-expressed an antisense RNA sequence to the transcribed stress response gene σ³² and OPH in Escherichia coli. The antisense a³² RNA was used to reduce the cell’s stress response, including proteolytic damage, to an expressed recombinant proteinaceous molecule. A six-fold enhanced specific activity of expressed OPH enzyme was seen (Srivastava, R. et al., 2000). In a further example, multiple OPH fusion proteins were expressed from the same vector using the same promoter but separate ribosome binding sites (Wu, C.-F. et al., 2001b).

As is well known to those of skill in the art, an expression vector generally comprises a plurality of functional nucleic acid sequences that either comprise a nucleic acid sequence with a molecular biological function in a host cell, such as a promoter, an enhancer, a ribosome binding site, a transcription terminator, etc, and/or encode a proteinaceous sequence, such as a leader peptide, a polypeptide sequence with enzymatic activity, a peptide or polypeptide with a binding property, etc. A nucleic acid sequence may comprise a “control sequence, which refers to a nucleic acid sequence necessary for the transcription and possibly translation of an operatively linked coding sequence in a particular host cell. As used herein, an operatively linked or operatively positioned nucleic acid sequence refers to the placement of one nucleic acid sequence into a functional relationship with another nucleic acid sequence. Vectors and

48ss expression vectors may further comprise one or more nucleic acid sequences that serve other functions as well and are described herein.

The various functional nucleic acid sequences that comprise an expression vector are operatively linked so to position the different nucleic acid sequences for optimal function in a host cell. In certain cases, the functional nucleic acid sequences may be contiguous such as placement of a nucleic acid sequence encoding a leader peptide sequence in correct amino acid frame with a nucleic acid sequence encoding a polypeptide comprising a polypeptide sequence with enzymatic activity. In other cases, the functional nucleic acid sequences may be non-contiguous such as placing a nucleic acid sequence comprising an enhancer distal to a nucleic acid sequence comprising such sequences as a promoter, a encoded proteinaceous molecule, a transcription termination sequence, etc. One or more nucleic acid sequences may be operatively linked using methods well known in the art, particularly ligation at restriction sites that may pre-exist in a nucleic acid sequence or be added through mutagenesis.

A promoter is a control sequence that is a region of a nucleic acid sequence at which initiation and rate of transcription are controlled. In the context of a nucleic acid sequence comprising a promoter and an additional nucleic acid sequence, particularly one encoding a gene or gene fragment’s product, the phrases operatively linked, operatively positioned, “under control, and ''under transcriptional control” mean that a promoter is in a correct functional location and/or orientation in relation to the additional nucleic acid sequence to control transcriptional initiation and/or expression of the additional nucleic acid sequence. A promoter may contain genetic elements at which regulatory proteins and molecules may bind such as RNA polymerase and other transcription factors. A promoter employed may be constitutive, tissue-specific, inducible, and/or useful under the appropriate conditions to direct high level expression of the introduced nucleic acid sequence, such as is advantageous in the large-scale production of a recombinant proteinaceous molecule. Examples of a promoter include a Zac, a (ac, an amp, a heat shock promoter of a P-element of Drosophila, a baculovirus polyhedron gene promoter, or a combination thereof. In a specific example, the nucleic acids encoding OPH have been expressed using the polyhedron promoter of a baculoviral expression vector (Dumas, D. P. et al1990 ״). In a further example, a Cochliobolus heterostrophus promoter, proml, has been used to express a nucleic acid encoding OPH (Dave, K. I. et al., 1994b).

The promoter may be endogenous or heterologous. An “endogenous promoter” comprises one naturally associated with a gene or sequence, as may be obtained by isolating the 5' non-coding sequences located upstream of the coding segment and/or exon. Alternatively, certain advantages will be gained by positioning the coding nucleic acid sequence under the

48tt

׳ע Ο control of a “heterologous promoter” or recombinant promoter,” which refers to a promoter that is not normally associated with a nucleic acid sequence in its natural environment.

A specific initiation signal also may be required for efficient translation of a coding sequence by the host cell. Such a signal may include an ATG initiation codon (start codon) and/or an adjacent sequence. Exogenous translational control signals, including the ATG initiation codon, may need to be provided. One of ordinary skill in the art would readily be capable of determining this and providing the necessary signals. It is well known that the initiation codon must be “in-frame״ with the reading frame of the desired coding sequence to ensure translation of the entire insert. The exogenous translational control signal and/or an initiation codon can be either natural or synthetic. The efficiency of expression may be enhanced by the inclusion of an appropriate transcription enhancer.

A promoter may or may not be used in conjunction with an “enhancer, which refers to a c/s-acting regulatory sequence involved in the transcriptional activation of a nucleic acid sequence. An enhancer may be one naturally associated with a nucleic acid sequence, located either downstream or upstream of that sequence. A recombinant or heterologous enhancer refers also to an enhancer not normally associated with a nucleic acid sequence in its natural environment. Such a promoter and/or enhancer may include a promoter and/or enhancer of another gene, a promoter and/or enhancer isolated from any other prokaryotic, viral, or eukaryotic cell, a promoter and/or enhancer not “naturally occurring, i.e., a promoter and/or enhancer comprising different elements of different transcriptional regulatory regions, and/or mutations that alter expression. In addition to producing a nucleic acid sequence comprising a promoter and/or enhancer synthetically, a sequence may be produced using recombinant cloning and/or nucleic acid amplification technology, including PCR™, in connection with the compositions disclosed herein (U.S. Patent 4,683,202, U.S. Patent 5,928,906).

It will be important to employ a promoter and/or enhancer that effectively directs the expression of the nucleic acid sequence in the cell type, chosen for expression. Those of skill in the art of molecular biology generally know the use of promoters, enhancers, and cell type combinations for expression. Furthermore, it is contemplated the control sequences that direct transcription and/or expression of sequences within non-nuclear organelles, including eukaryotic organelles such as mitochondria, chloroplasts, and the like, can be employed as well.

Vectors can include a multiple cloning site (“MCS), which is a nucleic acid region that contains multiple restriction enzyme sites, any of which can be used in conjunction with standard recombinant technology to digest the vector. Restriction enzyme digestion refers to catalytic

48uu cleavage of a nucleic acid molecule with an enzyme which functions only at specific locations in a nucleic acid molecule. Many of these restriction enzymes are commercially available. Use of such enzymes is widely understood by those of skill in the art. Frequently, a vector is linearized or fragmented using a restriction enzyme that cuts within the MCS to enable an exogenous nucleic acid sequence to be ligated to the vector. Ligation refers to the process of forming phosphodiester bonds between two nucleic acid fragments, which may or may not be contiguous with each other. Techniques involving restriction enzymes and ligation reactions are well known to those of skill in the art of recombinant technology.

A fusion protein, as used herein, is an expressed contiguous amino acid sequence comprising a proteinaceous molecule of interest and one or more additional peptide or polypeptide sequences. The additional peptide or polypeptide sequence generally provides an useful additional property to the fusion protein, including but not limited to, targeting the fusion protein to a particular location within or external to the host cell (e.g., a signal peptide); promoting the ease of purification and/or detection of the fusion protein (e.g., a tag, a fusion partner); promoting the ease of removal of one or more additional sequences from the peptide or polypeptide of interest (e.g., a protease cleavage site); and separating one or more sequences of the fusion protein to allow optimal activity or function of the sequence(s) (e.g., a linker sequence).

As used herein a tag is a peptide sequence operatively associated to the sequence of another peptide or polypeptide sequence. Examples of a tag include a His-tag, a strep-tag, a flag-tag, a T7-tag, a S-tag, a HSV-tag, a polyarginine-tag, a polycysteine-tag, a polyaspartic acid-tag, a polyphenylalanine-tag, or a combination thereof. A His-tag is 6 or 10 amino acids in length, and can be incorporated at the N-terminus, C-terminus or within an amino acid sequence for use in detection and purification. A His tag binds affinity columns comprising nickel, and is eluted using low pH conditions or with imidazole as a competitor (Unger, T. F., 1997). A strep-tag is 10 amino acids in length, and can be incorporated at the C-terminus. A strep-tag binds streptavidin or affinity resins that comprise streptavidin. A flag-tag is 8 amino acids in length, and can be incorporated at the N-terminus or C-terminus of an amino acid sequence for use in purification. A T7־tag is 11 or 16 amino acids in length, and can be incorporated at the N-terminus or within an amino acid sequence for use in purification. A S-tag is 15 amino acids in length, and can be incorporated at the N-terminus, C-terminus or within an amino acid sequence for use in detection and purification. A HSV-tag is 11 amino acids in length, and can be incorporated at the C-terminus of an amino acid sequence for use in purification. The HSV tag binds an anti-HSV antibody in purification procedures (Unger, T. F., 1997). A polyarginine-tag is 5 to 15 amino acids in length, and can be Incorporated at the C-terminus of an amino acid sequence for use in purification. A polycysteine-tag, is 4 amino acids in length, and can be

48w incorporated at the N-terminus of an amino acid sequence for.use in purification. A polyaspartic acid-tag can be 5 to 16 amino acids in length, and can be incorporated at the C-terminus of an amino acid sequence for use in purification. A polyphenylalanine-tag is 11 amino acids in length, and can be incorporated at the N-terminus of an amino acid sequence for use in purification.

In one example, a (His)6 tag sequence has been used to purify fusion proteins comprising GFP-OPH or OPH using immobilized metal affinity chromatography (IMAC) (Wu, C.-

F. et al., 2000b; Wu, C.-F. et al., 2002). In a further example, a (His)6 tag sequence followed by a thrombin cleavage site has been used to purify fusion proteins comprising squid-type DFPase using IMAC (Hartleib, J. and Ruterjans, H2001 ״a). In a further example, an OPH fusion protein comprising a C-terminal flag has been expressed (Wang, J. et al., 2001).

As used herein a fusion partner” is a polypeptide that is operatively associated to the sequence of another peptide or polypeptide of interest. Properties that a fusion partner can confer to a fusion protein include, but are not limited to, enhanced expression, enhanced solubility, ease of detection, and/or ease of purification of a fusion protein. Examples of a fusion partner include a thioredoxin, a cellulose-binding domain, a calmodulin binding domain, an avidin, a protein A, a protein G, a glutathione-S-transferase, a chitin-binding domain, an ELP, a maltose-binding domain, or a combination thereof. Thioredoxin can be incorporated at the N-terminus or C-terminus of an amino acid sequence for use in purification. A cellulose-binding domain binds a variety of resins comprising cellulose or chitin (Unger, T. F., 1997). A calmodulin-binding domain binds affinity resins comprising calmodulin in the presence of calcium, and allows elution of the fusion protein in the presence of ethylene glycol tetra acetic acid (“EGTA) (Unger, T. F., 1997). Avidin is useful in purification or detection. A protein A or a protein G binds a variety of anti-bodies for ease of purification. Protein A is generally bound to an IgG sepharose resin (Unger, T. F., 1997). Streptavidin is useful in purification or detection. Glutathione-S-transferase can be incorporated at the N-terminus of an amino acid sequence for use in detection or purification. Glutathione-S-transferase binds affinity resins comprising glutathione (Unger, T. F1997 ״). An elastin-like polypeptide comprises repeating sequences (e.g., 78 repeats) which reversibly converts itself, and thus the fusion protein, from an aqueous soluble polypeptide to an insoluble polypeptide above an empirically determined transition temperature. The transition temperature is affected by the number of repeats, and can be determined spectrographically using techniques known in the art, including measurements at 655 nano meters (nm) over a 4°C to 80°C range (Urry, D. W. 1992; Shimazu, M.etal., 2002). A chitin-binding domain preferable comprises an intein cleavage site sequence, and can be incorporated at the C-terminus for purification. The chitin-binding domain binds affinity resins comprising chitin, and an intein cleavage site sequence allows the self-cleavage in the presence

48ww

8/1 נ j of thiols at reduced temperature to release the peptide or polypeptide sequence of interest {Unger, T. F., 1997). A maltose-binding domain can be incorporated at the N-terminus or C-terminus of an amino acid sequence for use in detection or purification. A maltose-binding domain sequence usually further comprises a ten amino acid poly asparagine sequence between the maltose binding domain and the sequence of interest to aid the maltose-binding domain in binding affinity resins comprising amylose (Unger, T. F., 1997).

In an example, a fusion protein comprising an elastin-like polypeptide sequence and an OPH sequence has been expressed (Shimazu, M. et al., 2002). In a further example, a cellulosebinding domain-OPH fusion protein has also been recombinantly expressed (Richins, R.

D. et al., 2000). In an additional example, a maltose binding protein-E3 carboxylesterase fusion protein has been recombinantly expressed (Claudianos, C. et al., 1999)

A protease cleavage site promotes proteolytic removal of the fusion partner from the peptide or polypeptide of interest. Often, a fusion protein is bound to an affinity resin, and cleavage at the cleavage site promotes the ease of purification of a peptide or polypeptide of Interest with most or all of the tag or fusion partner sequence removed (Unger, T. F1997 ״). Protease cleavage sites are well known in the art, and examples of protease cleavage sites include the factor Xa cleavage site, which is four amino acids in length; the enterokinase cleavage site, which is five amino acids in length; the thrombin cleavage site, which is six amino acids in length; the rTEV protease cleavage site, which is seven amino acids in length; the 3C human rhino virus protease, which is eight amino acids in length; and the PreScission cleavage site, which is eight amino acids in length. In an example, an enterokinase recognition site was used to separate an OPH sequence from a fusion partner (Wu, C.-F. et al., 2000b; Wu, C.-

F. etal., 2001b).

In an eukaryotic expression system {e.g., a fungal expression system), the terminator region or “terminator may also comprise a specific DNA sequence that permits site-specific cleavage of the new transcript so as to expose a polyadenylation site. This signals a specialized endogenous polymerase to add a stretch of adenosine nucleotides (polyA) of about about 200 in number to the 3׳ end of the transcript. RNA molecules modified with this polyA tail appear to more stable and are translated more efficiently. Thus, in other embodiments involving an eukaryote, it is preferred that that terminator comprises a signal for the cleavage of the RNA, and it is more preferred that the terminator signal promote polyadenylation of the message. The terminator and/or polyadenylation site elements can serve to enhance message levels and/or to minimize read through from the cassette into other sequences.

48xx

’/3־ - י

A terminator contemplated for use in the invention include any known terminator of transcription described herein or known to one of ordinary skill in the art, including but not limited to, for example, a termination sequence of a gene, such as for example, a bovine growth hormone terminator or a viral termination sequence, such as for example a SV40 terminator. In certain embodiments, the termination signal may be a lack of transcribable or translatable sequence, such as due to a sequence truncation. In one example, a trpC terminator from Aspergillus nidulans has been used in the expression of recombinant OPH (Dave, K. I. et al., 1994b).

In expression, particularly eukaryotic expression, one will typically include a polyadenylation signal to effect proper polyadenylation of the transcript. The nature of the polyadenylation signal is not believed to be crucial to the successful practice of the invention, and/or any such sequence may be employed. Preferred embodiments include the SV40 polyadenylation signal and/or the bovine growth hormone polyadenylation signal, convenient and/or known to function well in various target cells. Polyadenylation may increase the stability of the transcript or may facilitate cytoplasmic transport.

In order to propagate a vector in a host cell, it may contain one or more origins of replication sites (“ori), which is a specific nucleic acid sequence at which replication is initiated. Alternatively an autonomously replicating sequence (ARS) can be employed if the host cell is yeast.

Various types of prokaryotic and/or eukaryotic expression vectors are known in the art. Examples of types of expression vectors include a bacterial artificial chromosome (“BAC”), a cosmid, a plasmid [e.g., a pMB1/colE1 derived plasmid such as pBR322, pUC18, a ΤΪ plasmid of Agrobacterium tumefaciens derived vector (Rogers, S. G. et al., 1987)], a virus (e.g., a bacteriophage such as a bacteriophage M13, an animal virus, a plant virus), or a yeast artificial chromosome (YAC). Some vectors, known herein as shuttle vectors may employ control sequences that allow it to be replicated and/or expressed in both prokaryotic and eukaryotic cells [e.g., a wheat dwarf virus (WDV) pW1-11 or pW1-GUS shuttle vector (Ugaki, M. et al., 1991)]. An expression vector operatively linked to a nucleic acid sequence encoding an enzymatic sequence of the present invention may be constructed using techniques known to those of skill in the art in light of the present disclosures [In “Molecular Cloning (Sambrook, J., and Russell,

D.W., Eds.) 3rd Edition, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press, 2001; In Current Protocols in Molecular Biology” (Chanda, V. B. Ed.) John Wiley &amp; Sons, 2002; In Current Protocols in Nucleic Acid Chemistry (Harkins, E. W. Ed.) John Wiley &amp; Sons, 2002; In

48yy “Current Protocols in Protein Science (Taylor, G. Ed.) John Wiley &amp; Sons, 2002; In “Current

Protocols In Cell Biology (Morgan, K. Ed.) John Wiley &amp; Sons, 2002].

Numerous expression systems exist that comprise at least a part or all of the compositions discussed above. Prokaryote- and/or eukaryote-based systems can be employed for use with the present invention to produce nucleic acid sequences, or their cognate polypeptides, proteins and peptides. Many such systems are widely available, including those provide by commercial vendors, as would be known to those of skill in the art. For example, an insect cell/baculovirus system can produce a high level of protein expression □f a heterologous nucleic acid sequence, such as described in U.S. Patent No. 5,871,986, 4,879,236, both incorporated herein by reference, and which can be bought, for example, under the name MAXBAC® 2.0 from INVITROGEN® and BacPack™ Baculovirus Expression System From Clontech®. In an addition example of an expression system include Stratagene®'s Complete Control™ Inducible Mammalian Expression System, which involves a synthetic ecdysone-inducible receptor, or its pET Expression System, an Escherichia coli expression system. Another example of an inducible expression system is available from Invitrogen®, which carries the T-REX™ (tetracycline-regulated expression) System, an inducible mammalian expression system that uses the full-length CMV promoter. Invitrogen® also provides a yeast expression system called the Pichia methanollca Expression System, which is designed for high-level production of recombinant proteins in the methylotrophic yeast Pichia methanolica. In a specific example, E3 carboxylesterase enzymatic sequences and phosphoric triester hydrolase functional equivalents have been recombinantly expressed in a BacPack™ Baculovirus Expression System From Clontech® (Newcomb, R. D. et al., 1997; Campbell, P. M. et a., 1998). In certain embodiments, a biomolecule may be expressed in a plant cell (e.g., a corn cell), using techniques such as those described in U.S. Patent Nos. 6,504,085, 6,136,320, 6,087,558, 6034,298, 5,914,123, and 5,804,694.

2. Prokaryotic Expression Vectors and Use

In preferred embodiments, a prokaryote such as a bacterium comprises a host cell. In specific aspects, the bacterium host cell comprises a Gram-negative bacterium cell. Various prokaryotic host cells have been used in the art with expression vectors, and it is contemplated that any prokaryotic host cell known in the art may be used to express a peptide or polypeptide comprising an enzyme sequence of the present invention.

An expression vector for use in prokaryotic cells generally comprises nucleic acid sequences such as, a promoter, a ribosome binding site (e.g., a Shine-Delgarno sequence), a start codon, a multiple cloning site, a fusion partner, a protease cleavage site, a stop codon, a

48zz transcription terminator, an origin of replication, a repressor, and/or any other additional nucleic acid sequence that would be used in such an expression vector, as would be known to one of ordinary skill in the art [Makrides. S. C., 1996; Hannig, G. and Makrides, S, C1998 ״; Stevens, R.

C., 2000; In “Molecular Cloning (Sambrook, J., and Russell, D.W., Eds.) 3rd Edition, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press, 2001; In “Current Protocols in Molecular Biology״ (Chanda, V. B. Ed.) John Wiley &amp; Sons, 2002; In “Current Protocols in Nucleic Acid Chemistry (Harkins, E. W. Ed.) John Wiley &amp; Sons, 2002; In “Current Protocols in Protein Science (Taylor, G. Ed.) John Wiley &amp; Sons, 2002; In “Current Protocols in Cell Biology (Morgan, K. Ed.) John Wiley &amp; Sons, 2002],

A promoter generally is positioned 10 to 100 nucleotides 5’ to a nucleic acid sequence comprising a ribosome binding site. Examples of promoters that have been used in a prokaryotic cell includes a T5 promoter, a /ac promoter, a tac promoter, a trc promoter, an araBAD promoter, a P_L promoter, a T7 promoter, a T7-lac operator promoter, and variations thereof. The T5 promoter is regulated by the lactose operator. A /ac promoter (e.g., a lac promoter, a /acUV5 promoter), a fac promoter (e.g״ a tad promoter, a tadl promoter), a T7־lac operator promoter or a trc promoter are each suppressed by a lad repressor, a more effective lad⁰ repressor or an even stronger /ac/⁰¹ repressor (Glascock, C. B. and Weickert, M. J., 1998). Isopropyl-p-D-thiogalactoside (“IPTG) is used to induce lac. tac, T7-lac operator and trc promoters. An araBAD promoter is suppressed by an araC repressor, and is induced by 1-arabinose. A P_L promoter or a T7 promoter are each suppressed by a Xclts857 repressor, and induced by a temperature of 42°C. Nalidixic acid may be used to induce a P_L promoter.

in an example, recombinant amino acid substitution mutants of OPH have been expressed in Escherichia coli using a lac promoter induced by IPTG (Watkins, L. M. etal., 1997b). In another example, recombinant wild type and a signal sequence truncation mutant of OPH was expressed in Pseudomonas putida under control of a ladac and tac promoters (Walker, A. W. and Keasling, J. D2002 ״). In a further example, an OPH-Lpp-OmpA fusion protein has been expressed in Escherichia coli strains JM105 and XL1-Blue using a constitutive Ipp-lac promoter or a tac promoter induced by IPTG and controlled by a fact⁰repressor (Richins. R. D. etal., 1997; Kaneva, I. etal, 1998; Mulchandani, A. etal., 1999b). In an additional example, a cellulose-binding domain-OPH fusion protein has also been recombinantly expressed under the control of a T7 promoter (Richins, R. D. etal., 2000). In a further example, recombinant Altermonas sp. JD6.5 OPAA has been expressed under the control of a trc promoter in Escherichia coli (Cheng, T.-C. et al., 1999). In an additional example, a (His)6 tag sequence-thrombin cleavage site-squid-type DFPase has been expressed using a Ptac promoter in Escherichia coli (Hartleib, J. and Ruterjans, H., 2001a).

aaa

A ribosome binding site is important for transcription initiation, and is usually positioned 4 to 14 nucleotides 5' from the start codon. A start codon signals initiation of transcription. A multiple cloning site comprises restriction sites for incorporation of a nucleic acid sequence encoding a peptide or polypeptide of interest.

A stop codon signals translation termination. The vectors or constructs of the present invention will generally comprise at least one termination signal. A “termination signal or “terminator is comprised of the DNA sequences involved in specific termination of an RNA transcript by an RNA polymerase. Thus, in certain embodiments a termination signal that ends the production of an RNA transcript is contemplated. A terminator may be necessary in vivo to achieve desirable message levels. A transcription terminator signals the end or transcription and often enhances mRNA stability. Examples of a transcription terminator include a rrnB T1 or a rrnB T2 transcription terminator (Unger, T. F., 1997). An origin of replication regulates the number of expression vector copies maintained in a transformed host cell.

A selectable marker usually provides a transformed cell resistance to an antibiotic. Examples of a selectable marker used in a prokaryotic expression vector include a β-lactamase, which provides resistance to antibiotic such as an ampicillin or a carbenicillin; a fet gene product, which provides resistance to a tetracycline, or a Km gene product, which provides resistance to a kanamycin. A repressor regulatory gene suppresses transcription from the promoter. Examples of repressor regulatory genes include the lad, lacf, or /ac/⁰¹ repressors (Glascock, C. B. and Weickert, M. J., 1998). Often, the host cell’s genome, or additional nucleic acid vector co-transfected into the host cell, may comprise one or more of these nucleic acid sequences, such as, for example, a repressor.

It is contemplated that an expression vector for a prokaryotic host cell will comprise a nucleic acid sequence that encodes a periplasmic space signal peptide. In preferred aspects, this nucleic acid sequence will be operatively linked to a nucleic acid sequence comprising an enzymatic peptide or polypeptide of the present invention, wherein the periplasmic space signal peptide directs the expressed fusion protein to be translocated into a prokaryotic host cell s periplasmic space. Fusion proteins secreted in the periplasmic space may be obtained through simplified purification protocols compared to non-secreted fusion proteins. A periplasmic space signal peptide are usually operatively linked at or near the N-terminus of an expressed fusion protein. Examples of a periplasmic space signal peptide include the Escherichia coli ompA, ompT, and male! leader peptide sequences and the T7 caspid protein leader peptide sequence (Unger, T. F., 1997).

48bbb

17365L ¹

Mutated and/or recombinantly altered bacterium that release a peptide or polypeptide comprising an enzyme sequence of the present invention into the environment may be particularly advantageous for purification and/or contact of enzyme with a target chemical substrate. It is contemplated that a strain of bacteria, such as, for example, a bacteriocin-release protein mutant strain of Escherichia coli, may be used to promote release of expressed proteins targeted to the periplasm into the extracellular environment (Van der Wai, F. J. et al., 1998). In other aspects, it is contemplated that a bacterium may be transfected with an expression vector that produces a gene and/or a gene fragment product that promotes the release of a protenaceous molecule of interest from the periplasm into the extracellular environment. For example, a plasmid encoding the third topological domain of TolA has been described as promoting the release of endogenous and recombinantly expressed proteins from the periplasm (Wan, E. W. and Baneyx, F., 1998).

D. Host Cells

Many host cells from various cell types and organisms are available and would be known to one of skill in the art. As used herein, the terms “cell,״ cell line, and “cell culture may be used interchangeably. All of these terms also include their progeny, which is any and all subsequent generations. It is understood that ail progeny may not be identical due to deliberate or inadvertent mutations. In the context of expressing a heterologous nucleic acid sequence, “host cell refers to a prokaryotic or eukaryotic cell, and it includes any transformable organism that is capable of replicating a vector and/or expressing a heterologous gene and/or gene fragment encoded by a vector. A host cell can, and has been, used as a recipient for vectors. A host ceil may be transfected” or “transformed, which refers to a process by which exogenous nucleic acid sequence is transferred or introduced into the host cell. A transformed ceil includes the primary subject cell and its progeny. Techniques for transforming a cell are extremely well known in the art, and include, for example calcium phosphate precipitation, cell sonication, diethylaminoethanol (DEAE”)-dextran, direct microinjection, DNA-ioaded liposomes, electroporation, gene bombardment using high velocity microprojectiles, receptor-mediated transfection, viral-mediated transfection, or a combination thereof [In Molecular Cloning” (Sambrook, J., and Russell, D.W., Eds.) 3rd Edition, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press, 2001; In Current Protocols in Molecular Biology” (Chanda, V. B. Ed.) John Wiley &amp; Sons, 2002].

Once a suitable expression vector is transformed into a cell, the cell may be grown in an appropriate environment, and in some cases, used to produce a tissue or whole multicellular organism. As used herein, the terms engineered and recombinant cells or host cells are intended to refer to a cell into which an exogenous nucleic acid sequence has been introduced.

8 ccc

365 /i ־.

Therefore, engineered cells are distinguishable from naturally occurring cells that do not contain a recombinantly introduced exogenous nucleic acid sequence. Engineered cells are thus cells having a nucleic acid sequence introduced through the hand of man. Recombinant cells include those having an introduced cDNA or genomic gene and/or a gene fragment positioned adjacent to a promoter not naturally associated with the particular introduced nucleic acid sequence, a gene, and/or a gene fragment. An enzyme or proteinaceous molecule produced from the introduced gene and/or gene fragment is referred to as a recombinant enzyme or recombinant proteinaceous molecule, respectively. All tissues, offspring, progeny or descendants of such a cell, tissue, and/or organism that comprise the transformed nucleic acid sequence thereof are considered part of the present invention.

Though it is possible to purify an expressed enzyme from cellular material, the discovery disclosed herein of the properties of an enzyme composition comprising, in preferred embodiments, an enzyme expressed and retained, whether naturally or through recombinant expression, within a cell. In preferred embodiments, an enzyme is produced using recombinant nucleic acid expression systems in the cell. Cells are known herein based on the type □f enzyme expressed within the cell, whether endogenous or recombinant, so that, for example, a ceil expressing an enzyme of interest would be known as an enzyme* cell, a cell expressing a phosphoric triester hydrolase would be known herein as a phosphoric triester hydrolase cell, efc. Additional examples of such nomenclature include an aryldialkylphosphatase* cell, an OPH* cell, an OPAA* cell, a human paraoxonase* cell, a carboxylase* cell, a prolidase* cell, an aminopeptideases* cell, a PepQ* cell, a mpd product* cell, a B esterase cell, an acetycholinesterase* cell, a butyrylcholinesterase* cell, diisopropyl-fluorophosphatase* cell, Mazur-type DFPase* cell, or a squid-type DFPase* cell, respectively denoting cells that comprise, an aryldialkylphosphatase, an OPH, a OPAA, a human paraoxonase, a carboxylase, a prolidase, an aminopeptidease, a PepQ, a mpd product, a B esterase, an acetycholinesterase, a butyrylcholinesterase, a diisopropyl-fluorophosphatase, a Mazur-type DFPase, or a squid-type DFPase, efc.

In preferred embodiments, an enzyme* cell comprises a bacterial cell, a yeast cell, an insect cell, a plant cell, or a combination thereof. In preferred aspects, the cell comprises a cell wall. Contemplated enzyme* cells that comprise cell walls include, but are not limited to, a bacterial cell, a fungal cell, a plant cell, or a combination thereof. In preferred facets, a microorganism comprises the enzyme* cell. Examples of contemplated microorganisms include a bacterium, a fungus, or a combination thereof. Examples of a bacterial host cell that have been used with expression vectors include an Aspergillus niger, a Bacillus (e.g., B. amytoliquefaciens, B. brevis, B. licheniformis, B. subtilis), an Escherichia coli, a Kluyveromyces lactis, a Moraxella

48ddd

36.

sp., a Pseudomonas (e.g., fluorescens, putida), Flavobacterium cell, a Piesiomonas cell, an

Afteromonas cell, or a combination thereof. Examples of a yeast cell include a Streptomyces lividans cell, a Gliociadium virens cell, a Saccharomyces cell, or a combination thereof.

Host cells may be derived from prokaryotes or eukaryotes, depending upon whether the desired result is replication of the vector or expression of part or all of the vector-encoded nucleic acid sequences. Numerous cell lines and cultures are available for use as a host ceil, and they can be obtained through the American Type Culture Collection, which is an organization that serves as an archive for living cultures and genetic materials. An appropriate host can be determined by one of skill in the art based on the vector backbone and the desired result. A plasmid or cosmid, for example, can be introduced into a prokaryote host cell for replication of many vectors. Examples of a bacterial cell used as a host cell for vector repiication and/or expression include DH5a, JM109, and KC8, as wall as a number of commercially available bacterial hosts such as Novablue™ Escherichia coli cells (Novagene®), SURE® Competent Cells and Solopack™ Gold Cells (Stratagene®). However, Escherichia coli cells have been the most common cell types used to express both wild type and mutant forms of OPH (Dumas, D. p. et al1989 ״a; Dave, K. I. et al., 1993; Lai, K. et al1994 ״; Wu, C.-F. et al., 2001a). In an example, the OPH I106A/F132A/H257Y and G60A mutants have been expressed in Escherichia coli BL-21 host cells (Kuo, J. M. and Raushel, F. M., 1994; Li, W.-S. et al., 2001). In a further example, maltose-binding domatn-E3 carboxylesterase and phosphoric triester hydrolase functional equivalents_have been expressed in Escherichia coli TB1 cells (Claudianos,

C. et aL, 1999). In another example, the OPH mutants designated W131F, F132Y, L136Y, L140Y, H257L, L271Y, F306A, and F306Y each have been expressed in Novablue™ Escherichia coli cells (Gopal, S. et aL, 2000). In an addition example, OPAA from Alteromonas sp JD6.5 has been recombinantly expressed in Escherichia coli cells (Hill, C, M., 2000). In a further example, recombinant Altermonas sp. JD6.5 OPAA has been expressed in Escherichia coli (Cheng, T.-

C. et aL, 1999). In a further example, the mpd gene has been recombinantly expressed in Escherichia coli, and the encoded enzyme demonstrated methyl parathion degradation activity (Zhongli, C. et aL, 2001). In an additional example, a recombinant squid-type DFPase fusion protein has been expressed Escherichia coli BL-21 cells (Hartleib, J. and Ruterjans, H., 2001a). Alternatively, bacterial cells such as Escherichia coli LE392 could be used as host cells for phage viruses. Of course, one of skill in the art may select a bacterium species to express a proteinaceous molecule due to a particular desirable property. In an example, Moraxella sp. that degrades p-nitrophenol, a toxic cleavage product of parathion and methyl parathion, has been used to recombinantly express an OPH-lnaV fusion protein. The resulting recombinant bacterial degrades both toxic OP compounds and their cleavage byproduct (Shimazu, M. et al., 2001b).

48eee ?658/1

Examples of eukaryotic host cells for replication and/or expression of a vector include yeast cells HeLa, NIH3T3, Jurkat, 293, Cos, CHO, Saos, and PC12. In an example, OPH has been expressed in the host yeast cells of Streptomyces iividans (Steiert, J. G. et al., 1989). In another example, OPH has been expressed in host insect cells, including Spodoptera frugiperda sf9 cells (Dumas, D. P. et al., 1989b; Dumas, D. P. et al., 1990). In a further example, OPH has been expressed in the cells of Drosophila melanogaster (Phillips, J. P. etal., 1990). In an additional example, OPH has been expressed in the fungus Gliocladium virens (Dave, K. I. etal., 1994b). In a further example, the gene for human paraoxonase, PON1, has been recombinantly expressed in human embryonic kidney cells (Josse, D. et aL, 2001; Josse,

D. etal., 1999). In a further example, E3 carboxylesterase and phosphoric triester hydrolase functional equivalents have been expressed in host insect Spodoptera frugiperda sf9 cells (Campbell, P. M. et al., 1998; Newcomb, R. D. et al., 1997). !ח an additional example, a phosphoric triester hydrolase functional equivalent of a butyrylcholinesterase has been expressed in Chinese hamster ovary (CHO) cells (Lockridge, O. et al., 1997). In certain embodiments, an eukaryotic cell that may be selected for expression is a plant cell, such as, for example, a corn cell.

E. Production of Expressed Proteinaceous Molecules

It is contemplated that any size flask or fermentor may be used to grow a tissue or organism that can express a recombinant proteinaceous molecule of the present invention. In certain embodiments, bulk production of compositions with enzymatic sequences is contemplated.

In an example, a fusion protein comprising, N-terminus to C-terminus, a (His)6 polyhistidine tag, a green fluorescent protein (GFP), an enterokinase recognition site, and a OPH lacking the 29 amino acid leader sequence, has been expressed in Escherichia coli. The GFP sequence produced fluorescence that was proportional both the quantity of the fusion protein, and the activity of the OPH sequence. The fusion protein was more soluble than OPH expressed without the added sequences, and was expressed within the ceils (Wu, C.-

F. et al., 2000b; Wu, C.-F. et al., 2001a).

It is contemplated that the temperature selected may influence the rate and/or quality of recombinant enzyme production. It is contemplated that in some embodiments, expression of an enzyme may be conducted at 4°C to 50°C, including ail intermediate ranges and combinations thereof. Such combinations may include a shift from one temperature (e.g., 37°C) to another temperature (e.g., 30°C) during the induction of the expression of proteinaceous molecule. For example, both eukaryotic and prokaryotic expression of OPH may be conducted at temperatures

48fff

30°C, which has increased the production of enzymatically active OPH by reducing protein misfolding and inclusion body formation in some instances (Chen-Goodspeed, M. etal., 2001b; Wang, J. et al., 2001; Omburo, G. A. et al., 1992; Rowland, S. S. et al., 1991). In an additional example, prokaryotic expression of recombinant squid-type DFPase fusion protein at 30°C also enhanced yields of active enzyme (Hartleib, J. and Ruterjans, H., 2001a). It is contemplated that fed batch growth conditions at 30°C, in a minimal media, using glycerol as a carbon source, will be suitable for expression of various enzymes.

F. Processing of Expressed Proteinaceous Molecules

After production of a biomoiecule by a living cell, the composition comprising the biomolecule may undergo one or more processing steps to prepare a biomolecule composition of the present invention. Examples of such steps include permeabilizing, disrupting, sterilizing, concentrating, drying, resuspending, or a combination thereof. Various embodiments of a biomolecule composition of the present invention are contemplated after one or more such processing steps. However, it is further contemplated that each processing step will increase economic costs and/or reduce total biomolecule yield, so that embodiments comprising fewer steps are preferred. It is further contemplated that the order of steps may be varied and still produce a biomoiecule composition of the present invention.

In certain embodiments, a biomoiecule composition of the present invention may comprise various cellular components (e.g., cell wall material, cell membrane material, nucleic acids, sugars, polysacharrides, peptides, polypeptides, proteins, lipids, etc). Such a biomoiecule composition of the present invention is known herein as a “crude cell preparation. A -“a crude cell preparation comprises the biomoiecule within or otherwise in contact with a cell and/or cellular debris. In certain aspects, it is contemplated that the total content of desired biomoiecule (θ g an active biomoiecule) may range from 0.0001% to 99.9999% of a crude cell preparation, including alt intermediate ranges and combinations thereof, by volume or dry weght, depending upon factors such as expression efficiency of the biomoiecule in the cell and the amount of processing and/or purification steps. A higher content of desired biomoiecule in the biomolecular composition is preferred. But. in certain embodiments, it is also preferred that the biomoiecule composition comprise cellular components, particularly cell wall and/or cell membrane material, to provide material that may be protective to the biomoiecule, enhances the particulate nature of the biomoiecule composition, or a combination thereof. Thus, the biomoiecule composition may comprise 0.0001% to 99.9999% of cellular components, including all intermediate ranges and combinations thereof, by volume or dry weight. However, in certain embodiments, lower ranges of cellular components is preferred, as the biomolecular composition would therefore comprise a greater percentage of a desired biomoiecule.

48ggg

In embodiments wherein the cellular material is derived from a microorganism, such ? through expression of the biomolecule by a microorganism, the biomolecular composition is known herein as a “microorganism based particulate materiai. The association of a biomolecule with a cell or cellular material is generally produced through endogenous expression, expression due to recombinant engineering, or a combination thereof. In preferred embodiments, a crude cell preparation comprises a biomolecule partly or whole encapsulated by a cell membrane and/or cell wall, whether naturally so and/or through recombinant engineering. Such a biomolecule (e.g., the active biomolecule) encapsulated within or as a part of a cell wall and/or cell membrane is referred to herein as a whole cell material” or “whole ceil particulate material .

It is contemplated that a biomolecule prepared as a crude cell preparation may have greater stability than a preparation wherein the biomolecule has been substantially separated from a cell membrane and/or cell wall. It is further contemplated that a biomolecule prepared as a crude ceil preparation, wherein the biomolecule is localized between the cell wall and cell membrane and/or within the cell so that the cell wall separates the biomolecule from the extracellular environment, may have greater stability than a preparation wherein the biomolecule has been substantially separated from a cell membrane and/or cell wall.

Additionally, it is contemplated that a biomolecule composition of the present invention may be encapsulated using a microencapsulation technique as would be known to one of ordinary skill in the art. Such encapsulation may enhance or confer the particulate nature of the biomolecule composition, provide protection to the biomolecule, increase the average particle size to a desired range, allow release of the biomolecule from the encapsulating material, alter surface charge, hydrophobicity, hydrophilicity, solubility and/or disperability of the particulate material, or a combination thereof. Examples of microencapulation (e.g., microsphere) compositions and techniques are described in Wang, Η. T. et al., J. of Controlled Resease 17:2325, 1991; and U.S. Patent Nos. 4,324,683, 4,839,046, 4,988,623, 5,026,650, 5153,131, 6,485,983, 5,627,021 and 6,020,312).

1. Cell Permeabilizatlon/Disruption

In preferred aspects, a biomolecular composition of the present invention comprises a crude cell preparation wherein the cell membrane and/or cell wall has been altered through a permeabllzating process, a disruption process, or a combination thereof. An example of such an altered crude cellular preparation includes disrupted cells, permeabilized cells, or a combination thereof. As used herein, a disrupted cell” is a crude cell preparation wherein wherein the cell membrane and/or cell wall has been altered through a disruption process. As used herein, a

48hhh permeabilized cell” is a crude cell preparation wherein the cell membrane and/or cell wall has been altered through a permeabilizating process. It is contemplated that a biomolecule composition of the present invention prepared as a crude cellular preparation may have greater stability than a preparation wherein the biomolecule has been substantially purified from the cell wall and/or membrane.

A processing step may comprise a permeabilizing step, wherein a celt is contacted with a permeabilizing agent such as dimethyl sulfoxide (DMSO), ethylenediaminetetraacetic acid (EDTA), tributyl phosphate, or a combination thereof. A permeabilizing step may increase the mass transport of a substrate into the interior of a cell, where an enzyme localized inside the cell can catalyze a chemical reaction with the substrate. (Martinez; Μ. B. etal., 1996; Martinez, M. B. etal., 2001; Hung, S.-C. and Liao, J. C., 1996). Cell permeabilizing using EDTA (Leduc, M. et al., 1985).

OP compound degradation rate has been limited by OPH intracellularly expressed in whole cells (Elashvili, I. and DeFrank, J. J., 1996; Elashvili, I. etal., 1998; Hung, S.-C. and Liao, J. C., 1996; Richins, R. etal., 1997). However, it is contemplated that a composition of the present invention comprising a whole cell particulate material will provide protection from diffusion of compounds that may damage a biomolecule, while allowing sufficient permeability to allow biomolecule function.

In some embodiments, a processing step comprises disrupting a cell. A cell may be disrupted by any method known in the art, including, for example, a chemical method, a mechanical method, a biological method, or a combination thereof. Examples of a chemical cell disruption method include suspension in a solvent for certain cellular components. In specific facets, such a solvent may comprise an organic solvent (e.g., acetone), a volatile solvent, or a combination thereof. In a particular facet, a cell be be disrupted by acetone (Wild, J. R. etal., 1986; Albizo, J. M. and White, W. E., 1986). In certain preferred facets, the cells are disrupted in a volatile solvent for ease in evaporation. Examples of a mechanical cell disruption method include pressure (e.g., processing through a French press), sonication, mechanical shearing, or a combination thereof. An example of a pressure cell disruption method includes processing through a French press. Examples of a biological cell disruption method include contacting the cell with one or more enzymes (e.g., lysozyme) that weaken, damage, and/or permeabilize a cell membrane, cell wall or combination thereof. Biological material comprising a proteinaceous molecule of the present invention may be homogenized, sheared, undergo one or more freeze thaw cycles, be subjected to enzymatic and/chemical digestion of cellular materials (e.g., cell walls, sugars, etc), undergo extraction with organic or aqueous solvents, etc, to weaken

48iii

.׳ /3658 interactions between the proteinaceous molecule and other cellular materials and/or partly purify the proteinaceous molecule. A processing step may comprise sonicating a composition comprising an enzyme. Other dissepting and drying will be done by freezedrying with or without a cryoprotector (typically a sugar).

2. Sterilization

A processing step may comprise sterilizing an enzyme composition of the present invention. Sterilizing kills living matter, and may be desirable as continued post expression growth of a host cell and/or a contaminating organism may detrimentally affect the composition. For example, one or more properties of a coating may be undesirably altered by the presence of a living organism. Additionally, sterilizing reduces the ability of a living recombinant organism to be introduced into the environment, when such an event is not desired. Sterilizing may be accomplished by any method known in the art. Examples of sterilizing may include contacting the living matter with a toxin, Irradiating the living matter, heating the living matter above 100°C, or a combination thereof. It is preferred that sterilizing comprises irradiating the living matter, as radiation generally does not leave a toxic residue, and is not contemplated to detrimentally affect the enzymes stability such as that which might occur during heating. Examples of radition include infrared (IR”) radiation, ionizing radiation, microwave radiation, ultra-violet (UV) radiation, particle radiation, or a combination thereof. Particle radiation, UV radiation and/or ionizing radiation are preferred, and particle radiation is particularly preferred. Examples of particle radiation include alpha radiation, electron beam/beta radiation, neutron radiation, proton radiation, or a combination thereof.

3. Concentrating a Biomolecule Composition

A processing step may comprise concentrating a biomolecule composition of the present invention. As used herein, “concentrating״ refers to any process wherein the volume of a composition is reduced. Often, undesired components that comprise the excess volume are removed, the desired composition is localized to a reduced volume, or a combination thereof.

For example, it is contemplated that a concentrating step may be used to reduce the amount of a growth and/or expression medium component from a composition of the present invention. It is contemplated that nutrients, salts and other chemicals that comprise a biological growth and/or expression medium may be unnecessary and/or unsuitable in a composition of the present invention, and reducing the amount of such compounds is preferred. A growth medium may promote undesirable microorganism growth in a composition of the present invention, while salts or other chemicals may undesirably alter the formulation of a coating.

48jjj

1/365'

Concentrating a biomolecule composition may be by any method known in the art, Including, for example, filtrating, a gravitational force, a gravimetric force, or a combination therof. An example of a gravitational force is normal gravity. An example of a gravimetric force is the force exerted during centrifugation. Often a gravitational or gravimetric force is used to concentrate a composition comprising the desired biomolecule from undesired components that are retained in the volume of a liquid medium. After ceils are localized to the bottom of a centrufugation devise, the media may be removed via such techniques as decanting, aspiration, etc.

4. Drying a Biomoiecule Composition

In additional embodiments, the disrupted cells and/or cell debris are dried, ground and/or milled to a powder. In specific facets, the cells added to the paint comprise disrupted cells, cell debris, and/or powder. The powder may be Preferrably stored at room temperature without need for dessication.

5. Resuspending Biomolecule Composition

A purification step may comprise resuspending a precipitated composition comprising an enzyme from cell debris.

The invention provides, in certain preferred embodiments, a composition comprising a coating and an enzyme prepared by the following steps: obtaining a culture of cells that express the enzyme; concentrating the cells and removing the culture media; disrupting the cell structure; drying the cells; and adding the cells to the coating. In some aspects, the composition is prepared by the additional step of suspending the disrupted cells in a solvent prior to adding the celts to the coating.

In certain aspects, the composition is prepared by adding the cell culture powder to glycerol, admixing with glycerol and/or suspending in glycerol. In other facets, the glycerol is at a concentration of about 50%. In specific facets, the cell culture powder comprised in glycerol at a concentration of about 3 mg of the milted powder to 3 ml of 50% glycerol. In certain facets, the composition is prepared by adding the powder comprised in glycerol to the paint at a concentration of about 3 ml glycerol comprising powder to 100 mt of paint. The powder may also be added to a liquid component such as glycerol prior to addition to the paint. The numbers are exemplary only and do not limit the use of the invention. The concentration was chosen merely to be compatible with the amount of substance that can be added to one example of paint without affecting the integrity of the paint itself. Any compatible amount may used within the scope of the present invention.

48kkk

17305S.

6. Temperatures

It is contemplated that in some embodiments, processing of an enzyme composition may be conducted at 4°C to 50°C, including all intermediate ranges and combinations thereof. In preferred embodiments, a processing step may comprise maintaining a composition comprising an enzyme at a temperature less than the optimum temperature for the activity of a living organism and/or enzyme that may detrimentially affect an enzyme of the present invention. Often 37°C is the maximum temperature for the processing of a eukarotic biomolecule (e.g., an enzyme). Thus temperatures less than 37°C are preferred, temperatures less than 30°C are more preferred, temperatures less than 20°C even more preferred, temperatures less than 10°C are particularly preferred, and temperatures of 4°C more preferred.

7. Other Processing Steps

In other embodiments, a proteinaceous molecule of the present invention may be a purified a proteinaceous molecule. A “purified proteinaceous molecule as used herein refers to any proteinaceous molecule of the present invention removed in any degree from other extraneous materials (e.g., cellular material, nutrient or culture medium used in growth and/or expression, etc). In certain aspects, removal of other extraneous material may produce a purified proteinaceous molecule of the present invention wherein its concentration has been enhanced 2to 10,000-fold or more, including all intermediate ranges and combinations thereof, from its original concentration in a material (e.g., a recombinant cell, a nutrient or culture medium, etc). In other embodiments, a purified proteinaceous molecule of the present invention may comprise 0.001% to 100%, including all intermediate ranges and combinations thereof of a composition comprising a proteinaceous molecule of the present invention. The degree or fold of purification may be determined using any method known to those of skill in the art or described herein. For example, it is contemplated that techniques such as measuring specific activity of a fraction by an assay described herein, relative to the specific activity of the source material, or fraction at an earlier step in purification, may be used.

Techniques for preparation of a proteinaceous molecule of the present invention are described herein. However, it is contemplated that one or more additional methods for purification of biologically produced molecule(s) that are known in the art or described herein may be applied to obtain a purified proteinaceous molecule of the present invention [Azzoni, A. R. et al., 2002; In Current Protocols in Molecular Biology (Chanda, V. B. Ed.) John Wiley &amp; Sons, 2002; In “Current Protocols in Nucleic Acid Chemistry (Harkins, E. W. Ed.) John Wiley &amp; Sons, 2002; In Current Protocols in Protein Science (Taylor, G. Ed.) John Wiley &amp; Sons, 2002; In Current Protocols in Cell Biology (Morgan, K. Ed.) John Wiley &amp; Sons, 2002; In ‘'Current Protocols in Pharmacology (Taylor, G. Ed.) John Wiley &amp; Sons, 2002; In “Current Protocols in

48111 ~ '־- — - —,r moieouia of the pre,an, invention may ba homogenlz^’ יי’״״״“““ interactions between the ρωίβίηΤοβουΓ T ° ^S°'^Ver,ts^etc&lt; ^t0 weaken the proteinaceous molecule. A procZfog^step⁰comprising an _en2ym־ ׳*״״״” ׳¹™ ”*־ ” .־ composition

- ו^{separa,s a}

LZ *־*¹״״™hy. ׳.W Performance liquid chromatograph| ״ ׳° - present invention _may molecule of e.g., affinity affinity chromatography), fast ־ HPLC ), ion-exchange _ is known. Blue) has been commonly _used to determine the purification of OPH, _as described (Kolakowski, J.

״» ^ra״־׳״—״ ״

SDS-PAGE and staining (_e._g., _C00massfe _. success of recombinant expression and/or

E. et al., 1997; Lai, K. et al., 1994).

In certain embodiments, an enzyme r ־ one or more enzyme crystals may be cross-linked for'from ( CLEC) (Hoskin, F. C. G. et al1999 ״).

may be in the form of a crystal. In other aspects, 1 an crosslinked enzyme crystal

6. Coatings composition that is converted to a או oatmg ) is a liquid, liquefiable or mastic application ־־ a thin ^d־“^ra“״^e “ ^־dh־r“'^fim ״״־

48mmm and combinations thereof. However, in most embodiments, it is contemplated that a coating will form a thin layer 15 urn to 150 urn thick, including all intermediate ranges and combinations thereof. Examples of a coating of the present invention include a clear coating or a paint.

A surface is the outer layer of any solid object. As would be known to those of ordinary skill in the art, the term substrate, in the context of a coating, is synonymous with the term surface. However, as “substrate has a different meaning to those of skill in arts of enzymology and coatings, the term “surface will be preferentially used herein for clarity. A surface wherein a coating has been applied, whether or not film formation has occurred, is known herein as a coated surface.”

As is known to those of ordinary skill in the art, a coating generally comprises one or more materials that contribute to the properties of the coating, the ability of a coating to be applied to a surface, the ability of the coating to undergo film formation, and/or the properties of the produced film. Examples of such coating components include a binder, a liquid component, a colorizing agent, an additive, or a combination thereof, and such materials are contemplated for used in a coating of the present invention. A coating typically comprises a material often referred to as a “binder, which is the primary material in a coating capable of film formation. Often the binder is the coating component that dominates conferring a physical and/or chemical property to a coating and/or film. Examples of properties of a binder typically affects include chemical reactivity, minimum film formation temperature, minimum T_g, volume fraction solids, a rheological property (e.g., viscosity), film moisture resistance, film UV resistance, film heat resistance, film weathering resistance, adherence, film hardness, film flexibility, or a combination thereof. Consequenfly, different categories of coatings may be identified herein by the binder used in the coating. For example, a binder may be an oil, a chlorinated rubber, or an acrylic, and examples of a coating comprising such binders include an oil coating, a chlorinated rubber-topcoat, an acrylic-lacquer, efc.

In most embodiments, a coating will comprise a liquid component (e.g., a solvent, a diluent, a thinner), which often confers and/or alters the coating's rheological properties (e.g., viscosity) to ease the application of the coating to a surface. In some embodiments, a coating will comprise a colorizing agent (e.g., a pigment), which usually functions to alter an optical property of a coating and/or film. In certain preferred embodiments, a microorganism based particulate material of the present invention is a colorizing agent. In particularly preferred embodiments, a colorizing agent comprising a microorganism based particulate material of the present invention is an extender, a pigment, or a combination thereof, in other preferred embodiments, a coating comprises a colorizing agent that comprises a microorganism based particulate material of the

48nnn present Invention. A coating wili often comprise an additive which is a composition incorporated into a coating to reduce and/or prevent the development of a physical, chemical, and/or aesthetic defect in the coating and/or film; confer some addition desired property to a coating and/or film; or a combination thereof. Examples of an additive include an accelerator, an adhesion promoter, an antioxidant, an antiskinning agent, a coalescing agent, a defoamer, a dispersant, a drier, an emulsifier, a fire retardant, a flow control agent, a gloss aid, a leveling agent, a marproofing agent, a slip agent, a thickener, a UV stabilizer, a viscosity control agent, a wetting agent, or a combination thereof. In certain preferred embodiments, a microorganism based particulate material of the present invention is an additive. In particularly preferred embodiments, an additive comprising a microorganism based particulate material of the present invention comprises a viscosity control agent, a dispersant, or a combination thereof. In other preferred embodiments, a coating comprises an additive that comprises a microorganism based particulate material of the present invention. A contaminant is a material that is unintentionally added to a coating, and may be volatile and/or non-volatile component of a coating and/or film. As would be known to those of ordinary skill in the art, a coating component may be categorized as possessing more than one defining characteristic, and thereby simultaneously functioning in a coating composition as a combination of a binder, a liquid component, a colorizing agent, and/or additive. Different coating compositions are described herein as examples of coatings with varying sets of properties.

In certain embodiments, a coating may be stored in a container (pot) prior to application. In certain aspects, the coating is a multi-pack coating which is a coating wherein different components are stored in a plurality of containers. Typically, this is done to reduce film formation during storage for certain types of coatings. The components are admixed prior to and/or during application. However, in certain embodiments, it is specifically contemplated that a coating comprising a microorganism based particulate material of the present invention is a multipack coating. In specific aspects, the coating is a two-pack coating, three-pack coating, four-pack coating, five-pack coating, or more wherein the coating components are stored in separate containers. In certain aspects, 0.001% to 100%, including all intermediate ranges and combinations thereof, of the microorganism based particulate material is stored in a separate container from a coating component, it is contemplated that separate storage may reduce undesirable microorganism growth in the coating component, damage to the microorganismbased particulate material of the present invention by the coating component, increase the storage life (pot life”) of a coating, reduce the amount of a preservative in a coating, or a combination thereof. In certain facets, it is contemplated that the coating components of a container holding the microorganism based particulate material of the present invention may further include a coating component such as a preservative, a wetting agent, a dispersing agent, a liquid component, a rheological modifier, or a combination thereof. It is contemplated that a

48000 i ;3058/1 preservative may reduce undesirable growth of a microorganism, whether the microorganism is derived from the microorganism based particulate material of the present invention or a contaminating microorganism. It is contemplated that a wetting agent, a dispersing agent, a liquid component, a rheological modifier, or a combination thereof, may promote ease of admixing of coating components in a multi-pack coating. In certain aspects, a three-pack coating or four-pack coating may be used, wherein the first container and the second container contain coating components separated to reduced film formation during storage, and a third container comprises 0.001% to 100%, including all intermediate ranges and combinations thereof, of the microorganism based particulate material. In certain facets, a multi-pack coating may be used to separate two or more preparations of the microorganism based particulate material of the present invention such as to reduce damage by different species of microorganisms to each other during storage.

A coating may be applied to a surface using any technique known in the art. A in the context of a coating, “application,” “apply, or “applying” is the process of transferring of a coating to a surface to produce a layer of coating upon the surface. As known herein, an “applicator is a devise that is used to apply the coating to a surface. Examples of an applicator include a brush, a roller, a pad, a rag, a spray applicator, etc. Application techniques that are contemplated as suitable for a user of the present invention of little or no particular skill include, for example, dipping, pouring, siphoning, brushing, rolling, padding, ragging, spraying, etc. Certain types of coatings may be applied using techniques contemplated as more suitable for a skilled artisan such as anodizing, electroplating, and/or laminating of a polymer film onto a surface.

In certain embodiments, the layer of coating undergoes film formation (curing, cure), which is the physical and/or chemical change of a coating to a solid that is a preferred solid when in the form of a layer upon the surface. In certain aspects, a coating may be prepared, applied and cured at an ambient condition, a baking condition, or a combination thereof. An ambient condition is a temperature range between -10°C to 40°C, including all intermediate ranges and combinations thereof. As used herein, a baking condition or “baking is contacting a coating with a temperature above 40°C and/or raising the temperature of a coating above 40°C, typically to promote film formation. Examples of baking the coating include contacting a coating and/or raising the temperature of coating to 40^DC to 300°C, or more, including all intermediate ranges and combinations thereof. Various coatings described herein or as would be known to one of ordinary skill in the art may be applied and/or cured at ambient conditions, baking conditions, or a combination thereof.

48ppp

058/1&lt; ־'

It is contemplated that in general embodiments, a coating comprising a microorganism based particulate material of the present invention may be prepared, applied and cured at any temperature range described herein or would be known to one of ordinary skill in the art in light of the present disclosures. An example of such a temperature range is -100°C to 300°C, or more, including all intermediate ranges and combinations thereof. However, a microorganism based particulate material may further comprise a desired biomolecule (e.g., a colorant, an enzyme), whether endogenously or recombinantly produced, that may have a reduced tolerance to temperature. It is contemplated that the preferred temperature that can be tolerated by a biomolecule will vary depending on the specific biomolecule used in a coating, and will generally be within the range of temperatures tolerated by the living organism from which the biomolecule was derived. For example, it is preferred for a coating comprising a microorganism based particulate material of the present invention, wherein the microorganism based material comprises an desired enzyme, that the coating is prepared, applied and cured at -100°C to 110C, including all intermediate ranges and combinations thereof. For example, it is contemplated that a temperature of -100°C to 40°C including all intermediate ranges and combinations thereof, will be suitable for many enzymes (e.g., a wild-type sequence and/or a functional equivalent) derived from an eukaryote, while temperatures up to, for example -100°C to 50°C including all intermediate ranges and combinations thereof, may be tolerated by enzymes derived from many prokaryotes.

The type of film formation that a coating may undergo depends upon the coating components. A coating may comprise, for example, volatile coating components, non-volatile coating components, or a combination thereof. In certain aspects, the physical process of film formation comprises loss of 1% to 100%, including all intermediate ranges and combinations thereof, of a volatile coating component. In general embodiments, a volatile component is lost by evaporation. In certain aspects, loss of a volatile coating component during film formation reaction is promoted by baking the coating. Examples of volatile coating components include a coalescing agent, a solvent, a thinner, a diluent, or a combination thereof. A non-volatile component of the coating remains upon the surface. In specific aspects, the non-volatile component forms a film. Examples of non-volatile coating components include a binder, a colorizing agent, a plasticizer, a coating additive, or a combination thereof. In specific aspects, a coating component may undergo a chemical change to form a film. In general embodiments, a binder undergoes a cross-linking (e.g., polymerization) reaction to produce a film. In general embodiments, a chemical film formation reaction occurs spontaneously under ambient conditions. In other aspects, a chemical film formation reaction is promoted by irradiating the coating, heating the coat, or a combination thereof. In some embodiments, irradiating the coating comprises exposing the coating to electromagnetic radiation, particle radiation, or a combination thereof.

48qqq ! 73658/ί

Examples of electromagnetic radiation used to irradiate a coating include UV radiation, infrared radiation, or a combination thereof. Examples of particle radiation used to irradiate a coating include electron-beam radiation. Often irradiating the coating induces an oxidative and/or free radical chemical reaction that cross-links of one or more coating components.

However, in some alternate embodiments, it is contemplated that a coating undergoes a reduced amount of film formation than such a solid film is not produced, or does not undergo film formation to a measurable extent during the period of time it is used on a surface. Such a coating is referred to herein as a non-film forming coating.” Such a non-film forming coating may be prepared, for example, by increasing the non-volatile component in a thermoplastic coating (e.g., increasing plasticizer content in a liquid component), reducing the amount of a coating component that contributes to the film formation chemical reaction (e.g., a binder, a catalyst), reducing the contact with an external a curing agent (e.g., radiation, baking), selection of a nonfilm formation binder produced from components that lack crosslinking moieties, selection of a non-film formation binder that lack sufficient size to undergo thermoplastic film formation, or a combination thereof. As used herein, a “non-film formation binder” refers to a molecule that is chemically similar to a binder, but lacks sufficient size and/or crosslinking moiety to undergo film formation. For example, a coating may be prepared by selection of an oil-based binder that lacks sufficient double bonds to undergo sufficient crosslinking reactions to produce a film. In another example, a non-film formation binder may be selected that lacks sufficient crosslinking moieties such as an epoxide, an isocyanate, a hydroxyl, a carboxyl, an amine, an amide, a silicon moiety, etc., to produce a film by thermosetting. Such a non-film formation binder may be prepared by chemical modification of a binder, such as, for example, a crosslinking reaction with a small molecule (e.g., less than 1 kDa) that comprises a moiety capable of reaction with a binder's crosslinking moiety, to produce a chemically blocked binder moiety that is inert to a further crosslinking reaction. In another example, a thermoplastic binder typically comprises a molecule 29 kDa to 1000 kDa or more in size. Film formation may be reduced or prevented by selection of a like molecule that is too small to effectively undergo thermoplastic film formation. An example would be selection of a non-film formation binder molecule between 1 kDa to 29 kDa in molecular weight, including all intermediate ranges and combinations thereof.

In other alternative embodiments, a coating may undergo film formation, but produce a film whose properties makes it more suited for a temporary use. Such a temporary film will generally possess a poor and/or low rating for a property that would confer longevity in use. For example, a film with a poor scrub resistance, a poor solvent resistance, a poor water resistance, a poor weathering property (e.g., UV resistance), a poor adhesion property, or a combination thereof, may be selected as a temporary film. In one aspect, a film may have poor adhesion for a

48rrr 'O6S8/1 surface, allowing ease of removal by stripping and/or peeling. In another example, a film may have a poor resistance to an environmental factor, and subsequently fail (e.g., crack, peel, chalk, etc.) to remain a viable film upon the surface. For example, a film that undergoes chalking is specifically contemplated. Chalking is the erosion a coating, typically by degradation of the binder due to various environmental forces (e.g., UV irradiation). It is contemplated that in some embodiments, chalking may be desirable, to expose remove a contaminant from the surface of a film and/or expose a component of the film (e.g., a biomolecular composition of the present invention) to the surface of the coating. A self-cleaning coating is a film with a desirable chalking property. It is further contemplated that in many aspects the layer of non-film forming coating, a temporary film and/or a self-cleaning film may be removed from a surface with ease. In such embodiments, a non-film forming coating, a temporary film, a self-cleaning film, or a combination thereof would be more suitable for a temporary use upon a surface, due to the ability to be applied as a layer and easily removed when its presence is no longer desired. In these embodiments, it is contemplated that the non-film forming coating, the temporary film, the selfcleaning film, or a combination thereof, is desired for a use upon a surface that lasts a temporary period □f time, such as, for example, 1 to 60 seconds, 1 to 24 hours, 1 to 7 days, 1 to 10 weeks, 1 to 6 months, including all intermediate ranges and combinations thereof, respectively.

In some embodiments, a plurality of coating layers, known herein as a multicoat system” (״multicoating system), may be applied upon a surface. The coating selected for use in a specific layer may differ from an additional layer of the multicoat system. This selection of coatings with differing components and/or properties is typically done to sequentially confer, in a desired pattern, the properties of differing coatings to a coated surface and/or multicoat system. Examples of a coating that may be selected for use, either alone or in a multicoat system, include a sealer, a water repellent, a primer, an undercoat, a topcoat, or a combination thereof. A sealer is coating applied to a surface to reduce or prevent absorption by the surface of a subsequent coating layer and/or a coating component thereof, and/or to prevent damage to the subsequent coating layer by the surface. A water repellant is a coating applied to a surface to repel water. A primer is a coating that is applied to increase adhesion between the surface and a subsequent layer. In typical embodiments a primer-coating, a sea I er-coating, a water repeilent-coating, or a combination thereof is applied to porous surface. Examples of a porous surface include drywall, wood, plaster, masonry, damaged and/or degraded film, corroded metal, or a combination thereof. In certain aspects, the porous surface is not coated or lacks a film prior to application of a primer, sealer, water repellent, or combination thereof. An undercoat is a coating applied to surface to provide a smooth surface for a subsequent coat. A topcoat (“finish) is a coating applied to a surface for a protective and/or decorative purpose. Of course, a sealer, water repellent, primer, undercoat, and/or topcoat may possess additional protective, decorative, and/or

48sss :73658/1 functional properties. Additionally, the surface a sealer, water repellent, primer, undercoat, and/or topcoat are applied to may be a coated surface such as a coating and/or film of a layer of the a multicoat system. In certain embodiments, a multicoat system may comprise any combination of a sealer, water repellent, primer, undercoat, and/or topcoat. For example, a multicoat system may comprise any of the following combinations: a sealer, a primer and a topcoat: a primer and topcoat; a water repellent, a primer, undercoat, and topcoat; an undercoat and topcoat; a sealer, an undercoat, and a topcoat; a sealer and topcoat; a water repellent and topcoat, etc. In particular aspects, a coating layer may comprise properties that would be a combination of those associated with different coating types such as a sealer, water repellent, primer, undercoat, and/or topcoat. In such instances, such a combination coating and/or film is designated by a backslash ’7“ separating the individual coating designations encompassed by the layer. Examples of such a coating layer comprising a plurality of functions include a sealer/primer coating, a sealer/primer/undercoat coating, a sealer/undercoat coating, a primer/undercoat coating, a water repellant/primer coating, an undercoat/topcoat coating, a primer/topcoat coating, a primer/undercoat/topcoat coating, etc. In embodiments wherein the coated surface comprises a particular type of coating, then the coated surface may be known herein by the type of coating such as a painted surface, a ״clear coated surface,” a “lacquered surface, a “varnished surface,” a water repellant/primered surface,” an primer/undercoat-topcoated surface,” etc.

In specific aspects, a multicoat system may comprise a plurality of layers of the same type, such as, for example, 1 to 10 layers, including all intermediate ranges and combinations thereof, of a sealer, water repellent, primer, undercoat, topcoat, or any combination thereof. In specific facets, a multicoat system comprises a plurality of layers of the same coating type, such as. for example, 1 to 10 layers, including all intermediate ranges and combinations thereof, of a sealer, water repellent, primer, undercoat, or topcoat. In embodiment where a coating does not comprise a multicoat system, but a single layer of coating applied to a surface, such a layer, regardless of typical function in a multicoat system, is regarded herein as a topcoat.

1. Paints

A paint is a pigmented liquid, liquefiable or mastic composition designed for application to a substrate in a thin layer which is converted to an opaque solid film after application. Used for protection, decoration or identification, or to serve some functional purpose such as the filling or concealing of surface irregularities, the modification of light and heat radiation characteristics, etc.’’ [^,,Paint and Coating Testing Manual, Fourteenth Edition of the Gardner-Sward Handbook (Koleske, J. V. Ed.), p. 696, 1995]. However, as certain coatings disclosed herein are non-film forming coatings, this definition is modified herein to encompass a coating with the same properties of a film forming paint, with the exception that it does not produce a solid film. In

48ttt

173658/ particular embodiments, a non-film forming paint possesses a hiding power sufficient to concealing surface feature comparable to an opaque film.

Hiding power is the ability of a coating and/or film to prevent light from being reflected from a surface, particularly to convey the suface's visual pattern. Opacity is the hiding power of a film. An example of hiding power would be the ability of a paint-coating to visually block the appearance of grain and color of a wooden surface, as opposed to a clear varnish-coating allowing the relatively unobstructed apperance of wood to pass through the coating. Standard techniques for determining the hiding power of a coating and/or film (e.g״ paint, a powder coating) are described, for example, in ASTM Book of Standards, Volume 06.01, Paint -- Tests for Chemical, Physical, and Optical Properties; Appearance, E284-02b, D344-97, D2805-96a, D2745-00 and D6762-02a 2002; “ASTM Book of Standards, Volume 06.02, Paint - Products and Applications; Protective Coatings; Pipeline Coatings,” D5007-99, D5150-92 and D6441-99, 2002; and Paint and Coating Testing Manual, Fourteenth Edition of the Gardner-Sward Handbook (Koleske, J. V. Ed.), pp. 481-506, 1995.

2. Clear-coatings

A clear-coating is a coating that is not opaque and/or does not produce an opaque solid film after application. A clear-coating and/or film may be transparent or semi-transparent (e.g., translucent). A clear-coating may be colored or non-colored. In certain embodiments, reducing the content of a pigment in a paint composition may produce a clear-coating. Additionally, a clear-coating may comprise a lacquer, a varnish, a shellac, a stain, a water repellent coating, or a combination thereof. Though some opaque coatings are referred to in the art as a lacquer, a varnish, a shellac, or a water repellent coating, all such opaque coatings are considered as paints herein (e.g״ a lacquer-paint, a varnish-paint, a shellac-paint, a water repellent paint).

a. Varnishes

A varnish is a thermosetting coating that converts to a transparent or translucent solid film after application. In general embodiments, a varnish is a wood-coating. A varnish comprises an oil and a dissolved binder. In general embodiments, the oil comprises a drying oil, wherein the drying oil functions as an additional binder. In other embodiments, the binder is solid at room temperature prior to dissolving into the oil and/or an additional liquid component of the varnish. Examples of a dissolvable binder include resins obtained from a natural source (e.g., a Congo resin, a copal resin, a damar resin, a kauri resin), a synthetic resin, or a combination thereof. In specific aspects, the additional liquid component comprises a solvent such as a hydrocarbon solvent. In some facets, the solvent is added to reduce viscosity of the varnish. A varnish may further comprise a coloring agent, including a pigment, for such purposes as conferring or altering

48uuu a color, gloss, sheen, or a combination thereof. A varnish undergoes thermosetting film formation by oxidative cross-linking. In certain aspects, a varnish may additionally undergo filmformation by evaporation of a volatile component. The dissolved binder generally functions to shorten the time to film-formation relative to certain measures (e.g., dryness, hardness), though the final cross-linking reaction time may not be significantly or measurably shortened. Standards for determining a varnish-coating and/or film's properties are described in, for example, “ASTM Book of Standards, Volume 06.03, Paint - Pigments, Drying Oils, Polymers, Resins, Naval Stores, Cellulosic Esters, and Ink Vehicles, D154-85, 2002.

b. Lacquers

A lacquer is a thermoplastic, solvent-borne coating that converts to a transparent or translucent solid film after application. In general embodiments, a lacquer is a wood-coating. A lacquer-coating comprises a thermoplastic binder dissolved in a liquid component comprising an active solvent. Examples of a thermoplastic binder include a cellulosic binder (e.g., nitrocellulose, cellulose acetate), a synthetic resin (e.g., an acrylic), or a combination thereof. In certain aspects, a liquid component comprises an active solvent, a latent solvent, diluent, a thinner, or a combination thereof. In certain embodiments, a lacquer is nonaqueous dispersion (NAD”) lacquer, wherein the content of solvent is not sufficient to fully dissolve the thermoplastic binder. In certain aspects, a lacquer may comprise an additional binder (e.g., an alkyd), a colorant, a plasticizer, or a combination thereof. Film formation of a lacquer occurs by loss of the volatile components, typically through evaporation.

Standards for a lacquer-coating and/or film's composition (e.g., a lacquer, a pigmentedlacquer, a nitrocellulose lacquer, a nitrocellulose-alkyd lacquer), physical and/or chemical properties (e.g., heat and cold resistance, hardness, film-formation time, stain resistance, particulate material dispersion), and procedures for testing a lacquers composition/properties, are described in, for example, in ASTM Book of Standards, Volume 06.02, Paint — Products and Applications; Protective Coatings; Pipeline Coatings, D333-01, D2337-01, D3133-01, D365-01, D2091-96, D2198-02, D2199-82, D2571-95 and D2338-02, 2002.

c. Shellacs

A shellac is similar to a lacquer, but the binder does not comprise a nitrocellulose binder, and the binder is soluble in alcohol, and the binder Is obtained from a natural source. A preferred binder comprises Laciffer lacca beetle secretion. In general embodiments, a shellac comprises a liquid component (e.g. alcohol). In specific aspects, the additional liquid component comprises a solvent. In some facets, the liquid component is added to reduce viscosity of the varnish, in other embodiments, a shellac undergoes rapid film formation. Standards for a shellac-coating

48vw and/or film’s composition and properties are described in, for example, ASTM Book of

Standards, Volume 06.03, Paint - Pigments, Drying Oils, Polymers, Resins, Naval Stores,

Cellulosic Esters, and Ink Vehicles, D29-98 and D360-89, 2002.

d. Stains

A stain a clear or semitransparent coating formulated to change the color of surface. In general embodiments, a stain is a wood-coating designed to color or protect a wood surface but not conceal the grain pattern or texture. A stain comprises a binder such as an oil, an alkyd, or a combination thereof. Often a stain comprises a low solid content. A low solids content for a wood stain is less than 20% volume of solids. The low solid content of a stain promotes the ability of the coating to penetrate the material of the wooden surface. This property is often used to, for example, to promote the incorporation of a fungicide that may be comprised within the stain into the wood. In certain alternative aspects, a stain comprises a high solids content stain, wherein the solid content is 20% or greater, may be used on a surface to produce a film possessing the property of little or no flaking. In other alternative aspects, a water-borne stain may be used such as a stain comprising a water-borne alkyd. A stain typically further comprises a liquid component (e.g., a solvent), a fungicide, a pigment, or a combination thereof. In other aspects, a stain comprises a water repellent hydrophobic compound so it functions as a water repellent-coating (stain/water repellent-coating). Examples of a water repellent hydrophobic compound a stain may comprise include a silicone oil, a wax, or a combination thereof. Examples of a fungicide include a copper soap, a zinc soap, or a combination thereof. Examples of a pigment include a pigment that is similar in color to wood. Examples of such pigments include a red pigment (e.g., a red iron oxide) a yellow pigment (e.g., a yellow iron oxide), or a combination thereof. Standards procedures for testing a stain’s (e.g., an exterior stain) properties, are described in, for example, in “ASTM Book of Standards, Volume 06.02, Paint — Products and Applications; Protective Coatings; Pipeline Coatings,” D6763-02, 2002.

e. Water repeilent-coatings

A water repellent-coating is a coating that comprises hydrophobic compounds that repel water. A water repellent-coating is typically applied to a surface susceptible to water damage, such as metal, masonry, wood, or a combination thereof. A water repellent-coating typically comprises a hydrophobic compound and a liquid component. In specific embodiments, a water repellent-coating comprises 1% to 65% hydrophobic compound, including all intermediate ranges and combinations thereof. Examples of a hydrophobic compound that may be selected include an acrylic, a siliconate, a metal-searate, a silane, a siloxane, a parafinnic wax, or a combination thereof. A water repellent may be a water-borne coating, or a solvent-borne coating. A solventborne water repellent-coating typically comprises a solvent that dissolves the hydrophobic

48www compound. Examples of solvents include an aliphatic, an aromatic, a chlorinated solvent, or a combination thereof.

in certain embodiments, a water repellent-coating, undergoes film formation, penetrates pores, or a combination thereof. In certain aspects, an acrylic-coating, a silicone-coating, or a combination thereof, undergoes film formation. In other aspects, a metal-searate, a silane, a siloxane, a parafinnic wax, or a combination thereof, penetrates pores in a surface. In some facets, a water repellent-coating (e.g., a silane, a siloxane) covalently bonds to a surface and/or pore (e.g., masonry). Standards for a water repellent-coating and/or film's composition and properties are described in, for example, “ASTM Book of Standards, Volume 06.02, Paint Products and Applications; Protective Coatings; Pipeline Coatings,” D2921-98, 2002; and in “Paint and Coating Testing Manual, Fourteenth Edition of the Gardner-Sward Handbook, (Koleske, J. V. Ed.), pp. 748-750, 1995. Alternatively, standards for a sealer-coating (e.g., a floor sealer) and/or film’s composition and properties are described in, for example, ASTM Book of Standards, Volume 06.02, Paint -- Products and Applications; Protective Coatings; Pipeline Coatings, D1546-96, 2002;

3. Coating Categories by Use

In light of the present disclosures, one of ordinary skill in the art may prepare and apply a coating of the present invention to any surface. However, it is preferred that the coating components and methods described herein are selected for a particular application to provide a coating and/or film with properties best suited for a particular use. For example, a coating used in an external environment would preferably comprise a coating component of superior UV resistance than a coating used in interior environment. In another example, a film used upon a surface of a washing machine would preferably comprise a component that confers superior moisture resistance than a component of a film for use upon a ceiling surface. In a further example, a coating applied to the surface of an assembly line manufactured product would preferably comprise components suitable for application by a spray applicator. Various properties of coating components are described herein to provide guidance to the selection of specific coating compositions with a suitable set of properties for a particular use.

A coating of the present invention may be classified by its preferred end use, including, for example, as an architectural coating, an industrial coating, a specification coating, or a combination thereof. An architectural coating is an organic coating intended for on-site application to interior or exterior surfaces of residential, commercial, institutional, or industrial buildings, in contrast to industrial coatings. They are protective and decorative finishes applied at ambient conditions״ [Paint and Coating Testing Manual, Fourteenth Edition of the Gardner48xxx

Sward Handbook (Koleske, J. V. Ed.), p. 686, 1995)] An industrial coating is a coating applied in a factory setting, typically for a protective and/or aesthetic purpose. A specification coating (“specification finish coating) Is a coating formulated to a precise statement of a set of requirements to be satisfied by a material, produce, system, or service that indicates the procedures for determining whether each of the requirements are satisfied [Paint and Coating Testing Manual, Fourteenth Edition of the Gardner-Sward Handbook (Koleske, J. V. Ed.), p. 891, 1995]. Often, a coating may be categorized as a combination of an architectural coating, an industrial coating, and/or a specification coating. For example, a coating for the metal surfaces of ships may be classified as specification coating, as specific criteria of water resistance and corrosion resistance are required in the film, but typically such a coating can be classified as an industrial coating, since it would typically be applied in a factory. Various examples of an architectural coating, an industrial coating and/or a specification coating and coating components are described herein. Additionally, architectural coatings, industrial coatings, specification coatings are known to those of ordinary skill in the art, and are described, for example, in Paint and Surface Coatings: Theory and Practice 2^״d Edition, pp. 190-192, 1999; in Paints, Coatings and Solvents 2^nd Edition, pp. 330-410, 1998; in “Organic Coatings: Science and Technology, Volume 1: Film Formation, Components, and Appearance” 2^nd Edition, pp. 138 and 317-318.

a. Architectural Coatings

An architectural coating (trade sale coating, “building coating,” decorative coating, house coating) is a coating suitable to coat surface materials commonly found as part of buildings and/or associated objects (e.g., furniture). Examples of a surface an architectural coating is typically applied to include, a plaster surface, a wood surface, a metal surface, a composite particle board surface, a plastic surface, a coated surface (e.g., a painted surface), a masonry surface, a floor, a wall, a ceiling, a roof, or a combination thereof. Additionally, an architectural coating may be applied to an interior surface, an exterior surface, or a combination thereof. An interior coating generally possesses properties such as minimal odor (e.g., no odor, very tow VOC), good blocking resistance, print resistance, good washability (e.g., wet abrasion resistance), or a combination thereof. An exterior coating typically is selected to possess good weathering properties. Examples of coating type commonly used as an architectural coating include an acrylic-coating, an alkyd-coating, a vinyl-coating, a urethane-coating, or a combination thereof. In certain aspects, a urethane-coating is applied to a piece of furniture. In other facets, an epoxy-coating, a urethane-coating, or a combination thereof, is applied to a floor. In some embodiments, an architectural coating is a multicoat system. In certain aspects, an architectural coating is a high performance architectural coating (HIPAC). A HIPAC is architectural coatings that produce a film with a combination of good abrasion resistance, staining resistance, chemical resistance, detergent resistance, and mildew resistance. Examples of binders suitable for

48yyy producing a HIPAC include a two-pack epoxide or urethane, or a moisture cured urethane. In general embodiments, an architectural coating comprises a liquid component, an additive, or a combination thereof. In certain aspects, an architectural coating is a water-borne coating or a solvent-borne coating. In other aspects, an architectural coating comprises a pigment, in preferred aspects, such an architectural coating is formulated to comprise a reduced amount or lack a toxic coating component. Examples of a toxic coating component include a heavy metal (e.g., lead), formaldehyde, a nonyl phenol ethoxylate surfactant, a crystalline silicate, or a combination thereof.

In certain embodiments, a water-borne coating has a density of 1.20 kg/L to 1.50 kg/L, including all intermediate ranges and combinations thereof, in other embodiments, a solventborne coating has a density of 0.90 kg/L to 1,2 kg/L, including all intermediate ranges and combinations thereof. The density of a coating can be empirically determined, for example, as described in ASTM Book of Standards, Volume 06.01, Paint -- Tests for Chemical, Physical, and Optical Properties; Appearance,” D1475-98, 2002. In certain embodiments, the course particle content of an architectural coating, by weight, is 0.5% or less. The coarse particle (e.g., coarse contaminants, pigment agglomerates) content of a coating can be empirically determined, for example, as described in “ASTM Book of Standards, Volume 06.03, Paint - Pigments, Drying Oils, Polymers, Resins, Naval Stores, Cellulosic Esters, and Ink Vehicles, D185-84, 2002. In some embodiments, the viscosity for an architectural coating at relatively low shear rates used during typical application, in Krebs Units (“Ku), is 72 Ku to 95 Ku, including all intermediate ranges and combinations thereof.

In typical use, an architectural coating is often stored in a container for months or even years prior to first use, and/or between different uses. In many embodiments, it will be preferred that a building coating will retain a desirable set properties of a coating, film formation, film, or a combination thereof, for a period of 12 months or greater in a container at ambient conditions. Properties that are preferred for storage include settling resistance, skinning resistance, coagulation resistance, viscosity alteration resistance, or a combination thereof. Storage properties can be empirically determined for a coating (e.g., an architectural coating) as described, for example, in ASTM Book of Standards, Volume 06.02, Paint - Products and Applications; Protective Coatings; Pipeline Coatings, D869-85 and D1849-95, 2002.

It is preferred that application and/or film formation of an architectural coating occurs at ambient conditions to provide ease of use to a casual user of the coating, as well as reduce potential damage to the target surface and the surrounding environment (e.g., unprotected people and objects). In general embodiments, it is preferred that an architectural coating does not

48zzz undergo film formation by a temperature greater than 40°C to reduce possible heat and fire damage. In other embodiments, it is preferred that an architectural coating is suitable to be applied by using hand-held applicator. Hand-held applicators are generally can be used without difficulty by most users of a coating, and examples include a brush, a roller, a sprayer (e.g., a spray can), or a combination thereof.

Specific procedures for determining the suitability of a coating and/or film for use as an architectural coating (e.g., a water-borne coating, a solvent-borne coating, an interior coating, an exterior paint, a latex paint), and specific assays for properties typically desired in an architectural coating (e.g., blocking resistance, hiding power, print resistance, washability, weatherability, corrosion resistance) have been described, for example, in ASTM Book of Standards, Volume 06.02, Paint - Products and Applications; Protective Coatings; Pipeline Coatings, D5324-98, D5146-98, D3730-98, D1848-88, D5150-92, D2064-91, D4946-89, D6583-00, D3258-00, and D3450-00, 2002; ASTM Book of Standards, Volume 06.01, Paint - Tests for Chemical, Physical, and Optical Properties; Appearance,” D660-93, D4214-98, D772-86, D662-93, and D661-93, 2002; and in “Paint and Coating Testing Manual, Fourteenth Edition of the Gardner-Sward Handbook (Koleske, J. V. Ed.), pp. 696-705, 1995.

(1) Wood Coatings

As is well known to those of ordinary skill in the art, a wood coating is often selected to protect the wood from damage, as welt as aesthetic purposes. For example, wood is susceptible to damage from bacteria and fungi. Examples of fungi that damage wood include Aureobasidium pullulans, and Ascomycotina, Deutermycotina, Basidiomycetes, Coniophora puteana, Serpula lacrymans, and Dacrymyces stillatus. It is preferred that a wooden surface is impregnated with a preservative such as a fungicide, prior to application of a coating of the present invention. However, most wood that is contemplated as a surface for a coating of the present invention is provided this way from wood suppliers. Specific procedures for determining the presence of a preservative and/or water repellent in wood have been described, for example, in “ASTM Book of Standards, Volume 06.02, Paint - Products and Applications; Protective Coatings; Pipeline Coatings, D2921-98, 2002.

Typically, wood surfaces are coated with a paint, a varnish, a stain, or a combination thereof. Often, the choice of coating is based on the ability of a coating to protect the wood from damage by moisture. Generally, a paint, a varnish, and a stain generally have progressively greater permeability to moisture, and moisture penetration of a wooden surface can which can lead to undesirable alterations in wood structure (e.g., splitting); undesirable alteration in piece of wood's dimension (“dimensional movement) such as shrinking, swelling, and/or warping;

48aaaa promote the growth of a microorganism such as fungi (e.g., wet rot, dry rot); or a combination thereof. Additionally, UV light irradiation damages a wood surface by depolymerizing lignin comprised in the wood. It is preferred that in embodiments wherein a wood surface is irradiated by UV light (e.g., sunlight), the wood coating comprises a UV protective agent such as a pigment that absorbs UV light. An example of a UV absorbing pigment includes a transparent iron oxide.

A preferred paint for use on a wood surface comprises an oil-paint, an alkyd-paint, or a combination thereof. A preferred alkyd-paint for use on a wood surface comprises a solventborne paint. A preferred paint system comprises a combination of a primer, an undercoat, and a topcoat. A film produced by a paint is often moisture impermeable. A film produced by paint upon a wooden surface may crack, flake, trap moisture that can encourage wood decay, be expensive to repair, or a combination thereof.

(2) Masonry Coatings

Masonry coatings refer to coatings used on a masonry surface, such as, for example, stone, brick, tile, cement-based materials (e.g., concrete, mortar), or a combination thereof. In general embodiments, a masonry coating is selected to confer resistance to water (e.g., salt water), resistance to acid conditions, alteration of appearance (e.g., color, brightness), or a combination thereof. Typically, a masonry coating comprises a multicoat system. In specific embodiments, a masonry multicoat system comprises a primer, a topcoat, or a combination thereof. Examples of a masonry primer include a rubber primer (e.g., a styrene-butadiene copolymer primer). In certain embodiments, a topcoat comprises a water-borne coating or a solvent borne coating. Examples of a water-borne coating that may be selected for a masonry topcoat include a latex coating, a water reducible polyvinyl acetate-coating, or a combination thereof. In certain aspects, a solvent-borne topcoat comprises a thermoplastic coating, a thermosetting coating, or a combination thereof. Examples of a thermosetting coating include an oil, an alkyd, a urethane, an epoxy, or a combination thereof. In certain aspects, a thermosetting coating is a multi-pack coating, such as, for example, an epoxy, a urethane, or a combination thereof. In specific aspects, a thermosetting coating undergoes film formation at ambient conditions, in other aspects, a thermosetting coating undergoes film formation at film formation at an elevated temperature such as a baking alkyd, a baking acrylic, a baking urethane, or a combination thereof. Examples of a thermoplastic coating include an acrylic, cellulosic, a rubberderivative, a vinyl, or a combination thereof. In specific aspects, a thermoplastic coating is a lacquer.

A masonry surface that is basic in pH, such as, for example, cement-based material and/or a calcareous stone (e.g., marble, limestone) may be damaging to certain coatings.

48bbbb

Specific procedures for determining the pH of a masonry surface have been described, for example, in “ASTM Book of Standards, Volume 06.02, Paint ־־ Products and Applications; Protective Coatings; Pipeline Coatings, D4262, 2002. Due to porosity and/or contact with an external environment, a masonry surface often accumulates dirt and other loose surface contaminants, which are preferably removed prior to application of a coating. Specific procedures for preparative cleaning (e.g., abrading, acid etching) of a masonry surface (e.g., sandstone, clay brick, concrete) have been described, for example, in “ASTM Book of Standards, Volume 06.02, Paint -- Products and Applications; Protective Coatings; Pipeline Coatings,” D4259-88, D426088D, 5107-90, D5703-95, D4261-83, and D4258-83, 2002. In certain embodiments, moisture at or near a masonry surface may be undesirable during application of a coating (e.g., a solventborne coating). Specific procedures for determining the presence of such moisture upon a masonry surface have been described, for example, in “ASTM Book of Standards, Volume 06.02, Paint - Products and Applications; Protective Coatings; Pipeline Coatings, D4263-83, 2002. Specific procedures for determining the suitability of a coating and/or film, particularly in conferring water resistance to a masonry surface, have been described, for example, in ASTM Book of Standards, Volume 06.02, Paint — Products and Applications; Protective Coatings; Pipeline Coatings, D6237-98, D4787-93, D5860-95, D6489-99, D6490-99, and D6532-00, 2002. Additional procedures for determining the suitability of a coating and/or film for use as a masonry coating have been described, for example, in Paint and Coating Testing Manual, Fourteenth Edition of the Gardner-Sward Handbook,” (Koleske, J. V. Ed.), pp. 725-730, 1995.

(3) Artist’s Coatings

Artist coatings refer to a coating used by artists for a decorative purpose. Often, an artist’s coating (e.g., paint) is selected for durability for decades or centuries at ambient conditions, usually indoors. Coatings such as an alkyd coating, an oil coating, an oleoresinous coating, an emulsion (e.g., acrylic emulsion) coating, or a combination thereof, are typically selected for use as an artist's coating. Specific standards for physical properties, chemical properties, and/or procedures for determining the suitability (e.g., lightfastness) of a coating and/or film for use as an artist's coating have been described, for example, in ASTM Book of Standards, Volume 06.02, Paint -- Products and Applications; Protective Coatings; Pipeline Coatings,” D4236-94, D5724-99, D4302-99, D4303-99, D4941-89, D5067-99, D5098-99, D538302, D5398-97, D5517-00, and D6801-023, 2002; and in Paint and Coating Testing Manual, Fourteenth Edition of the Gardner-Sward Handbook, (Koleske, J. V. Ed.), pp. 706-710, 1995.

b. Industrial Coatings

An industrial coating is a coating applied to a surface of a manufactured product in a factory setting. An industrial coating typically undergoes film formation to produce a film with a

48cccc protective and/or aesthetic purpose. Industrial coatings share some similarities to an architectural coating, such as comprising similar coating components, being applied to the same material types of surfaces, being applied to an interior surface, being applied to an exterior surface, or a combination thereof. Examples of coating types that are commonly used for an industrial coating Include an epoxy-coating, a urethane-coating, atkyd-coating, a vinyl-coating, chlorinated rubber-coating, or a combination thereof. Examples of a surface commonly coated by an industrial coating include metal (e.g., aluminum, zinc, copper, an alloy, efc); glass; plastic; cement; wood; paper; or a combination thereof. An industrial coating may be storage stable for 12 months or more, applied at ambient conditions, applied using a hand-held applicator, undergo film formation at ambient conditions, or a combination thereof.

However, an industrial coating often does not meet one or more of these characteristics previously described as preferred for an architectural coating. For example, an industrial coating may have a storage stability of only days, weeks, or months, as due to a more rapid use rate in coating factory prepared items. An industrial coating may be applied and/or undergo film formation at baking conditions. An industrial coating may be applied using techniques such as, for example, spraying by a robot, anodizing, electroplating, and/or laminating of a coating and/or film onto a surface. In some embodiments, an industrial coating undergoes film formation by irradiating the coating with non-visible light electromagnetic radiation and/or particle radiation such as UV radiation, infrared radiation, electron-beam radiation, ora combination thereof.

In certain embodiments, an industrial coating comprises an industrial maintenance coating, which is a coating that produces a protective film with excellent heat resistance (e.g., 121Ό or greater), solvent resistance (e.g., an industrial solvent, an industrial cleanser), water resistance (e.g., salt water, acidic water, alkali water), corrosion resistance, abrasion resistance (e.g., mechanical produced wear), or a combination thereof. An example of an industrial maintenance coating includes a high-temperature industrial maintenance coating, which is applied to a surface intermittently or continuously contacted with a temperature of 2040 or greater. An additional example of an industrial maintenance coating is an industrial maintenance anti-graffiti coating, which is a two-pack clear coating applied to an exterior surface that is intermittently contacted with a solvent and abrasion. Examples of coating types that are commonly used for an industrial maintenance coating include an epoxy-coating, a urethanecoating, alkyd-coating, a vinyl-coating, chlorinated rubber-coating, or a combination thereof.

Industrial coatings (e.g., coil coatings) and their use are well known to those of ordinary skill in the art (see, for example, in “Paint and surface coatings: Theory and Practice, 2^nd Edition, pp. 502-528, 1999; in Paints, Coatings and Solvents, 2^nd Edition, pp. 330-410, 1998; in “Organic

48dddd

Coatings: Science and Technology, Volume 1: Film Formation, Components, and Appearance,” 2^״d Edition, pp. 138, 317-318). Standard procedures for determining the properties of an industrial coating (e.g., an industrial wood coating, an industrial water-reducible coating) have been described, for example, in ASTM Book of Standards, Volume 06.02, Paint -- Products and Applications; Protective Coatings; Pipeline Coatings,” □4712-873, D6577-00a, D2336-99, D302398, D3794-00, □4147-99, and D5795-95, 2002.

(1) Automotive Coatings

Automotive coatings refer to coatings used on automotive vehicles, particularly those for civilian use. The manufacturers of a vehicle typically require that a coating conform to specific properties of weatherability (e.g., UV resistance) and/or appearance. Typically, an automotive coating comprises a multicoat system. In specific embodiments, an automotive multcoat system comprises a primer, a topcoat, or a combination thereof. Examples of an automotive primer include a nonweatherable primer, which lack sufficient UV resistance for single layer use, or a weatherable primer, which possesses sufficient UV resistance to be used without an additional layer. Examples of a topcoat include an interior topcoat, an exterior topcoat, or a combination thereof.

Examples of a nonweatherable automotive primer include a primer applied by electrodeposition, a conductive (“electrostatic”) primer, or a nonconductive primer. In certain embodiments, a primer is applied by electrodeposition, wherein a metal surface is immersed in a primer, and electrical current promotes application of a primer component (e.g., a binder) to the surface. An example of a metal primer suitable for electrodeposition application includes a primer comprising an epoxy binder comprising an amino moiety, a blocked isocyanate urethane binder, and a 75% to 95% aqueous liquid component, tn other embodiments, a primer is a conductive primer, which allows additional coating layers to be applied using electrostatic techniques. A conductive primer typically is applied to a plastic surface, including a flexible plastic surface or a nonflexible plastic surface. Such primers vary in their respective flexibility property to better suit use upon the surface. An example of a flexible plastic conductive primer includes a primer comprising polyester binder, a melamine binder and a conductive carbon black pigment. An example of a nonflexible plastic primer includes a primer that comprises an epoxy ester binder and/or an alkyd binder, a melamine binder and conductive carbon black pigment. In certain embodiments, a melamine binder may be partly or fully replaced with an aromatic isocyanate urethane binder, wherein the coating is a two-pack coating. A nonconductive primer is similar to a conductive primer, except the carbon-black pigment is absent or reduced in content. In certain embodiments, a nonconductive primer is a metal primer, a plastic primer, or a combination

48eeee thereof. In specific aspects, the nonconductive primer comprises a pigment for colorizing purposes.

Examples of a weatherable automotive primer include a primer/topcoat or a conductive primer. An example of a primer/topcoat includes a flexible plastic primer, with suitable weathering properties (e.g., UV resistance) to function as a single layer topcoat. Examples of a flexible plastic primer include a primer comprising an acrylic and/or polyester binder and a melamine binder. In certain embodiments, a melamine binder may be partly or fully replaced with an aliphatic isocyanate urethane binder, wherein the coating is a two-pack coating. A weatherable conductive primer typically is similar to a weatherable primer/topcoat, including a conductive pigment. In specific aspects, a weatherable automotive primer comprises a pigment for colorizing purposes.

An interior automotive topcoat typically is applied to a metal surface, a plastic surface, a wood surface, or a combination thereof. In certain aspects, an interior automotive topcoat is part of a multicoat system further comprising a primer. Examples of an interior automotive topcoat include a coating comprising a urethane binder, an acrylic binder, or a combination thereof.

An exterior automotive topcoat is typically is applied to a metal surface, a plastic surface, or a combination thereof. In certain aspects, an exterior automotive topcoat is part of a multicoat system further comprising a primer, sealer, undercoat, or a combination thereof. In certain embodiments, an exterior automotive topcoat comprises a binder capable of thermosetting in combination with a melamine binder. Examples of such a thermosetting binder include an acrylic binder, an alkyd binder, a urethane binder, polyester binder, or a combination thereof. In certain embodiments, a melamine binder may be partly or fully replaced with an urethane binder, wherein the coating is a two-pack coating. In typical embodiments, an exterior automotive topcoat further comprises a light stabilizer, a UV absorber, or a combination thereof. In general aspects, an exterior automotive topcoat further comprises a pigment.

Specific procedures for determining the suitability of a coating (e.g., a nonconductive coating) and/or film for use as an automotive coating, including spray application suitability, coating VOC content and film properties (e.g., corrosion resistance, weathering) have been described, for example, in ASTM Book of Standards, Volume 06.01, Paint - Tests for Chemical, Physical, and Optical Properties; Appearance,” D5087-02, D6266-00, and D6675-01, 2002; and ASTM Book of Standards, Volume 06.02, Paint - Products and Applications; Protective Coatings; Pipeline Coatings,” D5066-91, D5009-02, D5162-01, and D6486-01, 2002; and in

48ffff “Paint and Coating Testing Manual, Fourteenth Edition of the Gardner-Sward Handbook, (Koleske, J. V. Ed.), pp. 711-716, 1995.

(2) Can Coatings

Can coatings refer to coatings used on a container (e.g., an aluminum container, a steel container), for food, chemicals, or a combination thereof. The manufacturers of a can typically require that a coating conform to specific properties of corrosion resistance, inertness (e.g., to prevent flavor alterations in food, a chemical reaction with a container's contents, etc), appearance, durability, or a combination thereof. Typically, a can coating comprises an acryliccoating, an alkyd-coating, an epoxy-coating, a phenolic-coatlng, a polyester-coating, a poly(vinyl chloride)-coating, or combination thereof. Though a can may be made of the same or similar material, different surfaces of a can may require coatings of differing properties of inertness, durability and/or appearance. For example, a coating for a surface of the interior of a can that contacts the container's contents may ba selected for a chemical inertness property, a coating for a surface at the end of a can may be selected for a physical durability property, or a coating for a surface on the exterior of a can may be selected for an aesthetic property. To meet the varying can surface requirements, a can coating may comprise a multicoat system. In specific embodiments, a can multicoat system comprises a primer, a topcoat, or a combination thereof. In certain embodiments, an epoxy-coating, a poly(vinyl chloride-coating), or a combination thereof is selected as a primer for a surface at the end of a can. In other embodiments, an oleoresinouscoating, a phenolic-coatlng, or a combination thereof is selected as a primer for a surface in the interior of a can. In some aspects, a water-borne epoxy and acrylic-coating is selected as a topcoat for a surface of an interior of a can. In addition embodiments, an acrylic-coating, an alkyd-coating, a polyester-coating, or a combination thereof is selected as an exterior coating. In certain facets, a can coating (e.g., a primer, a topcoat) will further comprise an amino resin, a phenolic resin, or a combination thereof for cross-linking in a thermosetting film formation reaction. In certain embodiments, a can coating is applied to a surface by spray application. In other embodiments, a can coating undergoes film formation by UV irradiation. Specific procedures for determining the suitability of a coating and/or film for use as a can coating, have been described, for example, in Paint and Coating Testing Manual, Fourteenth Edition of the Gardner-Sward Handbook, (Koleske, J. V. Ed.), pp. 717-724, 1995.

(3) Sealant Coatings

Sealant coatings refer to coatings used to fill a joint to reduce or prevent passage of a gas (e.g., air), water, a small material (e.g., dust), a temperature change, or a combination thereof. A sealant coating (sealant) may be thought of as a coating that bridges by contact two or more surfaces. A joint is a gap or opening between two or more surfaces, which may or may

48gggg not be of the same material type (e.g., metal, wood, glass, masonry, plastic, etc). In typical embodiments, a joint has a width, depth, breadth, or a combination thereof, of 0.64 mm to 5.10 mm, including all intermediate ranges and combinations thereof.

In certain embodiments, a sealant coating comprises an oil, a butyl, an acrylic, a blocked styrene, a polysulfide, a urethane, a siltcone, or a combination thereof. A sealent may be a solvent-borne coating or a water-borne coating (e.g., a latex). In certain aspects, a sealant comprises a latex (e.g., an acrylic latex). In other embodiments, a sealant is selected for flexibility, as one or more of the joint surfaces may move during normal use. Examples of a flexible sealant include a silicone, a butyl, an acrylic, a blocked styrene, an acrylic latex, or a combination thereof. An oil sealent typically comprises a drying oil, an extender pigment, a thixotrope, and a drier. A solvent-borne butyl sealent typically comprises a polyisobytylene and/or a polybutene, an extender pigment (e.g., talc, calcium carbonate), a liquid component, and an additive (e.g., an adhesion promoter, an antioxidant, a thixotrope). A solvent-borne acrylic sealent typically comprises a polymethylacrylate (e.g., polyethyl, polybutyl), a colorant, a thixotrope, an additive, and a liquid component. A solvent-borne blocked styrene sealant typically comprises styrene, styrene-butadiene, isoprene, or a combination thereof, and a liquid component. A solvent-bome acrylic sealant, blocked styrene sealant, or a combination thereof typically is selected for aspects wherein UV resistance is desired. A urethane sealant may be a one-pack or two-pack coating. A solvent-borne one-pack urethane sealant typically comprises an urethane that comprises a hydroxyl moiety, a filler, a thixotrope, an additive, an adhesion promoter, and a liquid component. A solvent-borne two-pack urethane sealent typically comprises a polyether that comprises an isocyanate moiety in one-pack and a binder comprising a hydroxyl moiety in a second pack. A solvent-borne two-pack urethane sealent typically also comprises a filler, an adhesion promoter, an additive (e.g., a light stabilizer), or a combination thereof. In certain aspects, a solvent-borne urethane sealent is selected for a sealent with a good abrasion resistance. A polysuifide sealant may be a one-pack or two-pack coating. A solventborne one-pack polysulfide sealant typically comprises an urethane that comprises a hydroxyl moiety, a filler, a thixotrope, an additive, an adhesion promoter, and a liquid component. A solvent-borne two-pack polysuifide seaient typically comprises a first pack, which typically comprises a polysulfide, an opacifing pigment, a colorizer (e.g., a pigment), clay, a thixotrope (e.g., a mineral), and a liquid component; and a second pack, which typically comprises a curing agent (e.g., lead peroxide), an adhesion promoter, an extender pigment, and a light stabilizer. A silicone sealant typically comprises a polydimethyllsiloxane and a methyltriacetoxy silane, a methyltrimethoxysilane, a methyltricyclorhexylaminosilane, or a combination thereof. A waterborne acrylic latex sealant typically comprises a thermoplastic acrylic, a filler, a surfactant, a thixotrope, an additive, and a liquid component. Procedures for determining the suitability of a

48hhhh coating and/or film for use as an sealant coating have been described, for example, in Paint and Coating Testing Manual, Fourteenth Edition of the Gardner-Sward Handbook, (Koleske, J.

V. Ed.), pp. 735-740, 1995.

(4) Marine Coatings

A marine coating is a coating used on a surface that contacts water, or a surface that is part of a structure continually near water (e.g., a ship, a dock, an drilling platform for fossil fuels, etc). Typically, such surfaces comprise metal, such as aluminum, high tensile steel, mild steel, or a combination thereof. For embodiments wherein a surface contacts water, the type of marine coating is selected to resist fouling, corrosion, or a combination thereof. Fouling is an accumulation of aquatic organisms, including microorganisms, upon a marine surface. Fouling can damage a film, and as many marine coatings are formulated with a preservative, an anticorrosion property (e.g., an anticorrosion pigment), or a combination thereof, as such damage often leads to corrosion of metal surfaces. Additionally, a marine coating may be selected to resist fire, such as a coating applied to a surface of a ship. Further properties that are often desirable for a marine coating include chemical resistance, impact resistance, abrasion resistance, friction resistance, acoustic camouflage, electromagnetic camouflage, or a combination thereof.

To achieve the various properties of a marine coating, often a multicoat system is used. For metal surfaces, a primer known as a blast primer is typically applied to the surface within seconds of blast cleaning. Examples of a blast primer include a polyvinyl butyral (PVB) and phenolic resin coating, a two-pack epoxy coating, or a two-pack zinc and ethyl silicate coating. A marine metal surface undercoat or topcoat typically comprises an alkyd coating, a bitumen coating, a polyvinyl coating, or a combination thereof. Marine coatings and their use are well known to those of ordinary skill in the art (see, for example, in Paint and Surface Coatings: Theory and Practice,” 2^nd Edition, pp. 529-549, 1999; in Paints, Coatings and Solvents, 2^ndEdition, pp. 252-258, 1998; in Organic Coatings: Science and Technology, Volume 1: Film Formation, Components, and Appearance, 2^nd Edition, pp. 138, 317-318). Specific procedures for determining the purity/properties of a marine coating, anti-fouling coating, or coating component thereof (e.g., cuprous oxide, copper powder, organotin) under marine conditions (e.g., submergence, water based erosion, seawater biofouling resistance, barnacle adhesion resistance) and/or film have been described, for example, in ASTM Book of Standards, Volume 06.02, Paint — Products and Applications; Protective Coatings; Pipeline Coatings, D3623-78a, D4938-89, D4939-89, D5108-90, D5479-94, D6442-99, D6632-01, D4940-98, and D5618-94, 2002; and “ASTM Book of Standards, Volume 06.03, Paint -- Pigments, Drying Oils, Polymers, Resins, Naval Stores, Cellulosic Esters, and Ink Vehicles, D912-81 and D964-65, 2002.

48iiii

c. Specification Coatings

It is contemplated that, in light of the present disclosures, a specification coating may be formulated by selection of coating components one of ordinary skill in the art to fulfill a set of requirements prescribed by a consumer. Examples a specification finish coating include a military specified coating, a Federal agency specified coating (e.g., Department of Transportation), a state specified coating, or a combination thereof. A specification coating such as a CARC, a camouflage coating, or a combination thereof would be preferred in certain embodiments for incorporation of a biomolecule composition of the present invention. A camouflage coating is a coating that is formulated with materials (e.g., pigments) that reduce the visible differences between the appearance of a coated surface from the surrounding enviroment. Often, as would be known to one of ordinary skill in the art, a camouflage coating is formulated to reduce the detection of an coated surface by devise that measures nonvisible light (e.g., infrared radiation). Various sources of specification coating requirements are described in, for example, “Paint and Coating Testing Manual, Fourteenth Edition of the Gardner-Sward Handbook, (Koleske, J. V. Ed.), pp. 891-893, 1995).

(1) Pipeline Coatings

An example of a specification coating is a pipeline (e.g., a metal pipeline) coating used to convey a fossil fuel. A pipeline coating must possess corrosion resistance, and an example of a pipeline coating includes a coal tar-coating, a polyethylene-coating, an epoxy powder-coating, or a combination thereof. A coal tar-coating may comprise, for example, a coal tar mastic-coating, a coal tar epoxide-coating, a coal tar urethane-coating, a coal tar enamel-coating, or a combination thereof. A coal tar mastic-coating typically comprises an extender, a vicosifier, or a combination thereof. In general aspects, a coal tar mastic-coating layer is 127 mm to 160 mm thick, including all intermediate ranges and combinations thereof, in embodiments wherein superior water resistance is desired, a coal tar epoxide-coating may be selected. In embodiments wherein rapid film formation is desired (e.g., pipeline repair), a coal tar urethane-coating may be selected. In embodiments wherein good water resistance, heat resistance up to B2°C, bacteria! resistance, poor UV resistance, or a combination thereof, is suitable, a coal tar enamel may be selected. In embodiments wherein cathodic protection, physical durability, or a combination thereof is desired, an epoxide powder-coating may be selected. In certain embodiments, an electrostatic spray applicator may be used to apply the powder coating. In certain embodiments, a pipeline coating comprises a multicoat system. In specific aspects, a pipeline multicoat system comprises an epoxy powder primer, a two-pack epoxy primer, a chlorinated rubber primer, or a combination thereof and a polyethylene topcoat. Specific procedures for determining the suitability of a coating and/or film for use.as a pipeline coating, including coating storage stability (e.g., settling)

48jjjj and film properties (e.g., abrasion resistance, water resistance, flexibility, weathering, film thickness, impact resistance, chemical resistance, cathodic disbonding resistance, heat resistance) have been described, for example, in “ASTM Book of Standards, Volume 06.02, Paint - Products and Applications; Protective Coatings; Pipeline Coatings,” G6-88, G9-87, G10-83, G11-88, G12-83, G13-89, G20-88, G70-81, G8-96, G17-88, G18-88, G19-88, G42-96, G55-88, G62-87, G80-88, G95-87, and D6676-01e1, 2002; and in Paint and Coating Testing Manual, Fourteenth Edition of the Gardner-Sward Handbook,” (Koleske, J. V. Ed.), pp. 731-734, 1995.

(2) Traffic Marker Coatings

A traffic marker coating is a coating (e.g., a paint) used to very visibly conveys information on a surface usually subjected to weathering and abrasion (e.g., a pavement). A traffic marker coating may be a solvent-borne coating or a water-borne coating. Examples of a solvent-borne traffic marker coating include an alkyd, a chlorinated rubber, or a combination thereof, in certain aspects, a solvent-borne coating is applied by spray application. In some embodiments, a traffic marker coating is a two-pack coating, such as, for example, an epoxycoating, a polyester-coating, or a combination thereof. In other embodiments, a traffic marker coating comprises a thermoplastic coating, a thermosetting coating, or a combination thereof. Examples of a combination thermoplastic/thermosetting coating include a solvent-borne alkyd and/or solvent-borne chlorinated rubber-coating. Examples of a thermoplastic coating include a maleic-modifled glycerol ester-coating, a hydrocarbon-coating, or a combination thereof. In certain aspects, a thermoplastic coating comprises a liquid component, wherein the liquid component comprises a plasticizer, a pigment, and an additive (e.g., a glass bead).

Specific procedures for determining the suitability of a coating and/or film for use as a traffic marker paint, including coating storage stability (e.g., settling), glass bead properties (e.g., reflectance), film durability (e.g., adhesion, pigment retention, solvent resistance, fuel resistance) and particularly relevant film visual properties (e.g., retroreflectance, fluorescence) have been described, for example, in ASTM Book of Standards, Volume 06,02, Paint — Products and Applications; Protective Coatings; Pipeline Coatings, D713-90, D868-85, D969-85, D1309-93, D2205-85, D2743-68, D2792-69, D4796-88, D4797-88, D1155-89, D1214-89, and D4960-89, 2002; in ASTM Book of Standards, Volume 06.01, Paint -- Tests for Chemical, Physical, and Optical Properties; Appearance, F923-00, E1501-99e1, E1696-02, E1709-00e1, E1710-97, E1743-96, E2176-01, E808-01, E809-02, E810-01, E811-95, D4061-94, E2177-01, E991-98, and E1247-92, 2002; and in Paint and Coating Testing Manual, Fourteenth Edition of the GardnerSward Handbook, (Koleske, J. V. Ed.), pp. 741-747,1995.

48kkkk (3) Aircraft Coatings

An aircraft coating protects and/or decorates a surface (e.g., metal, plastic) of an aircraft. Typically, an aircraft coating is selected for excellent weathering properties, excellent heat and cold resistance (e.g., -54°C to 177°C), or a combination thereof. Specific procedures for determining the suitability of a coating and/or film for use as aircraft coating, are described in, for example, in Paint and Coating Testing Manual, Fourteenth Edition of the Gardner-Sward Handbook, (Koleske, J. V. Ed.), pp. 683-695, 1995.

(4) Nuclear Power Plant Coatings

An additional example of a specification coating is a coating for a nuclear power plant, which generally must possess particular properties (e.g., gamma radiation resistance, chemical resistance), as described in “ASTM Book of Standards, Volume 06.02, Paint — Products and Applications; Protective Coatings; Pipeline Coatings, D5962-96, D5163-91, D5139-90, D514400, D4286-90, D3843-00, D3911-95, D3912-95, D4082-02, D4537-91, D5498-01, and D4538-95, 2002.

H. Coating Components in addition to the disclosures herein, the preparation and/or chemical syntheses of coating components, other than the microbial-based particulate matter of the present invention disclosed herein, are well known to those ordinary skill in the art [see, for example, Paint and Coating Testing Manual, Fourteenth Edition of the Gardner-Sward Handbook, (Koleske, J. V., Ed.) (1995); Paint and Surface Coatings, Theory and Practice, Second Edition, (Lambourne, R. and Strivens, T. A״ Eds.) (1999); Wicks, Jr., Z. W״ Jones, F. N״ Pappas, S. P. 'Organic Coatings, Science and Technology, Volume 1: Film Formation, Components, and Appearance, (1992); Wicks, Jr״ Z. W״ Jones, F. N., Pappas, S. P. Organic Coatings, Science and Technology, Volume 2: Applications, Properties and Performance, (1992); Paints, Coatings and Solvents, Second. Completely Revised Edition,״ (Stoye, D. and Freitag, W., Eds.) (1998); ASTM Book of Standards, Volume 06.01, Paint -- Tests for Chemical, Physical, and Optical Properties, Appearance, (2002); ASTM Book of Standards, Volume 06.02, Paint -- Products and Applications; Protective Coatings; Pipeline Coatings, (2002); ASTM Book of Standards, Volume 06.03, Paint -- Pigments, Drying Oils, Polymers, Resins, Naval Stores, Cellulosic Esters, and Ink Vehicles, (2002); and “ASTM Book of Standards, Volume 06.04, Paint - Solvents; Aromatic Hydrocarbons,” (2002)].

However, as would be known to one of ordinary skill in the art, coating components are typically obtained from commercial vendors, which is a preferred method of obtaining a coating component due to ease and reduced cost. As would be known to one of ordinary skill in the art,

481111 texts as, for example, Flick, E. W. Handbook of Paint Raw Materials, Second Edition,1989 ״, describes over 4,000 coating components (e.g., an antifoamer, an antiskinning agent, a bactericide, a binder, a defoamer, a dispersant, a drier, an extender, a filler, a flame/fire retardant, a flatting agent, a fungicide, a latex emulsion, an oil, a pigment, a preservative, a resin, a rheological/viscosity control agent, a silicone additive, a surfactant, a titanium dioxide, etc) provided by commercial vendors; and Ash, M. and Ash, I. Handbook of Paint and Coating Raw Materials, Second Edition, 1996, which describes over 18,000 coating components (e.g., an accelerator, an adhesion promoter, an antioxidant, an antiskinning agent, a binder, a coalescing agent, a defoamer, a diluent, a dispersant, a drier, an emulsifier, a fire retardant, a flow control agent, a gloss aid, a leveling agent, a marproofing agent, a pigment, a slip agent, a thickener, a UV stabilizer, viscosity control agent, a wetting agent, etc) provided by commercial vendors.

Specific commercial vendors are referred to herein as examples, and include Acima™ AG, Im Ochsensand, CH-9470 Buchs / SG; Air Products and Chemicals, Inc., 7201 Hamilton Boulevard, Allentown, PA 18195-1501; Avecia Inc., 1405 Foulk Road, PO Box 15457, Wilmington, DE 19850-5457, U.S.A.; Bayer Corporation, 100 Bayer Rd., Pittsburgh, PA 152059741, U.S.A.; Buckman Laboratories, Inc., 1256 North McLean Blvd., Memphis, TN 38108-0305,

U.S.A.; BYK-Chemie GmbH, Abelstrasse 45, P.O. Box 100245, D-46462 Wesel, Germany; Ciba Specialty Chemicals, 540 White Plains Road, P.O. Box 2005, Tarrytown, NY 10591-9005, U.S.A.; Clariant LSM (America) Inc., 200 Rodney Building, 3411 Silverside Road, Wilmington, Delaware 19810 U.S.A.; Cognis Corporation, 5051 Estecreek Drive, Cincinnati, OH 45232-1446, U.S.A.; Condea Sen/o LLC., 4081 B Hadley Road, South Plainfield, NJ 07080-1114, U.S.A.; Cray Valley Limited, Waterloo Works, Machen, Caerphilly CF83 8YN United Kingdom; Dexter Chemical

L.L.C., 845 Edgewater Road Bronx, NY 10474, U.S.A.; Dow Chemical Company, 2030 Dow Center, Midland, Michigan 48674 U.S.A.; Elementis Specialties, Inc., PO Box 700, 329 Wyckoffs Mill Road, Hightstown, NJ 08520 U.S.A.; Goldschmidt Chemical Corp., 914 East Randolph Road PO Box 1299 Hopewell, VA 23860 U.S.A.; Hercules Incorporated, 1313 North Market Street, Wilmington, DE 19894-0001, U.S.A.; International Specialty Products, 1361 Alps Road, Wayne, New Jersey 07470, U.S.A.; Octel-Starreon LLC USA, North American Headquarters, 8375 South Willow Street, Littleton, Colorado 80124, U.S.A.; Rohm and Haas Company, 100 Independence Mall West, Philadelphia, PA 19106-2399, U.S.A.; Solvay Advanced Functional Minerals, Via Varesina 2-4, 1-21021 Angera (VA); Troy Corporation, 8 Vreeland Road, PO Box 955, Florham park, New Jersey, 07932 U.S.A.; R. T. Vanderbilt Company, Inc., 30 Winfield Street, Norwalk, CT 06855, U.S.A; Union Carbide Chemicals and Plastics Co., Inc., 39 Old Ridgebury Road, Danbury, CT 06817-0001, U.S.A.

48mmm m

1. Binders

A binder (“polymer,” resin, film former) is a molecule capable of film formation. Film formation is a physical and/or chemical change of a binder In a coating, wherein the change converts the coating into a film. Often, a binder converts into a film through a polymerization reaction, wherein a first binder molecule covalently bonds with at least a second binder molecule to form a larger molecule, known as a “polymer. As this process is repeated a plurality of times, the composition converts from a coating comprising a binder into a film comprising a polymer.

A binder may comprise a monomer, an oligomer, a polymer, or a combination thereof. A monomer is a single unit of a chemical species that can undergo a polymerization reaction. However, a binder itself is often a polymer, as such larger binder molecules are more suitable for formulation into a coating capable of both being easily applied to a surface and undergoing an additional polymerization reaction to produce a film. An oligomer comprises 2 to 25 polymerized monomers, including all intermediate ranges and combinations thereof.

A homopolymer is a polymer that comprises monomers of the same chemical species. A copolymer is a polymer that comprises monomers of at least two different chemical species. A linear polymer is an unbranched chain of monomers. A branched polymer is a branched (“forked) chain of monomers. A network (“cross-linked) polymer is a branched polymer wherein at least one branch forms an interconnecting covalent bond with at least one additional polymer molecule.

A thermoplastic binder and/or coating reversibly softens and/or liquefies when heated. Film formation for a thermoplastic coating generally comprises a physical process, typically the loss of the volatile (e.g., !!quid) component from a coating. As a volatile component is removed, a solid film may be produced through entanglement of the binder molecules. In many aspects, a thermoplastic binder is generally a higher molecular mass than a comparable thermosetting binder. In many aspects, a thermoplastic film is often susceptible to damage by a volatile component that can be absorbed by the film, which can soften and/or physically expand the film. In certain facets, a thermoplastic film may be removed from a surface by use of a volatile component. However, In many aspects, damage to a thermoplastic film may be repaired by appiication of a thermoplastic coating into the damaged areas and subsequent film formation.

A thermosetting binder undergoes film formation by a chemical process, typically the cross-linking of a binder into a network polymer. In certain embodiments, a thermosetting binder does not possess significant thermoplastic properties.

48nnnn

The glass transition temperature is the temperature wherein the rate of increase of the volume of a binder or a film changes. Binders and films often do not convert from solid to liquid (melt”) at a specific temperature (T_m”), but rather possess a specific glass transition temperature wherein there is an increase in the rate of volume expansion with increasing temperature. At temperatures above the glass transition temperature, a binder or film becomes increasingly rubbery in texture until it becomes a viscous liquid. In certain embodiments described herein, a binder, particularly a thermoplastic binder, may be selected by its glass transition temperature, which provides guidance to the temperature range of film formation, as well as thermal and/or heat resistance of a film. The lower the T_fl, the “softer the resin, and generally, the film produced from such a resin. A softer film typically possesses greater flexibility (e.g., crack resistance) and/or poorer resistance to dirt accumulation than a harder film.

In certain embodiments, a coating comprises a low molecular weight polymer, a high molecular weight polymer, or a combination thereof. Examples of a low molecular weight polymer include an alkyd, an amino resin, a chlorinated rubber, an epoxide resin, an oleoresinous binder, a phenolic resin, a urethane, a polyester, an urethane oil, or a combination thereof. Examples of a high molecular weight polymer include a latex, a nitrocellulose, a non-aqueous dispersion polymer (NAS), a solution acrylic, a solution vinyl, or a combination thereof. Examples of a latex include an acrylic, a polyvinyl acetate (PVA), a styrene/butadiene, or a combination thereof.

In addition to the disclosures herein, a binder, methods of binder preparation, commercial vendors of binder, and techniques for using an binder in a coating known to those of ordinary skill in the art may be applied in the practice of the present invention (see, for example, Flick, E. W. “Handbook of Paint Raw Materials, Second Edition,” pp. 287-805 and 879-998, 1989; in “Paint and Coating Testing Manual, Fourteenth Edition of the Gardner-Sward Handbook,” (Koleske, J. V. Ed.), pp. 23-29, 39-67, 74-84, 87, 268-285, 410, 539-540, 732, 735-736, 741, 770, 806-807, 845-849, and 859-861, 1995; in “Paint and Surface Coatings, Theory and Practice, Second Edition, (Lambourne, R. and Strivens, T. A., Eds.), pp. 2-3, 7-10, 21, 24-40, 40-54, 60-71, 76, 8186, 352, 358, 381-394, 396, 398, 405, 433-448, 494-497, 500, 537-540, 700-702, and 734, 1999; Wicks, Jr., Z. W., Jones, F. N., Pappas, S. P. “Organic Coatings, Science and Technology, Volume 1: Film Formation, Components, and Appearance, pp. 39, 49-57, 62, 65-67, 67, 76-80, 83, 91, 104-118, 155, 168, 178, 182-183, 200, 202-203, 209, 214-216, 220 and 250, 162-186, 215-216 and 232, 59-60, 183-184, 133-143, 39, 144-161, 203, 219220־ and 239, 23, 110, 120132, 122-130, 198, 202209 ,203־ and 220, 60-62, 83-103, 164-167, 173, 177-178, 184-187, 195, 206, and 216-219, 1992; Wicks, Jr., Z. W., Jones, F. N., Pappas, S. P. Organic Coatings, Science and Technology, Volume 2: Applications, Properties and Performance, pp. 13-14, 18480000

19, 26, 33-34, 36, 41, 57, 77, 92, 95, 116-119, 143-145, 156, 161-165, 179-180, 191-193, 197203, 210-211, 213-214, 216, 219-222, 230-239, 260-263, 269-271, 276-284, 288-293, 301-307, 310, 315-316, 319-321, and 325-346, 1992; and in Paints, Coatings and Solvents, Second, Completely Revised Edition, (Stoye, D. and Freitag, W., Eds.) pp. 5,11-22, 37-50, 54-55, 72, 8087, 96-98, 108, 126, and 136, 1998.

a. Oil-Based Binders

Certain binders, such as, for example, an oil (e.g., a drying oil), an alkyd, an oleoresinous binder, a fatty acid epoxide ester, or a combination thereof, are prepared and/or synthesized from an oil and/or a fatty acid, and undergo film formation by thermosetting oxidative cross-linking of fatty acids, and will be referred to herein as an oil-based binder. These types of binders often possess similar properties (e.g., solubility, viscosity). An oil-based binder coating often further comprises a drier, an antiskinning agent, an alkylphenolic resin, a pigment, an extender, a liquid component (e.g., a solvent), ora combination thereof. A drier, such as a primary drier, secondary drier, or a combination thereof, may be selected to promote film formation. In certain facets, an oil-based binder coating may comprise an anti-skinnlng agent, which is typically used to control undesirable film-formation caused by a primary drier and/or oxidation. A liquid component may be selected, for example, to alter a rheological property (e.g., flow), wetting and/or dispersion of particulate material, or a combination thereof. In certain embodiments, a liquid component comprises a hydrocarbon. In particular embodiments, the hydrocarbon comprises an aliphatic hydrocarbon, an aromatic hydrocarbon (e.g., toluene, xylene), or a combination thereof. In some facets, the liquid component comprises, by weight, 5% to 20% of an oil-based binder coating, including alt intermediate ranges and combinations thereof.

In alternative embodiments, an oil-based temporary coating (e.g. a non-film forming coating) may be produced, for example, by inclusion of an antioxidant, reduction of the amount of a drier, selection of a oil-based binder that comprises fewer or no double bonds, or a combination thereof.

An oil-based binder coating may be selected for embodiments wherein a relatively low viscosity is desired, such as, for example, application to a corroded metal surface, a porous surface (e.g., wood), or a combination thereof, due to the penetration power of a low viscosity coating. In certain facets, it is preferred that application of an oil-binder coating produces a layer is less than 25 pm on vertical surfaces and 40 pm on horizontal surfaces to reduce shrinkage, wrinkling. Additionally, in aspects wherein the profile of the wood surface is to be retained, a such a thin film thickness is preferred. In specific aspects, an oil-binder coating may be selected as a wood stains, a topcoat, or a combination thereof. In particular facets, a wood stain

48pppp comprises an oi! (e.g., linseed oil) coating, an alkyd, or a combination thereof. Often, wood coating comprises a lightstabilizer (e.g., UV absorber).

(1) Oils

An oil is a polyol esterified to at least one fatty acid. A polyol (“polyalcohol,״ “polyhydric alcohol) is an alcohol comprising more than one hydroxyl moiety per molecule. In certain embodiments, an oil comprises an acylglycerol esterified to one fatty acid (monacylglycerol ), two fatty acids (“diacylglycerol), or three fatty acids (“triacylglycerol, “triglyceride”). Typically, however, an oil will comprise a triacylglycerol. A fatty acid is an organic compound comprising a hydrocarbon chain that includes a terminal carboxyl moiety. A fatty acid may be unsaturated, monounsaturated, and polyunsaturated referring to whether the hydrocarbon chain possess no carbon double bonds, one carbon double bond, or a plurality of carbon double bonds (e.g., 2, 3, 4, 5, 6, 7, or 8 double bonds), respectively.

In typical use in a coating, a plurality of fatty acids forms covalent cross-linking bonds to produce a film in coatings comprising oil binders and/or other binders comprising a fatty acid. Usually oxidation through contact with atmospheric oxygen is used to promote film formation. Exposure to light also enhances film formation. The ability of an oil to undergo film formation by chemical cross-linking is related to the content of chemically reactive double bonds available in its fatty acids. Oils are generally a mixture of chemical species, comprising different combinations of fatty acids esterified to glycerol. The overall types and percentages of particular fatty acids that are comprised in oils affect the ability of the oil to be used as a binder. Oils can be classified as a drying oil, a semi-drying oil, or a non-drying oil depending upon the ability of the oil to cross-link into a dry film without additives (e.g., driers) at room temperature and atmospheric oxygen. A drying oil forms a dry film to touch upon cross-linking, a semi-drying oil forms a sticky ('¹tacky') film to touch upon cross-linking, while a non-drying oil does not produce a tacky or dry film upon cross-linking. In certain facets, it is contemplated that film-formation of a non-chemically modified oil-binder coating will typically take from 12 hours to 24 hours at room temperature, air, and lighting. Procedures for selection and testing of drying oils for a coating are described in, for example, ASTM Book of Standards, Volume 06.03, Paint - Pigments, Drying Oils, Polymers, Resins, Naval Stores, Cellulosic Esters, and Ink Vehicles, D555-84, 2002.

Drying oils comprise at least one polyunsaturated fatty acid to promote cross-linking. Polyunsaturated fatty acids (polyenoic fatty acids) include, but are not limited to, 7,10,13hexadecatrienoic (“16:3 n-3’); linoleic [9,12-octadecadienoic, Ί8:2(π-6)־]; y-linolenic [6,9,12״octadecatrienoic,” 18:3״(n-6)]; a trienoic 20:3(n-9); dihomo- y-linolenic [8,11,14-eicosatrienoic,” “20:3(n-6)l; arachidonic [5,8,11,14-eicosatetraenoic, 20:4(n-6)“]; a licanic, (4-oxo 9c11t13t48qqqq

18:37,10,13,16 ;(״-docosatetraenoic [“22:4(n-6)”]; 4,7,10,13,16-docosapentaenoic [22:5(n-6)]; a-linolenic [“9,12,15-octadecatrienolc,” “18:3(n-3)״]; stearidonic [6,9,12,15-octadecatetraenoic, “18:4(n8,11,14,1 ;[”(3־ ?-eicosatetraenoic [“20:4(n5,8,11,14,17 ;[״(3־-elcosapentaenoic [ΈΡΑ,״ 20:5(n-3)”]; 7,10,13,16,19-docosapentaenolc [״DPA,” “22:5(n-3)”]; 4,7,10,13,16,19docosahexaenoic [“DHA,” “22:6(n-3)5,8,11 ;[״-eicosatrienoic [Mead acid, “20:3(n-9)”l; taxoleic (־all-cis-5,9-18:2״); pinolenic (“all-cis-5,9,12-18:3״); sciadonic (״all-cis-5,11,14-20:3״); dihomotaxoleic (7,11-20:2); cis9־, cis-15 octadecadienoic (9,15-18:2); retinoic; or a combination thereof.

Drying oils can be further characterized as non-conjugated or conjugated drying oils depending upon whether their most abundant fatty acid comprises a polymethylene-interrupted double bond or a conjugated double bond, respectively. A polymethylene-interrupted double bond is two double bonds separated by two or more methylene moieties. A polymethyleneinterrupted fatty acid is a fatty acid comprising such a configuration of double bonds. Examples of polymethylene-interrupted fatty acids include taxoleic, pinolenic, sciadonic, dihomotaxoleic, cis9, cis-15 octadecadienoic, retinoic, or a combination thereof.

A conjugated double bond is a moiety wherein a single methylene moiety connects pair of carbon chain double bonds. A conjugated fatty acid is a fatty acid comprising such a pair of double bonds. A conjugated double bond is more prone to cross-linking reactions than nonconjugated double bonds. A conjugated diene fatty acid, a conjugated triene fatty acid or a conjugated tetraene fatty acid, possesses only two, three or four conjugated double bonds, respectively. An example of a common conjugated diene fatty acid is a conjugated linoleic. Examples of a conjugated triene fatty acid include an octadecatrienoic, a licanic, □r a combination thereof. Examples of an octadecatrienoic acid include an a-eleostearic comprising the 9c, 11t,13t isomer, a calendic comprising a 8t,10t,12c isomer, a catalpic comprising the 9c,11t,13c isomer, or a combination thereof. An example of a conjugated tetraene fatty acid is a-parinaric comprising the 9c,111,13t, 15c isomer, and β-parinaric comprising the 9t,111,13t,15t isomer, or a combination thereof.

Oils for use in coatings are generally obtained from renewable biological sources, such as plants, fish or a combination thereof. Examples of plant oils commonly used in coatings or coating components include cottonseed oil, linseed oil, oiticica oil, safflower oil, soybean oil, sunflower oil, tall oil, rosin, tung oil, or a combination thereof. An example of a fish oil commonly used in coatings or coating components include caster oil. A colder environment generally promotes a higher polyunsaturated fatty acid content in an organism (e.g., sunflowers). Cottonseed oil comprises about 36% saturated fatty acids, 24% oleic, and 40% linoleic. Castor

48rrrr oil comprises about 3% saturated fatty acids, 7% oleic, 3% linoleic, and 87% ricinoleic (“12hydroxy-9-octadecenoic). Linseed oil comprises about 10% saturated fatty acids, 20% to 24% oleic (“cis-9-octadecenoic), 14% to 19% linoleic, and 48% to 54% linolenic. Oiticica oil comprises about 16% saturated fatty acids, 6% oleic, and 78% licanic. Safflower oil comprises about 11% saturated fatty acids, 13% oieic, 75% linoleic, and 1% linolenic. Soybean oil comprises about 14% to 15% saturated fatty acids, 22% to 28% oleic, 52% to 55% linoleic, and 5% to 9% linolenic. Tall oil, which is a product of paper production and generally is not in the form of a triglyceride, often comprises about 3% saturated fatty acids, 30% to 35% oleic, 35% to 40% linoleic, 2% to 5% linolenic, and 10% to 15% of a combination of pinolenic and conjugated linoleic. Rosin is a combination of acidic compounds isolated during paper production, such as, for example, abietic acid, neoabietic acid, dihydroabietic acid, tetraabietic acid, isodextropimaric acid, dextropimaric acid, dehydroabietic acid, and levopimaric acid. Tung oil comprises about 5% saturated fatty acids, 8% oleic, 4% linoleic, 3% linolenic, and 80% a־elestearic. Standards for physical properties, chemical properties, and/or procedures for testing the purity/properties of various oils (e.g., caster, linseed, oiticica, safflower, soybean, sunflower, tall, tung, rosin, dehydrated caster, boiled linseed, a drying oil, a fish oil, a heat-bodied drying oil) for use in a coating are described, for example in “ASTM Book of Standards, Volume 06.03, Paint Pigments, Drying Oils, Polymers, Resins, Naval Stores, Cellulosic Esters, and Ink Vehicles,” D555-84. D960-02a, D961-86. D234-82, D601-87, D1392-92, D1462-92, D12-88, D1981-02, D5768-95, D3169-89, D260-86, D124-88, D803-02, D1541-97, D1358-86, D1950-86, D1951-86, D1952-86, D1954-86, D1958-86, D464-95, D465-01, D1959-97, D1960-86, D1962-85, D1964-85, D1965-87, D1966-69, D1967-86, D3725-78, D1466-86, D890-98, D1957-86, D1963-85, D597400, D1131-97, D1240-02,. D889-99, D509-98, D269-97, D1065-96 ,and D804-02, 2002.

In certain embodiments, an oil comprises a chemically modified oil, which is an oil altered by a reaction thought to promote limited cross-linking. Generally, such a modified oil possesses an altered property, such as a higher viscosity, which may be more suitable for a particular coating application. Examples of a chemically modified oil include a bodied oil, a blown oil, a dimer acid, or a combination thereof. A bodied oil (heat bodied oil,״ “stand oil) is produced, for example, by heating a nonconjugated oil (e.g., 320°C) or a conjugated oil (e.g., 240°C) in an chemically unreactive atmosphere to promote limited cross-linking. A blown oil is produced, for example, by passing air through a drying oil at, for example, 150°C. A dimer acid is produced, for example, by acid catalyzed dimerization or oligomerization of a polyunsaturated acid.

In certain embodiments, an oil comprises a synthetic conjugated oil, which is an oil altered by a reaction thought to produce a conjugated double bond in a fatty acid of the oil. Conjugated fatty acids have been produced from nonconjugated fatty acids by alkaline hydroxide

48ssss catalyzed reactions. However, a synthetic conjugated oil is generally semi-drying in air catalyzed film formation at room temperature, and a coating comprising such an oil is typically cured by baking. Additionally richinoleic acid, which is prevalent in castor oil, can be dehydrogenated to produce a mixture of conjugated and non-conjugated fatty acids. Dehydrogenated castor oil comprises about 2% to 4% saturated fatty acids, 6% to 8% oleic, 48% to 50% linoleic, and 40% to 42% conjugated linoleic.

Certain other compounds comprising a fatty acid and polyol are classified herein as an oil for use as a binder such as a high ester oil, a maleated oil, or a combination thereof. A high ester oil comprises a polyol capable of comprising greater than three fatty acid esters per molecule and at least one fatty acid ester. However, a high ester oil comprising four or more fatty acid esters per molecule is preferred. Examples of such a polyol include a pentaerythritiol, a dipentaerythritiol, a tripentaerythritiol, or a styrene/ailyl alcohol copolymer. These high ester oils generally form films more rapidly than acylglycerol based oil, as the opportunity for cross-linking reactions between fatty acids increases with the number of fatty acids attached to a single polyol. A maleated oil is an oil modified by a chemical reaction with maleic anhydride. Maleic acid and an unsaturated or polyunsaturated fatty acid react to produce a fatty acid with additional acid moieties. A maleated oil generally is more hydrophilic and/or has a faster film formation time than a comparative non-maleated oil.

(2) Alkyd Resins

In certain embodiments, a binder can comprise an alkyd resin. In general embodiments, an alkyd-coatlng may be selected as an architectural coating, a metal coating, a plastic coating, a wood coating, or a combination thereof. In certain aspects, an alkyd coating may be selected for use as a primer, an undercoat, a topcoat, or a combination thereof. In particular aspects, an. alkyd coating comprises a pigment, an additive, or a combination thereof.

An alkyd resin comprises a polyester prepared from a polyol, a fatty acid, and a polybasic (polyfunctional) organic acid or acid anhydride. An alkyd resin is generally produced by first preparing monoacylpolyol, which is a polyol esterified to one fatty acid. The monoacylpolyol is polymerized by ester linkages with a polybasic acid to produce an alkyd resin of desired viscosity in a solvent. Examples of a polyol include 1,3-butylene glycol; diethylene glycol; dipentaerythritol; ethylene glycol; glycerol; hexylene glycol; methyl glucoside; neopentyl glycol; pentaerythritol; pentanediol; propylene glycol; sorbitol; triethylene glycol; trimethylol ethane; trimethylol propane; trimethylpentanediol; or a combination thereof. In certain aspects, a polyol comprises ethylene glycol; glycerol; neopentyl glycol; pentaerythritol; trimethylpentanediol; or a combination thereof. Examples of a polybasic acid or an acid anhydride include adipic acid, azelaic acid, chlorendic

48tttt anhydride, citric acid, fumaric acid, isophthalic acid, mafeic anhydride, phthalic anhydride, sebacic acid, succinic acid, trimelletic anhydride, or a combination thereof. In certain aspects, a polybasic acid or an acid anhydride comprises isophthalic acid, maleic anhydride, phthalic anhydride, trimelletic anhydride, or a combination thereof. Examples of a fatty acid include abiatic, benzoic, caproic, caprylic, lauric, linoleic, linolenic, oleic, a tertiary-butyl benzoic acid, a fatty acid from an oil/fat (e.g., castor, coconut, cottonseed, tall, tallow), or a combination thereof. In certain aspects, a fatty acid comprises benzoic, a fatty acid from tall oil, or a combination thereof. In specific aspects, an oil is used in the reaction directly as a source of a fatty acid and/or a polyol. Examples of an oil include castor oil, coconut oil, corn oil, cottonseed oil, dehydrated castor oil, linseed oil, safflower oil, soybean oil, tung oil, walnut oil, sunflower oil, menhaden oil, palm oil, or a combination thereof. In some aspects, an oil comprises coconut oil, linseed oil, soybean oil, or a combination thereof.

In addition to the standards and analysis techniques previously described for an oil, standards for physical properties, chemical properties, and/or procedures for testing the purity/properties of various fatty acids (e.g., coconut, corn, cottonseed, dehydrated caster, linseed, soybean, tall oil fatty acids, rosin fatty acids) and a polyol (e.g., pentaerythntol, hexylene glycol, ethylene glycol, diethylene glycol, propylene glycol, dipropylene glycol) and acid anhydrides (e.g., phthalic anhydride, maleic anhydride) for use in an alkyd or other coating components are described, for example, in “ASTM Book of Standards, Volume 06.03, Paint Pigments, Drying Oils, Polymers, Resins, Naval Stores, Cellulosic Esters, and Ink Vehicles,' D1537-60, D1538-60, D1539-60, D1841-63, D1842-63, D1843-63, D5768-95, D1981-02, D198285, D1980-87, D804-02, D1957-86, D464-95, D465-01, D1963-85, D5974-00, D1466-86, D280092, D1585-96, D1467-89, and D1983-90, 2002; and in ASTM Book of Standards, Volume 06.04, Paint - Solvents; Aromatic Hydrocarbons, D2403-96, D3504-96, D2930-94, D3366-95, D343899, D2195-00, D2636-01, D2693-02, D2694-91, D5164-91, D1257-90, and D1258-95, 2002. Further, the composition, properties and/or purity of an alkyd resin and/or a solution comprising an alkyd resin selected for use in a coating such as phthalic anhydride content, isophthahc acid content, unsaponifiable matter content, fatty acid content/identification, polyhydric alcohol content/identification, glycerol, ethylene glycol and/or pentaerythirol content, and silicon content can be empirically determined by procedures known to those of ordinary skill in the art (see, for example, “ASTM Book of Standards, Volume 06.03, Paint - Pigments, Drying Oils, Polymers, Resins, Naval Stores, Cellulosic Esters, and Ink Vehicles, D2689-88, D563-88, D2690-98, D2998-89, D1306-88, D1397-93, D1398-93, D2455-89, D1639-90, D1615-60, and D2456-91, 2002).

48uuuu (i) Oil Length Alkyd Binders

In specific embodiments, an alkyd resin may be selected based on the materials used in its preparation, which typically affect the alkyd's properties. In general aspects, an alkyd resin is often classified and/or selected for use in a particular application by its oil content, as the oil content affects the alkyd resin properties. Oil content is the amount of oil relative to the solventfree alkyd resin. Based on oil content, an alkyd resin may be classified as a very long oil alkyd resin, a long oil alkyd resin, a medium oil alkyd resin, or a short oil alkyd resin. Generally, the greater the oil content classification of an alkyd resin that Is comprised in a coating, the greater the ease of brush application, the slower the rate of film formation, the greater the film’s flexibility, the poorer the chemical resistance of the film, the poorer the retention of gloss in exterior environments, or a combination thereof. A short oil alkyd, a medium oil alkyd, a long oil alkyd, and a very long oil alkyd has an oil content range of 1% to 40%, 40% to 60%, 60% to 70%, and 70% to 85%, respectively, including all intermediate ranges and combinations thereof, respectively. In typical embodiments, a short oil alkyd, a medium oil alkyd, a long oil alkyd, and a very long oil alkyd resin and/or coating comprise 50%, 45% to 50%, 60% to 70%, or 85% to 100% nonvolatile component, respectively.

In certain embodiments, a short oil alkyd coating may be selected as an industrial coating. In certain aspects, a short oil alkyd is synthesized from an oil, wherein the oil comprises castor, dehydrated castor, coconut, linseed, soybean, tall, or a combination thereof. In some aspects, the oil of a short oil alkyd comprises a saturated fatty acid. Examples of a saturated fatty acid include, but are not limited to, caproic (hexanoic, 6:0); caprylic (“octanoic, “8:0); lauric (dodecanoic, “12:0); or a combination thereof. In particular facets, a short oil alkyd coating comprises a solvent, wherein the solvent comprises an aromatic hydrocarbon, isobutanol, VMP naphtha, xylene, or a combination thereof. In other facets, the aromatic solvent comprises a high boiling aromatic solvent. In some aspects, a short oil alkyd is insoluble or poorly soluble in an aliphatic hydrocarbon. In further embodiments, a short oil alkyd coating undergoes film formation by baking.

in certain embodiments, a medium oil alkyd coating may be selected as a farm implement coating, a railway equipment coating, a maintenance coating, or a combination thereof. In certain aspects, a medium oil alkyd is synthesized from an oil, wherein the oil comprises linseed, safflower, soybean, sunflower, tall, or a combination thereof. In some aspects, the oil of a medium oil alkyd comprises a monounsaturated fatty acid (e.g., oleic acid). In particular facets, a medium oil alkyd coating comprises a solvent, wherein the solvent comprises an aliphatic hydrocarbon, an aromatic hydrocarbon, or a combination thereof.

48vwv

In certain embodiments, a tall oil alkyd coating may be selected as an architectural coating, a maintenance coating, a primer, a topcoat, or a combination thereof. In certain aspects, a tall oil alkyd is synthesized from an oil, wherein the oil comprises linseed, safflower, soybean, sunflower, tail, □r a combination thereof. In some aspects, the oil of a long oil alkyd comprises a polyunsaturated fatty acid. In particular facets, a tall oil alkyd coating comprises a solvent, wherein the solvent comprises an aliphatic hydrocarbon.

In certain embodiments, a very long oil alkyd coating may be selected as a latex architectural coating, a wood stain, or a combination thereof. In certain aspects, a very long oil alkyd is synthesized from an oil, wherein the oil comprises linseed, soybean, tall, or a combination thereof. In some aspects, the oil of a long oil alkyd comprises a polyunsaturated fatty acid. In particular facets, a very long oil alkyd coating comprises a solvent, wherein the solvent comprises an aliphatic hydrocarbon.

(ii) High Solid Alkyd Coatings

A high solid alkyd possesses a reduced viscosity, a lower average molecular weight, or a combination thereof. A high solid alkyd may be selected for embodiments wherein a reduced quantity liquid content (e.g., solvent) of a coating is desired. In some embodiments, a high solid alkyd coating comprises an enamel coating. In other aspects, a high solid long or very long oil alkyd coating comprises an architectural coating. In further aspects, a high solid medium oil alkyd coating comprises a transportation coating. In further aspects, a high solid short oil alkyd coating comprises an industrial coating. Additional, various chemical moieties may be incorporated in an alkyd to modify a property. Examples of such moieties include an acrylic, a benzoic acid, an epoxide, an isocyanate, a phenolic, a polyamide, a rosin, a silicon, a styrene (e.g., a paramethyl styrene), a vinyl toluene, or a combination thereof. In certain embodiments, a benzoic acid modified high solid alkyd coating comprises a coating for a tool. In other embodiments, a phenolic modified high solid alkyd coating comprises a primer. A silicone modified alkyd coating may be selected for improved weather resistance, heat resistance, or a combination thereof. In specific aspects, a silicone modified alkyd coating may comprise an additional binder capable of cross-linking with the silicone moiety (e.g., a melamine formaldehyde resin). In specific facets, a silicone modified alkyd coating may be selected as a coil coating, an architectural coating, a metal coating, an exterior coating, or a combination thereof. In certain facets, a high solid siliconmodified alkyd coating may substitute an oxygenated compound (e.g., a ketone, an ester) for an aromatic hydrocarbon liquid component. However, a high solid silicon-modified alkyd coating, to achieve cross-linking during film-formation, should comprise an additional binder capable of cross-linking. In further embodiments, a silicone modified high solid alkyd coating comprises a maintenance coating, a topcoat, or a combination thereof.

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(iti) Uralkyd Coatings

An uralkyd binder (uralkyd,” “urethane alkyd, “urethane oil,” “urethane modified alkyd״) is an alkyd binder, with the modification that compound comprising plurality of diisocyanate moieties partly or fully replacing the dibasic acid (e.g., phthalic anhydride) in the synthesis reactions. Examples of an isocyanate comprising compounds include a 1,6-hexamethylene diisocyanate (“HDI”), a toluene diisocyanate (TDI”), or a combination thereof. An uralkyd binder may be selected for embodiments wherein a superior abrasion resistance, superior resistance to hydrolysis, or a combination thereof, relative to an alkyd, is desired in a film. However, an uralkyd binder prepared using ΤΌΙ often has greater viscosity in a coating, inferior color retention in a film, or a combination thereof, relative to an alkyd binder. Additionally, an uralkyd binder prepared using an aliphatic isocyanate generally possesses superior color retention to an uralkyd prepared from TDI. An uralkyd coating tends to undergo film formation faster than a comparable alkyd binder, due to a generally greater number of available conjugated double bonds, an increased T_ain an uralkyd binder prepared using an aromatic isocyanate, or a combination thereof. A film comprising an uralkyd binder tends to develop a yellow to brown color. An uralkyd binder is often used in preparation of an architectural coating such as a varnish, an automotive refinish coating, or a combination thereof. Examples of a surface where an uralkyd coating may be applied include a furniture surface, a wood surface, or a floor surface.

(iv) Water-Borne Alkyd Coatings

In general embodiments, an alkyd coating is a solvent-borne coating. However, an alkyd (e.g., a chemically modified alkyd) may be combined with a coupling solvent and water to produce a water-borne alkyd coating. Examples of a coupling solvent that may confer water reducibility to an alkyd resin includes ethylene glucol monobutyether, propylene glycol monoethylether, propylene glycol monopropylether, an alcohol whose carbon content is four carbon atoms (e.g., s-butanol), or a combination thereof. In certain embodiments, a water-borne long oil alkyd coating may be selected as a stain, an enamel, or a combination thereof. In other embodiments, a water-borne medium oil alkyd coating may be selected as an enamel, an industrial coating, or a combination thereof. In further facets, a water-borne medium oil alkyd coating may undergo film formation by air oxidation, in other embodiments, a water-borne short oil alkyd coating may be selected as an enamel, an industrial coating, or a combination thereof. In further facets, a water-borne short oil alkyd coating may undergo film formation by baking.

(3) Oleoreslnous Binders

An oleoreslnous binder is a type of binder prepared from heating a resin and an oil. Examples of a resin typically used in the preparation of an oleoresinous binder include resins

48xxxx obtained from a biological source (e.g., a wood resin, a bitumen resin); a fossil source (e.g., copal resin, a Kauri gum resin, a rosin resin, a shellac resin); a synthetic source (e.g., a rosin derivative resin, a phenolic resin, an epoxy resin); or a combination thereof. An example of an oil typically used in the preparation of an oleoresinous binder includes a vegetable oil, particularly an oil that Is comprises a polyunsaturated fatty acid such as tung, linseed, or a combination thereof. The type of resin and oil used can identify an oleoresinous binder such as a copal-tung oleoresinous binder, a rosin-linseed oleoresinous binder, etc. An oleoresinous binder generally are used in clear varnishes such as a lacquer, as well as in applications as a primer, an undercoat, a marine coating, or a combination thereof. In addition to the standards and analysis techniques previously described for an oil, standards for physical properties, chemical properties, and/or procedures for testing the purity/properties (e.g., glass transition temperature, molecular weight, color stability) of a hydrocarbon resin (e.g., a synthetic source resin) for use in an oleoresinous binder or other coating component are described, for example, in ASTM Book of Standards, Volume 06.03, Paint ״ Pigments, Drying Oils, Polymers, Resins, Naval Stores, Cellulosic Esters, and Ink Vehicles, E28-99, D6090-99, D6440-01, D6493-99, D6579-00, D660400, and D6605-00, 2002.

Similar to alkyd resins, oleoresinous binders can be categorized by oil length as a short oil or long oil oleoresinous binder, depending whether oil length is 1% to 67% or 67% to 99% oil, including all intermediate ranges and combinations thereof, respectively. Short oil oleoresinous binders generally dry fast and form relatively harder, less flexible films, and are used, for example, for floor varnishes. Long oil oleoresinous binders generally dry slower and form relatively more flexible films, and are used, for example, as an undercoat, exterior varnish, or combination thereof.

(4) Fatty Acid Epoxy Esters

In certain facets, an epoxy coating may be cured by fatty acid oxidation rather than epoxide moiety or hydroxyl moiety cross-linking reactions. A fatty acid epoxide ester resin is an ester of an epoxide resin and a fatty acid, which can be used to produce an ambient cure coating that undergoes film formation by oxidative reactions as an oil-based coating. In certain embodiments, an epoxy resin may be selected with an epoxy equivalent weight of 800 to 1000, including all intermediate ranges and combinations thereof. Short, medium, and long oil epoxide ester resins comprise 30% to 50%, 50% to 70%, or 70% to 90% fatty acid esterification, including all intermediate ranges and combinations thereof, respectively, with similar, though sometimes superior, properties relative to an analogous alkyd. An epoxide ester resin is inferior in chemical resistance than a film produced by an epoxy and a curing agent comprising an amine. An epoxy

48yyyy ester resin may be selected as a substitute for an alkyd, a marine coating, an industrial maintenance coating, a floor topcoat, or a combination thereof.

b. Polyester Resins

A polyester resin (polyester, “oil-free alkyd) is a polyester chemical, other than an alkyd resin, capable as use as a binder. A polyester resin is chemically very similar to an alkyd, though the oil content is 0%. Consequently, a polyester-coating does not form cross-linking bonds by fatty acids oxidation during thermosetting film formation, but rather is combined with an additional binder to form a cross-linked film. The selection of a polyester and additional binder combination is generally determined by the polyester's crosslinkable moieties. For example, a hydroxyterminated polyester is a polyester produced by an esterification reaction comprising a molar excess of a polyol, and may be crosslinked with a urethane, an amino resin, or a combination thereof. A hydroxy-terminated polyester’s hydroxyl moiety may react with a urethane's isocyanate moiety such as at ambient conditions or low-bake conditions, while such a polyester generally undergoes film formation at baking temperatures with an amino resin. In another example, a carboxylic acid-terminated polyester” is a polyester produced by an esterification reaction comprising an molar excess of a polycarboxylic acid, and may be crosslinked with a urethane, an amino resin, a 2-hydroxylakylamide, or a combination thereof.

In general embodiments, a polyester-coating possesses superior color retention, flexibility, hardness, weathering, or a combination thereof, relative to an alkyd-coating. In some embodiments, a polyester resin may be selected to produce a coating for a metal surface. Generally, a polyester-coating possesses a superior adhesion property on a metal surface than a thermosetting acrylic-coating. Often, a polyester-coating is a thermosetting coating, particularly in embodiments for use upon a metal surface. However, a polyester-coating generally comprises an ester linkage that is susceptible to hydrolysis, therefore, applications wherein such a polyestercoating contacts water is less preferred.

A polyester resin is generally prepared by an acid catalyzed esterification of a polyacid (e.g., a polycarboxylic acid, an aromatic polyacid) and a polyalcohol. A polyacid (polybasic acid”) is a chemical comprising more than one acid moiety. Typically, a polyacid used in the preparation of a polyester comprise two acidic moieties, such as, for example, an aromatic dibasic acid, an anhydride of an aromatic dibasic acid, an aliphatic dibasic acid, or a combination thereof. Usually, a polyester resin comprises a plurality of polycarboxylic acids and/or polyalcohols, and such a polyester resin is known herein as a copolyester resin. Examples of polycarboxylic acids commonly used to prepare a polyester resin includes adipic acid (“AA); azelic acid (AZA); dimerized fatty acid; dodecanoic acid; hexahydrophthalic anhydride (HHPA);

48zzzz isophthalic acid (IPA”); phthalic anhydride (“PA”); sebacid acid; terephthalic acid; trimelhtic anhydride; or a combination thereof. Examples of a polyalcohol commonly used to prepare a polyester resin include 1.2-propanediol; 1,4-butanediol; 1,4-cyclohexanedimethanol (CHDM);

1,6-hexanediol (HD”); diethylene glycol; ethylene glycol; glycerol; neopentyl glycol (“NPG); pentaerythitol (“PE); trimethylolpropane (TMP”); or a combination thereof. in certain embodiments, a polyester may be selected that has been synthesized by an acid catalyzed esterification reaction between a plurality of polyalcohols comprising two hydroxy moieties (a “diol), a polyalcohol comprising three hydroxy moieties (a “triol), and a dibasic acid. An example of a diol includes 1,4-cyclohexanedimethanoi; 1,6-hexanediol; neopentyl glycol; or a combination thereof. An example of a triol includes trimethylolpropane. An example of a polyol comprising four hydroxy moieties (a ¹׳tetraol״) includes pentaerythitol. In addition to the standards and analysis techniques previously described for an oil, an alkyd, a polyol, an acid anhydride standards for physical properties, chemical properties, and/or procedures for testing the purity/properties of an polyester are described, for example, in “ASTM Book of Standards, Volume 06.03, Paint - Pigments, Drying Oils, Polymers, Resins, Naval Stores, Cellulosic Esters, and Ink Vehicles,” D2690-98 and D3733-93, 2002.

The selection of a polyacid and/or a polyalcohol often affects a property of the polyester resin, such as the resistance of the polyester resin to hydrolysis, and similarly the water resistance of a coating and/or film comprising such a polyester resin. In embodiments wherein a polyester-coating is desired with a superior water resistance property relative to other types of polyester-coatings, it is preferred that the coating comprises a polyester prepared with a polyol that is more difficult to esterify, and thus generally more difficult to hydrolyze. Examples of such polyols include neopentyl glycol, trimethylolpropanel ,4-cyclohexanedimethanol, or a combination thereof.

In general embodiments, a polyester-coating is a solvent-borne coating. However, a polyester suitable for a water-borne coating is known to one of ordinary skill in the art. A waterborne polyester-coating generally comprises a polyester resin, wherein the acid number of the polyester resin is 40 to 60 Including all intermediate ranges and combinations thereof, and wherein the acid moieties have been neutralized by an amine, and wherein the coating comprises liquid component that comprises a co-solvent. An additional water-borne binder (e.g., an an amino resin) may be used to produce thermosetting film formation. In specific aspects, a waterborne polyester-coating produces a film of excellent hardness, gloss, flexibility, or a combination thereof.

48aaaaa

In alternative embodiments, a polyester temporary coating (e.g, a non-film forming coating) may be produced, for example, by selection of a polyester that that comprises fewer or no crosslinkable moieties, selection of an addition binder that comprises fewer or no crossiinkable moieties, reducing the concentration of the polyester or additional binder, or a combination thereof.

c. Modified Cellulose Binders

In some embodiments, a chemically modified cellulose molecule ( modified cellulose, “cellulosic״) may be used as a coating component (e.g., a binder). Cellulose is a polymer of anhydroglucose monomers that is insoluble in water and organic solvents. Various chemically modified forms of cellulose with enhanced solubility have been used as a coating component. Examples of chemically modified cellulose (“modified cellulose,' ‘cellulosic ) include a cellulose ester, a nitrocellulose, or a combination thereof. Examples of a cellulose ester include cellulose acetate (“CA), cellulose butyrate, cellulose acetate butyrate (“CAB), cellulose acetate propionate (“CAP), a hydroxy ethyl cellulose, a carboxy methyl cellulose, cellulose acetobutyrate, ethyl cellulose, or a combination thereof. A cellulose ester coating typically produces films with excellent flame resistance, toughness, clarity, or a combination thereof. In certain embodiments, a cellulose ester coating is selected as a topcoat, a clear coating, a lacquer, or a combination thereof. A cellulose ester is often selected for embodiments wherein the coating comprises an automotive coating, a furniture coating, a wood surface coating, cable coating, or a combination thereof. A cellulose ester coating may be a thermoplastic coating, a thermosetting coating, or a combination thereof.

A cellulose ester may be selected by the properties associated with the degree and/or type of esterification. Typically, solubility in a liquid component and/or combinability with an addition binder is increased by partial esterification of an anhydroglucose’s hydroxy moieties. For example, for a cellulose acetate butyrate, properties such as compatibility, diluent tolerance, flexibility (e.g־, lower T_g), moisture resistance, solubility, or a combination thereof, increases with greater butyrate esterification. However, decreased hydroxyl content alters properties in a cellulose ester. For example, a cellulose acetate butyrate comprising a hydroxy content of 1% or below has limited solubility in most solvents, while a hydroxy content of 5% or greater allows solubility in many alcohols, and the increased number of hydroxy moieties allows a greater degree of cross-linking reactions with binders such as, for example, an amino binder, an an acrylic binder, urethane binder, or a combination thereof. A cellulose acetate butyrate acryliccoating may be selected as lacquers, an automotive coating, a coating comprising a metallic pigment (e.g., aluminum), or a combination thereof. A cellulose acetate butyrate acrylic-coating may comprise a liquid component that comprises greater amounts of an aromatic hydrocarbon

48bbbbb solvent with the selection of a CAB with greater butyrate ester content. Though not a cellulosic, sucrose esters may be similarly used as cellulose ester, particularly CAB.

In some embodiments, in a cellulose ester comprising an acetyl ester (e.g., comprises cellulose acetate, cellulose acetate butyrate, cellulose acetate propionate), the acetyl content will range from 0.1% to 40.5% acetate, including all intermediate ranges and combinations thereof. In certain aspects, the acetyl content of a cellulose acetate, a cellulose acetate butyrate, or a cellulose acetate propionate will range from 39.0% to 40.5%, 1.0% to 30.0%, or 0.3% to 3.0%, respectively, including all intermediate ranges and combinations thereof, respectively. In many aspects, in a cellulose ester comprising a butyryl ester (e.g., cellulose acetate butyrate), the butyryl content will range from 15.0% to 55.0% butyryl, including all intermediate ranges and combinations thereof. In other aspects, in a cellulose ester comprising a propionyl ester (e.g., cellulose acetate propionate), the propionyl content will range from 40.0% to 47.0% propionyl, including all intermediate ranges and combinations thereof. In other embodiments, the hydroxyl content of a cellulose acetate, a cellulose acetate butyrate, or a cellulose acetate propionate will range from 0% to 5.0%, including all intermediate ranges and combinations thereof.

A nitrocellulose (cellulose nitrate) resin comprises a cellulose molecule wherein a hydroxyl moiety has been nitrated. A nitrocellulose for use In a coating typically comprises an average of 2.15 to 2.25 nitrates per anhydrogiucose monomer, and is soluble in an ester, a ketone, or a combination thereof. Additionally, nitrocellulose is soluble in a combination of a ketone, an ester, and an alcohol and/or hydrocarbon. A nitrocellulose may be selected as a lacquer, an automotive primer, automotive topcoat, a wood topcoat, or a combination thereof. Nitrocellulose coatings are typically a thermoplastic coating.

Standard procedures for determining physical and/or chemical properties (e.g., acetyl content, ash, apparent acetyl content, butyryl content, carbohydrate content, carboxyl content, color and haze, combined acetyl, free acidity, heat stability, hydroxyl content, intrinsic viscosity, solution viscosity, moisture content, propionyl content, sulfur content, sulfate content, metal content), of a cellulose and/or a modified cellulose (e.g., cellulose acetate, cellulose acetate propionate, cellulose acetate butyrate, methylcellulose, sodium carboxy methylcellulose, ethylcellulose, hydroxypropyl methylcellulose, hydroxyethylcellulose, hydroxypropylcellulose) have been described, for example, in “ASTM Book of Standards, Volume 06.03, Paint Pigments, Drying Oils, Polymers, Resins, Naval Stores, Cellulosic Esters, and Ink Vehicles, D1695-96 D817-96, D871-96. D1347-72, D1439-97, D914-00, D2363-79, D2364-01, D5400-93, D1343-95, D1795-96, D2929-89, D3971-89, D4085-93, D1926-00, D4794-94, D3876-96, D351689, D5897-96, D5896-96, D6188-97, D1348-94, and D1696-95, 2002. Specific procedures for

48ccccc determining purity/properties of a nitrocellulose (e.g., nitrogen content) have been described, for example, in “ASTM Book of Standards, Volume 06.03, Paint - Pigments, Drying Oils, Polymers, Resins, Naval Stores, Cellulosic Esters, and Ink Vehicles, D301-95 and D4795-94, 2002.

In alternative embodiments, a modified cellulose temporary coating (e.g, a non-film forming coating) may be produced, for example, by selection of a modified cellulose that that comprises fewer or no crosslinkable moieties, selection of an addition binder that comprises fewer or no crosslinkable moieties, reducing the concentration of the modified cellulose or additional binder, or a combination thereof.

d. Polyamide and Amidoamlne Binders

A polyamide (fatty nitrogen compound, fatty nitrogen product) is a reaction product of a polyamlne and a dimerized and/or trlmerized fatty acid. In typical embodiments, a polyamide is an oligomer. An amide resin comprises a terminal amine moiety capable of cross-linking with an epoxy moiety, and it is particularly preferred that a polyamide binder is combined with an epoxide binder. In other aspects, a polyamide may be considered an additive (e.g., a curing agent, a hardening agent, a coreactant) of an epoxide coating. A polyamine-epoxy coating may be used as an industrial coating (e.g., an industrial maintenance coating), a marine coating, or a combination thereof. A polyamide-epoxide coating may be applied to a surface such as, for example, wood, masonry, metal (e.g., steel), or a combination thereof. However, it is preferred that any surface is thoroughly cleaned prior to application to promote adhesion. Such surface preparation are well known to those of ordinary skill in the art, and include, for example, removal of rust, degraded film, grease, etc. A polyamide-epoxy coating typically is a solvent-borne coating. Examples of solvents for a polyamide include an alcohol, an aromatic hydrocarbon, a glycol ether, a ketone, or a combination thereof. In certain embodiments, a polyamide-epoxy coating may comprise a two-pack coating, wherein coating component(s) comprising the polyamide resin are stored in one container, and coating components comprising the epoxy resin are stored in a second container. Such a two-pack coating is admixed immediately before application, as the stoichiometric mix ratio of resin is formulated to promote a rapid cure. However, in other embodiments, a polyamide-epoxy coating may be a single container coating. Such a soivent-borne polyamine-epoxy coating may be formulated for a storage life of a year or more. An aluminum and or stainless steel container is suitable, though a carbon steel container may alter coating and/or film color. However, such a coating typically undergoes film formation in stages, wherein the liquid component is physically lost by evaporation while thermosetting produces a physically durable film in about 8 to 10 hours, a chemically resistant film in three to four days, and final cross-linking completed in about three weeks. In some embodiments, a polyamine-epoxy coating may undergo chalking upon exterior weathering.

48ddddd

Though a polyamide is prepared from a fatty acid, it is not classified as an oil-based binder herein due to the chemistry of film formation for polyamide binder. The dimerized (dibasic”) or tnmerized fatty acid generally comprises a polyunsaturated fatty acid, a monounsaturated fatty acid, or a combination thereof. In certain aspects, the fatty acid is a linseed oil fatty acid, soybean oil fatty acid, tall oil fatty acid, or a combination thereof. In specific facets, the fatty acid is an 18-carbon fatty acid. However, to reduce the volatile organic compounds of solvent-borne coating, a polyamide binder may be partly or fully substituted, such as 0% to 100% substitution, including all intermediate ranges and combinations thereof, with an amidoamine binder. An amidomine binder differs from a polyamide binder by the use of a fatty acid rather than a dimerized fatty acid in the synthesis of the resin. The selection of the polyamine in the preparation of a polyamide can affect the properties of the polyamide. The polyamine may be linear (e.g., diethylenetriamine), branched or cyclic (e.g., aminoethylpiperazine). Standards for physical properties, chemical properties, and/or procedures for testing the purity/properties (e.g., amine value) of a polyamide and/or an amidoamine are described, for example, in ASTM Book of Standards, Volume 06.03, Paint - Pigments, Drying Oils, Polymers, Resins, Naval Stores, Cellulosic Esters, and Ink Vehicles, D2071-87, D2073-92, D2082-92, D2072-92, D2074-92, D2075-92, D2076-92. D2077-92, D2078-86, D2079-92. D208092, D2081-92, and D2083-92, 2002.

In general embodiments, a polyamine comprises a polyethylene amine. A polyamide produced from diethytenetriamine can be prepared to comprise a varying amount, typically 35% to 85%, including all intermediate ranges and combinations thereof, of an imidazoline moiety. In other embodiments, the amount of amine moiety capable of cross-linking with an epoxy moiety may vary from 100 to 400 amine value, including all intermediate ranges and combinations thereof. However, the amine value is converted into units known as ”active hydrogen equivalent weight,” which varies from 550 to 140, including all intermediate ranges and combinations thereof for comparison to the epoxy resins epoxide equivalent weight for determining the stoichiometric mix ratio of a polyamide-epoxy combination. The stoichiometric mix ratio affects coating and film properties. As the polyamide to epoxy stoichiometric mix ratio increases from a ratio of less than one to a ratio of greater than one, properties such as excellent impact resistance, excellent chemical resistance, or a combination thereof, decrease while film flexibility Increases. Examples of polyamide to epoxy stoichiometric mix ratio include 2:1 to 1:2, including all intermediate ranges and combinations thereof.

In alternative embodiments, a polyamide and/or amidoamine temporary coating (e.g, a non-film forming coating) may be produced, for example, by selection of a polyamide and/or

48eeeee amidoamine that that comprises fewer or no crosslinkable moieties, selection of an addition binder that comprises fewer or no crosslinkable moieties, reducing the concentration of the polyamide and/or amidoamine or additional binder, selection of a stoichiometric ratio that is less suitable for crosslinking reactions, or a combination thereof.

e. Amino Resins

An amino resin (“amino binder,” “aminoplast, “nitrogen resin) is a reaction product of formaldehyde, an alcohol and a nitrogen compound such as, for example, urea, melamine (“1:3:5 triamino triazine”), benzoguanamine, glucoluril, or a combination thereof. An amino resin may be used to a thermosetting coating. An amino resin comprises an alkoxymethyl moiety capable of cross-linking with a hydroxyl moiety of an additional binder such as an acrylic binder, an alkyd resin, a polyester binder, or a combination thereof, and it is preferred that an amino resin is combined with a binder that comprises a hydroxyl moiety in a coating. In aspects wherein the coating comprises an amino resin and an alkyd resin, it is preferred that the amino:alkyd resin ratio is 1:1 to 1:5, including all intermediate ranges and combinations thereof. An amino resin coating typically is a solvent-borne coating. Examples of solvents for an amino resin include an alcohol (e.g., butanol, isobutanol, methanol, isopropanol), a ketone, hydroxyl functional glycol ether, or a combination thereof. Additionally, an amino resin generally possesses limited solubility in a hydrocarbon (e.g., xylene), which may be added to a solvent-borne coatings liquid component. In certain aspects, an amino resin coating may be a water-borne coating, wherein water is a solvent for an amino resin comprising a plurality of methylol moieties. In other embodiments, a water-borne amino resin coating may comprise a water-reducible coating, particularly wherein the liquid component comprises a glycol ether, an alcohol, or a combination thereof. In certain embodiments, an amino coating comprises an acid catalyst.

An amino resin coating generally is cured by baking at a temperature of 82 C and 204 C, including all intermediate ranges and combinations thereof. Baking generally promotes reactions between amino resins, though it does improve the reaction rate between an amino resin and an additional binder. It is preferred that in embodiments wherein the coating comprises an additional binder, the additional resin comprises less hydroxyl moieties and/or the amino resin is polar amino resin (e.g., a conventional amino resin) a when cured by baking than embodiments wherein an acid catalyst is used. An amino resin coating undergoes rapid film formation, typically lasting 30 seconds and 30 minutes, wherein a higher temperature and/or acid catalyst shortens film formation time. An amino resin prepared from urea is generally undergoes film formation faster than an amino resin prepared from melamine. However, an amino resin coating generally produces an alcohol (e.g., methanol, butanol) and formaldehyde during film formation as byproducts.

48fffff

An amino resin for use in a coating may be classified by content of a liquid component (e.g., a solvent) as a high solids amino resin or a conventional amino resin. The liquid component is generally used to reduce the viscosity of the resin for coating preparation. A high solids amino resin comprises 80% to 100%. by weight, an amino resin, with the balance a liquid component. A high solids amino resin is are relatively less polar, less polymeric, lower in viscosity, or a combination thereof, relative to a conventional amino resin. The lower viscosity allows the use of little or no liquid component. Additionally, a high solids amino resin may be water-soluble and/or water reducible. A conventional amino resin comprises less than 80% amino resin, by weight, with the balance a liquid component. Properties of a high solids or conventional amino resin selected for use in a coating such as the amount of amino resin and liquid component, the amount of unreacted formaldehyde in the resin preparation, the viscosity of the resin, the ability of the resin to accept additional liquid component as a solvent, can be empirically determined by procedures known to those of ordinary skill in the art (see, for example, ASTM Book of Standards, Volume 06.03, Paint - Pigments, Drying Oils, Polymers, Resins, Naval Stores, Cellulosic Esters, and Ink Vehicles,״ D4277-83, D1545-98, D1979-97, and D119893, 2002; and “ASTM Book of Standards, Volume 06.01, Paint - Tests for Chemical, Physical, and Optical Properties; Appearance, D2369-01e1,2002).

In embodiments wherein an amino resin coating comprise an amino resin prepared from urea, the coating may be used as wood coating (e.g., furniture coating), an industrial coating (e.g., an appliance coating), an automotive primer, a clear coating, or a combination thereof. However, an amino resin film, wherein the resin was prepared from urea, generally produces a film with poor resistance to moisture, and is preferred as an internal coating and/or as part of a multicoat system. In certain embodiments, an amino resin prepared from melamine, generally produces films with good resistance to moisture, temperature, UV irradiation, or a combination thereof. A melamine-based amino coating may be applied to a metal surface, in specific aspects, such a melamine amino resin coating may be an automotive coating, a coil coating, a metal container coating, or a combination thereof. In embodiments wherein an amino resin coating comprise an amino resin prepared from benzoguanamine, the film produced generally possesses poor weathering resistance, good corrosion resistance, water resistance, detergent resistance, flexibility, hardness, or a combination thereof. A benzoguanamine amino resin may be used as an industrial coating, particularly for indoor applications (e.g., an appliance coating). In embodiments wherein an amino resin coating comprise an amino resin prepared from, giycoluril, a higher baking temperature and/or acid catalyst may be used during film formation, but less byproducts may be released. A glycoluril-based amino-coating typically produces a film with

48ggggg excellent corrosion resistance, humidity resistance, or a combination thereof. A glycoluril-based amino-coating may be selected as a metal coating.

In alternative embodiments, an amino resin temporary coating (e.g, a non-film forming coating) may be produced, for example, by selection of an amino resin that that comprises fewer or no crosslinkable moieties, selection of an addition binder that comprises fewer or no crosslinkable moieties, reducing the concentration of the amino resin and/or additional binder, selection of a binder ratio that is less suitable for crosslinking reactions, using a bake cured amino resin coating at temperatures less than is needed for curing (e.g., ambient conditions) or a combination thereof.

f. Urethane Binders

A urethane binder (polyurethane binder, urethane,“ polyurethane) is a binder comprising prepared from compounds that comprise an isocyanate moiety. The urethane binder's urethane moiety can form intemolecular hydrogen bonds between urethane binder polymers, and these non-covalent bonds confer useful properties in a coating or film comprising an urethane binder. The hydrogen bonds can be broken by mechanical stress, but will reform, thereby conferring a property of abrasion resistance. Additionally, a urethane binder can form some hydrogen bonds with water, conferring a plasticizing property to the coating. In certain embodiments, a urethane binder comprises an isocyanate moiety. The isocyanate moiety is highly reactive (e.g., crosslinkable) with a moiety comprising a chemically reactive hydrogen. Examples of a chemically reactive hydrogen moiety include a hydroxyl moiety, an amine moiety, or a combination thereof. Examples of an additional binder include a polyol, an amine, an epoxide, silicone, vinyl, phenolic, or a combination thereof. In certain embodiments, a urethane coating is a thermosetting coating. In specific aspects, a urethane coating comprises a catalyst (e.g., dibutyltin dilaurate, stannous octoate, zinc octoate). In specific facets, the coating comprises 10 to 100 parts per million catalyst, including all intermediate ranges and combinations thereof, in some embodiments, such a coating will undergo film formation at ambient conditions or slightly greater temperatures. A binder comprising an isocyanate moiety is often selected to produce a coating with durability in an external environment. A urethane coating typically possesses good flexibility, toughness, abrasion resistance, chemical resistance, water resistance, or a combination thereof. An aliphatic urethane coating may be selected for the additional property of good lightfastness.

In general embodiments, a urethane binder may be selected based on the materials used in its preparation, which typically affect the urethane binder's properties. An example of a urethane binder includes an aromatic isocyanate urethane binder, an aliphatic isocyanate

48hhhhh seleL L ’ “׳⁸⁰״י״ ״⁰³¹’״ '^S0Cya^ate «“״ ־׳־ ״־י״“ ™״ft״־ for embodiments ״herein a superior exterior durability, cotor stability. _g00d lightfast־־־״ ־ a combination thereof re,*־ fo an aromatic isocyanate bind־, is desirad isocyanate urethane binder includes a hydrogenated bls(4-lsocyanatoph״־yl)metha״e ( 4,4d1cyclohexylmethane dilsocyanat־,- HMDI), HDI. a combination of 2 2 4-trtmethvl hexametbylene dlisocyanata and 2.4.4-trimethyt bexamathyiene dilsocyanat־ (TMHDI) 1 4 cyclohexane diisocyanate (CHOt), _isopboron־ diisocyanate ' ζ ־ י ־^thy cycfohexyl isocyanate, iPOt). or a combination thereof. ,״ _{aspec|s ־} specs, a DI derived binder may be prepared from a 1,6-hexamethytene diisocyanate isocyanurate, »herein such ־ HDI derived binder produces a coating with genaraiiy superior beat resistance and/or exterior dumbiiity Is desired mtetive to other HDI derived binders. As ״ouid be

0> ץ one of ordinary ־« ,, _{tb8 art}. _{standards physfca}, ־ d/or procedures for testing the punty/properties of urethane precursor components (eg.' tot־״־״) and urethane resins (e.g. Isocyanate moieties) for use in e coaling are described exampi״. ־ ASTM Book 0, Standards. Vo,״me 06.04. Paint _ _S0,y_{ents; Aromatic} p₀,_״ Η⁰⁰²' ”^AS™ ^B°°^k ״ ^S,a^dards'^Voluma °^{6 03}^Pain' ־־ ^piE׳’^M5־»«י ׳ Oils

Jymers, Reams. Naval Stems. Ceiiuiosic Esters, and Ink Vehicles. 03432-89 _a״_d D2S72.S7’

In «״»״ _smtodime״,_s. ״ _{urethanB coating a uretha״a} ,־action. _{An axampte ls a molsturecure ״}J ״ ־ isocyanate moiehr. Contact between _a״ _lsooyana,_e °״ ™ moiety cap־,,־ of bonding with _{an 150cya}״_{ate mp|־ty p},^״־ a_t 1 רהכהז״ τ״^οη ״׳^{c־rtain asp־c,s}^a ־³.־ ^־״ at too C 140 C. mciudmg _־ll intermediate _{ranges and com}b_ina״o״s, crosslinking reactions between the linear polymers. ,״ certain embodiments, a moist״r־-c״re urethane coating is a soivent-borne _m־ting. ,״ _{spec(fc ־sp־־fe ־}J coatmg compnses a dehydrator, in genera, aspect־. moistum-cum urethane coating ty_{pfcally 15 a}ne-pack coating, prepared for storage of the coating in anhydrous conditions.

In certam embodiments, an ״,ethane coating comprises a blocked Isocyanate umthane binder, where,, the isocyanate moiety has been chemicaliy modltied by a hydrogen do־״, to be ™צ ^{COn,aCl־d Wi,h a baRlna tempara,Ure}^Su* « “׳״»< -y־>־״־ urethane coating ,־ a one-pack coating, as it is designed fo, stabliity at ambient conditions. Additionatty. a blocked isocyanate urethane coating may be a powder coating.

48iiiii

In certain embodiments, a urethane coating comprises an additional binder. In certain embodiments, a urethane may be combined with a binder such as an amine, an epoxide, silicone, vinyl, phenolic, a polyol, or a combination thereof, wherein the binder comprises a reactive hydrogen moiety. In specific embodiments, selection of a second binder to crosslink with the urethane binder affects coating and/or film properties. In certain aspects, a coating comprising a urethane and an epoxide, vinyl, phenolic, or a combination thereof produces a film with good chemical resistance. In other aspects, a coating comprising a urethane and a silicone produces a coating with good thermal resistance. In some aspects, a coating comprises a urethane and a polyol. A primary hydroxyl moiety, secondary hydroxy! moiety, and tertiary hydroxyl moiety of a polyol are respectively the fastest, moderate, and slowest to react with a urethane. Steric hindrance from a neighboring moiety may slow the reaction with a hydroxyl moiety. In an additional example, use of a polyol may increase flexibility of a urethane coating. Often, a selected polyol has a molecular weight from 200 Da to 3000 Da, including all intermediate ranges and combinations thereof. Generally, a lower molecular weight polyol increases the hardness property, lowers the flexibility property, or a combination thereof, of a urethane polyol film. Examples of a polyol include a glycol, a triol (e.g., 1,4-butane-diol, diethylene glycol, trimethylolpropane), a tetraol, a polyester polyol, a polyether polyol, an acrylic polyol, a polylactone polyol, or a combination thereof. Examples of a polyether polyol include a poly (propylene oxide) homopolymer polyol, a poly (propylene oxide) and ethylene oxide copolymer polyol, or a combination thereof.

In certain embodiments, a urethane binder comprises a thermoplastic urethane binder. Typically, a thermoplastic urethane binder is from 40 kDa to 100 kDa, including all intermediate ranges and combinations thereof. In particular aspects, a thermoplastic urethane binder comprises little or no isocyanate moieties. in general aspects, a thermoplastic urethane coating is a solvent borne coating. In specific facets, a thermoplastic urethane coating is a lacquer, a high gloss coating, or a combination thereof.

In certain embodiments, a urethane binder is an urethane acrylate (acrylated urethane ) binder. An urethane acrylate binder generally comprises an acrylate moiety at an end of the polymeric binder. The acrylate moiety is typically part of an acrylate monomer, wherein the monomer comprises a hydroxyl moiety (e.g., a 2-hydroxy-ethyl acrylate). An urethane acrylate coating generally comprises another binder for crosslinking reactions. Examples of a suitable binder include a triacrylate (e.g., teimethylolpropane). A urethane acrylate coating generally also comprises a viscosifier, wherein the viscosifier reduces viscosity. Examples of such a viscosifer include an acrylate monomer, a N-vinyl pyrrolidone, or a combination thereof. A urethane acrylate coating is cured by irradiation. Examples of irradiation include UV light, electron beam,

48jjjjj or a combination thereof. In embodiments wherein UV light is a curing agent, a urethane acrylate coating typically comprises a photoinitiator. Examples of a suitable initiator include 2,2,diethoxyacetophenone, a combination of benzophenone and an amine synergist, or a combination thereof. In specific facets, an urethana acrylate coating is applied to a plastic surface. In other facets, an urethane acrylate coating floor coating, an electronic circuit board coating, or a combination thereof.

In alternative embodiments, a urethane temporary coating (e.g, a non-film forming coating) may be produced, for example, by selection of a urethane resin that that comprises fewer or no crosslinkable moieties, selection of an addition binder that comprises fewer or no crosslinkable moieties, reducing the concentration of the a urethane resin and/or additional binder, using a bake cured a urethane resin coating at temperatures less than is needed for curing (e.g., ambient conditions), selection of size range for a thermoplastic urethane resin coating that is less suitable for film formation (e.g., 1 kDa to 40 kDa), or a combination thereof.

(1) Water-Borne Urethanes

The previous discussion of urethane coatings focused on solvent-borne urethane coating. A water-borne urethane coating typically is comprises a water-dispersible urethane binder such as a cationic modified urethane binder and/or anionic modified urethane binder. A cationic modified urethane binder is a urethane binder chemically modified by an diol comprising an amine, such as, for example, diethanolamine, methyl diethanolamine, N,N-bis(hydroxyethyl)-aaminopyridine, lysine, N-hydroxyethylpiperidine, or a combination thereof. An anionic modified urethane binder is a urethane binder chemically modified by an diol comprising a carboxylic acid such as dimethylolpropionic acid (2,2-bis(hydroxymethyl) propionic acid), dihydroxybenzoic acid, and/or a sulfonic acid (e.g., 2-hydroxymethyl-3-hydroxy-propanesulfonic acid), or a combination thereof.

(2) Urethane Powder Coatings

A urethane powder coating refers to a polyester and/or acrylic coating, wherein the binder has been modified to comprise a urethane moiety. Such a coating is typically a thermosetting, bake cured coating, an industrial coating (e.g., an appliance coating), or a .combination thereof.

g. Phenolic Resins

A phenolic resin (phenolic binder,” “phenolic) is reaction product of a phenolic compound and an aldehyde. A preferred aldehyde is formaldehyde, and such a phenolic resin is known as a phenolic formaldehyde resin (“PF resin). The properties of a phenolic resin are affected by the phenolic compound and reaction conditions used during synthesis. A resole resin

48kkkkk (“resole phenolic) is prepared by a reaction of a molar excess of a phenolic compound with formaldehyde under alkaline conditions. A novolac resin (novolac phenolic) is prepared by a reaction of a molar excess of formaldehyde with a phenolic compound under acidic conditions. Examples of phenolic compounds used in preparing a phenolic resin include phenol; orthocresol ( o-cresol); metacresol, paracresol (p-cresol”); a xylenol (e.g., 4-xylenol); bisphenol-A [“2,2-bis (4-hydroxylphenyl) propane”; ^,,diphenylol propane״); p-phenylphenol; p-tert-butylphenol; p-tertamyfphenol; p-tert-octyl phenol; p-nonylphenol; or a combination thereof. As would be known to one of ordinary skill in the art, standards for physical properties, chemical properties, and/or procedures for testing the purity/properties of various compounds used in phenolic resins (e.g., bisphenol A, a phenol, a cresol, formaldehyde) for use in a coating are described, for example in ASTM Book of Standards, Volume 06.04, Paint — Solvents; Aromatic Hydrocarbons, D6143-97, D3852-99, D4789-94, D2194-02, D2087-97, D2378-02, D2379-99, D2380-99, D1631-99, D614297. D4493-94, D4297-99, and D4961-99, 2002. As would be known to one of ordinary skill in the art, standards for physical properties, chemical properties, and/or procedures for testing the purity/properties of phenolic resins for use in a coating are described, for example in ASTM Book of Standards, Volume 06.03, Paint - Pigments, Drying Oils, Polymers, Resins, Naval Stores, Cellulosic Esters, and Ink Vehicles,״ D1312-93, D4639-86, D4706-93, D4613-86 and D4640-86, 2002.

In alternative embodiments, a phenolic resin temporary coating (e.g, a non-film forming coating) may be produced, for example, by selection of a phenolic resin that that comprises fewer or no crosslinkable moieties, selection of an addition binder that comprises fewer or no crosslinkable moieties, reducing the concentration of the a phenolic resin and/or additional binder, using a bake cured a phenolic resin coating at temperatures less than is needed for curing (e.g., ambient conditions), or a combination thereof.

(1) Resole

A resole resin is the more commonly used PF resin. A solvent-borne phenolic formaldehyde coating typically comprises an alcohol, an ester, a glycol ether, a ketone, or a combination thereof, as a PF solvent. However, a phenolic resin prepared from phenolic compound comprising an alkyd moiety, such as, for example, p-tert-butylphenol p-tert-amylphenol p-tert-octyl phenol, or a combination thereof, typically has solubility in an aromatic compound and/or able to tolerate an aliphatic diluent. Often, a phenoiic-resin coating comprises an additional binder such as an alkyd resin, an amino resin, a blown oil, an epoxy resin, a polyamide, a polyvinyl resin [e.g., polyfvinyl butyral)], or a combination thereof. An example of a phenolicresin coating includes a varnish, an industrial coating, or a combination thereof. A phenolic resincoating may be selected for embodiments wherein a film possessing solvent resistance, corrosion

4811111 resistant, of a combination thereof, is desired. Examples of surfaces wherein such properties are often desirable include a surface of a metallic container (e.g., a can, a pipeline, a drum, a tank), a coil coating, or a combination thereof. In specific aspects, a phenolic coating produces a film 0.2 to 1.0 mil thick, including all intermediate ranges and combinations thereof. In specific aspects, coating comprising a phenolic-binder and additional binder undergoes thermosetting cross-linking reactions between the binders during film formation. In certain embodiments, a phenolic-resin coating undergoes cure by baking, such as, for example, 135 C to 204 C, including all intermediate ranges and combinations thereof. In specific aspects, a baking cure time is one minute to four hours, with shorter cure times at high temperatures. A phenolic-resin film generally possesses excellent hardness property (e.g., glass-like), excellent resistance to solvents, water, acids, salt, electricity, heat resistance, as well as thermal resistance up to 370°C for a period of minutes.

However, a phenolic-resin film is poorly resistant to alkali unless made from a coating that also comprised an epoxy binder. In certain embodiments, a phenolic-epoxy coating comprises a binder ratio of 15:85 to 50:50 phenolic binder:epoxy binder, including all intermediate ranges and combinations thereof. In certain aspects, a phenolic-epoxy coating possesses superior flexibility, toughness, or a combination thereof relative to a phenolic coating. In specific facets, a phenolic-epoxy coating is cured at 200°C for 10 to 12 minutes.

In other aspects, a phenolic coating comprises a blown oil, an alkyd, or a combination thereof. In some aspects, such a coating comprises a phenolic resin prepared from p-tertbutylphenol p-tert-amylphenol p-fert-octyl phenol, or a combination thereof. In specific aspects, such a coating is applied to electrical coil, electrical equipment, or a combination thereof.

(2) Novolak

In other aspects, wherein a film is desired, it novolak coating may be used. However, a novolak resin is generally a non-film forming resin, in is particularly preferred that the coating comprise an epoxy resin. It is also preferred that the coating comprise a basic catalyst. A film produced from such a novolak-epoxy coating typically possesses good resistance to chemicals, water, heat, or a combination thereof, in specific facets, a novolak-epoxy coating may be a high solids coating, a powder coating, a pipeline coating, or a combination thereof.

A novolak resin prepared from phenolic compound comprising an alkyd moiety such as ptert-butylphenol p-tert-amylphenol ρ-tert-octyl phenol, or a combination thereof, typically has solubility in an oil. Additionally, a PF resin may be modified by reaction with an oil to produce an oil modified PF resin, which is also oil soluble. An alkyd phenol-formaldehyde resin, an oil

48mm m mm צי&lt;&amp;&lt;^ךד I modified phenol-formaldehyde resin, is generally a non-film forming resin. A coating capable of producing a film may be formulated by combining such a resin with a drying oil, an alkyd, or a combination thereof, in specific aspects, an alkyd phenol-formaldehyde resin, an oil modified phenol-formaldehyde resin undergoes cross-linking with an oil and/or an alkyd. Such a coating may further comprise a liquid component (e.g., a solvent), a drier, a UV absorber, an anti-skinning agent, or a combination thereof. In certain facets, such a coating undergoes film formation under ambient conditions or by baking. In particular aspects, such a coating comprises a varnish, a wood coating, or a combination thereof. In specific facets, such a coating comprises a pigment.

h. Epoxy Resins

An epoxy resin (epoxy binder, epoxy) is a compound comprising an epoxide (oxirane) moiety. An epoxide resin may be used in a thermosetting coating, thermoplastic coating, or a combination thereof. An epoxide coating typically is a solvent borne coating, though examples of a water-borne and powder epoxy coating are described herein. An epoxide coating generally possesses excellent properties of adhesion, corrosion resistance, chemical resistance, or a combination thereof. An epoxide coating may be selected for various surfaces, particularly a metal surface.

An epoxide resin (e.g., a bisphenol A epoxy resin) generally comprises one or two epoxide moieties per resin molecule. An epoxide resin may additionally comprise a monomer, oligomer, or polymer of repeating chemical units, each generally lacking an epoxide moiety, but comprising a hydroxy moiety. The number of monomer(s) present is expressed “n value, wherein an average increase of one monomer per epoxide resin molecule increase the n value by one. The chemical and/or physical properties of an epoxide resin are affected by the n value. For example, as the n value increases, the chemical reactions selected for film formation in a thermosetting coating may become more dominated by reactions with the increasing numbers of hydroxyl moieties, and less dominated by the epoxide moieties. Often, an epoxide resin is classified by an epoxide equivalent weight, which is the grams of resin required to provide 1 M epoxide moiety equivalent. In certain embodiments, the epoxide equivalent weight is 182 to 3050, including ail intermediate ranges and combinations thereof. Additionally, an epoxide resin may be used in a thermoplastic coating, particularly wherein the π value is greater than 25. In certain embodiments, an epoxide resin may possess an n value of 0 to 250, including all intermediate ranges and combinations thereof. As would be known to one of ordinary skill in the art, standards for physical properties, chemical properties, and/or procedures for testing the purity/properties of epoxy resins (e.g., epoxy moiety content) for use in a coating are described, for example in “ASTM Book of Standards, Volume 06.03, Paint - Pigments, Drying Oils,

48nnnnn

Polymers, Resins, Nava! Stores, Cellulosic Esters, and Ink Vehicles, D4142-89, D1652-97, D1726-90, D1847-93, and D4301-84, 2002.

An epoxide moiety is chemically reactive with a variety of other moieties, such as, for example, an amine, a carboxyl, a hydroxyl or a phenol. An epoxide coating may comprise an additional binder capable of undergoing a cross-linking reaction with the epoxide during film formation. Various such additional binders are known to those of ordinary skill in the art, and are often referred to as a curing agent or hardener.” The selection of a curing agent and/or an epoxide can affect whether the coating undergoes film formation at ambient conditions or by baking.

In alternative embodiments, an epoxide resin temporary coating (e.g, a non-film forming coating) may be produced, for example, by selection of an epoxide resin that that comprises fewer or no crosslinkable moieties, selection of an addition binder that comprises fewer or no crosslinkable moieties, reducing the concentration of the an epoxide resin and/or additional binder, using a bake cured an epoxide resin at temperatures less than is needed for curing (e.g., ambient conditions), not irradiating the coating, or a combination thereof.

(1) Ambient Condition Curing Epoxies

In certain embodiments, a curing agent suitable for curing at ambient conditions comprises an amine moiety such as a polyamine adduct, which is an epoxy resin modified to comprise an amine moiety, a polyamide, a ketimine, an aliphatic amine, or a combination thereof. Examples of an aliphatic amine include ethylene diamine (EDA), diethylene triamine (DETA”), triethylene tetraamine (“TETA”), or a combination thereof. Selection of a polyamine adduct generally produces a film with excellent solvent resistance, corrosion resistance, acid resistance, flexibility, impact resistance, or a combination thereof. Selection of a polyamide generally produces a film with superior adhesion, particularly to a moist or poorly prepared surface, good solvent resistance, excellent corrosion resistance, good acid resistance, superior flexibility retention, superior impact resistance retention, or a combination thereof. A ketimine is a reaction product of a primary amine and a ketone, and produces a coating and/or film with similar properties as a polyamine or amine adduct. However, the pot life is longer with a ketimine, and moisture (e.g., atmospheric humidity) activates this cure agent. Examples of an epoxide selected for curing at ambient conditions includes a low mass epoxide resins with an n value from 0 to 2.0, including all intermediate ranges and combinations thereof. In certain embodiments, an epoxy resin may be selected with an epoxy equivalent weight of 182 to 1750, including all Intermediate ranges and combinations thereof. In specific aspects, the greater the n value of an epoxide resin, the longer the pot life in a two-pack coating, the greater the coating leveling property, the lower

4800000 the film solvent resistance, the lower the film chemical resistance, the greater the film flexibility, or a combination thereof. In certain aspects, an ambient curing epoxide coating is a two-pack coating, wherein the epoxide resin is in one container and the curing agent tn a second container. In typical aspects, the pot life upon admixing the coating components is two hours to two days. An ambient cure epoxide may be selected for an industrial coating {e.g., industrial maintenance coating), a marine coating, an aircraft primer, a pipeline coating, a HIPAC, or a combination thereof.

(2) Bake Curing Epoxies

In other embodiments, a curing agent suitable for curing by baking includes an amino resin (e.g., a urea or melamine-based amino resin), a phenolic resin, or a combination thereof. Since baking is generally needed to promote film formation, an epoxy coating comprising such a curing agent typically is a one-pack coating. In certain embodiments, an epoxy resin may be selected with an epoxy equivalent weight of 1750 to 3050, including all intermediate ranges and combinations thereof. An epoxy resin coating that comprises an amino resin cure agent typically is selected for a lower cure temperature. Such a coating may be selected as a can coating, a metal coating, an industrial coating {e.g., equipment, appliances), or a combination thereof. An epoxy coating comprises an phenolic resin cure agent typically possesses greater chemical resistance and/or solvent resistance, and is typically selected for a can coating, a pipeline coating, a wire coating, an Industrial primer, or a combination thereof. Examples of an epoxide selected for curing by baking Includes a higher mass epoxide resins with an n value from 9.0 to 12.0, including all intermediate ranges and combinations thereof. In certain embodiments, a heat-cured epoxy coating is a water-borne coating. Such a water-borne coating comprises a higher mass epoxide resin modified to comprise a terpolymer that comprises monomers of styrene, methacrylic, acrylate, or a combination thereof, and an amino resin, a phenolic resin, or a combination thereof. Such a water-borne coating is typically selected as a can coating.

(3) Electrodeposition Epoxies

Another example of a water-borne epoxide coating is an electrodeposition epoxy coating. In certain embodiments, an epoxy resin may be selected with an epoxy equivalent weight of 500 to 1500, including all intermediate ranges and combinations thereof. An anionic and/or cationic epoxy resin is electrically attracted to a surface for application. The surface removed from the coating bath, and the coating is baked cured into a film upon the surface. Such a water-borne coating may be selected for an automotive primer, described elsewhere herein.

48ppppp (4) Powder Coating Epoxles

Ao epoxy coating may be a powder coating, wherein the various nonvolatile coating components are admixed. Examples of typical admixed components include an epoxy resin, a curing agent, and a pigment, an additive, or a combination thereof. In certain embodiments, an epoxy resin may be selected with an epoxy equivalent weight of 550 to 750, including all intermediate ranges and combinations thereof. The mixture is then melted, cooled, and powderized. The powder coating Is typically applied by attraction to an electrostatic charge of a surface. The thermosetting coating is cured by baking. An epoxy powder coating may be selected as a pipe coating, an electrical devise coating, an industrial coating {e.g., appliance coating, automotive coating, furniture coating), or a combination thereof.

(5) Cycloaliphatic Epoxles

A cycloaliphatic epoxy binder possesses a ring structure, rather than the linear structure for the epoxy embodiments described above. Examples of a cycloaliphatic epoxide is ERL-4221 (“3,4-epoxycyclohexylmethyl 3,4-epoxycyclohexane carboxylate), which has an epoxy equivalent weight of 131 to 143, bis(3,4-epoxycyclohexylmethyl) adipate, which has an epoxy equivalent weight of 190 to 210, 2-(3,4-epoxycyclohexyl-5,5-spiro-3,4-epoxy)cyclohexane-m-dioxane, which has an epoxy equivalent weight of 133-154, 1-vinyl-epoxy3,4־-epoxycyclohexane, which has an epoxy equivalent weight of 70 to 74, or a combination thereof. Usually, a cycloaliphatic epoxy coating is combined with another binder, such as a polyol, a polyol modified to comprise a carboxyl moiety, or a combination thereof. An acid may be used to initiate crosslinking, particularly with a polyol. A cycloaliphatic epoxy polyol coating may comprise a triflic acid salt (e.g., diethylammonium triflate) to produce a one-pack coating with a pot life of up to eight months. In certain embodiments, a cycloaliphatic epoxy coating is a UV radiation cured coating, wherein the coating comprises a compound that converts to a strong acid upon UV irradiation (e.g., an onium salt). In certain aspects, a UV radiation cured cycloaliphatic epoxy coating is a one-pack coating. A UV radiation cured cycloaliphatic epoxy coating generally possesses excellent flame resistance, water resistance, or a combination thereof, and may be selected as a can coating or an electrical equipment coating. A compound comprising a carboxyl moiety (e.g., a carboxyl modified polyol) readily crosslinks with a cycloaliphatic epoxy binder. However, such a cycloaliphatic epoxy coating comprising such an additional binder generally has a short pot life (e.g., less than eight hours). In certain aspects, a cycloaliphatic epoxy carboxylic acid binder coating is a two-pack coating. A cycloaliphatic epoxy carboxylic acid polyol coating generally possesses excellent adhesion, toughness, gloss, hardness, solvent resistance, or a combination thereof.

48qqqqq

I. Polyhydroxyether Binders

A polyhydroxyether binder (“polyhydroxyether resin, “phenoxy binder, phenoxy) chemically resembles a bisphenol A epoxy resin, though a poiyhydroxyether binder lacks an epoxide moiety, and about 30 kDa in size. A poiyhydroxyether coating is typically a thermoplastic coating. The poiyhydroxyether binder comprises a hydroxyl moiety, and can be cross-linked with an additional binder such as an epoxide, a polyurethane comprising an isocyanate moiety, an amino resin, or a combination thereof. A thermosetting poiyhydroxyether coating typically possesses excellent physical resistance properties, excellent chemical resistance, modest solvent resistance, or a combination thereof, in alternative embodiments, a poiyhydroxyether binder temporary coating (e.g, a non-film forming coating) may be produced, for example, by selection of a poiyhydroxyether binder that that comprises fewer or no crosslinkable moieties, selection of an addition binder that comprises fewer or no crosslinkable moieties, reducing the concentration of the a poiyhydroxyether binder and/or additional binder, or a combination thereof.

j. Acrylic Resins

An acrylic resin (acrylic polymer,״ acrylic binder, “acrylic) is a binder comprising a polymer of an acrylate ester monomer, a methacrylate ester monomer, or combination thereof. An acrylic-coating generally possesses a superior property of water resistance and/or exterior use durability than a polyester-coating. Other properties that an acrylic-coating typically possesses include color stability, chemical resistance, resistance to a UV light, or a combination thereof. An acrylic resin may further comprise an additional monomer to confer a desirable property to the resin, coating and/or film. For example, a styrene, a vinyltoluene, or a combination thereof, generally improve alkali resistance. Examples of such properties include the acrylic resin's chemical reactivity (e.g., cross-linkability), acidity, alkalinity, hydrophobicity, hydrophilicity, glass transition temperature, or a combination thereof. However, a thermoplastic acrylic film generally possesses poor solvent (e.g., acetone, toluene) resistance. Like other thermoplastic films, a thermoplastic acrylic film is generally easy to repair by application of additional acrylic coating to an area of solvent damage. An acrylic-coating is often suitable for various surfaces (e.g., metal), and examples of such coatings include an aerosol lacquer, an automotive coating, an architectural coating, a clear coating, a coating for external environment, an industrial coating, or a combination thereof. An acrylic resin may be used to prepare a thermoplastic coating, a thermosetting coating, or a combination thereof. In certain aspects, an acrylic-coating is selected for use as a thermosetting coating, particularly in embodiments for use upon a metal surface. Acrylic resins generally are soluble in a solvent with a similar solubility parameter. Examples of solvents typically used to dissolve an acrylic resin include an aromatic hydrocarbon (e.g., toluene, a xylene); a ketone (e.g., methyl ethyl ketone), an ester, or a combination thereof.

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The thermoplastic and/or thermosetting properties of an acrylic resin are related to the monomers that are comprised in the selected resin. Examples of an acrylate ester monomer include a butylacrylate, an ethylacrylate (“EA”), ethylhexylacrylate (“EHA), or a combination thereof. Examples of a methacrylate ester monomer include a butylmethacrylate (BMA”), an ethylmethacrylate, a methylmethacrylate (“MMA), or a combination thereof. Standards for physical properties, chemical properties, and/or procedures for empirically determining the purity/properties of various acrylic monomers (e.g., acrylate esters, 2-ethylhexyl acrylate, n-butyl acrylate, ethyl acrylate, methacrylic acid, acrylic acid, methyl acrylate) are known to those of ordinary skill in the art (see, for example, “ASTM Book of Standards, Volume 06.04, Paint Solvents; Aromatic Hydrocarbons, D3362-93, D3125-97, D4415-91, D3541-91, D3547-91, D3548-99, D3845-96, D4416-89, and D4709-02, 2002).

In alternative embodiments, an acrylic resin temporary coating (e.g, a non-film forming coating) may be produced, for example, by selection of an acrylic resin that that comprises fewer or no crosslinkable moieties, selection of an addition binder that comprises fewer or סח crosslinkable moieties, reducing the concentration of the an acrylic resin and/or additional binder, using a bake cured an acrylic resin coating at temperatures less than is needed for curing (e.g., ambient conditions), selection of size range for a thermoplastic acrylic resin coating that is less suitable for film formation (e.g., 1 kDa t075 kDa), selection of a thermoplastic acrylic resin with T_athat is lower than the temperature ranges herein and/or 20°C lower than the temperature range of use, or a combination thereof.

(1) Thermoplastic Acrylic Resins

A strait acrylic resin (strait acrylic polymer,’’ strait acrylic binder) is a homopolymer or copolymer comprising an acrylate ester monomer and/or a methacrylate ester monomer. A strait acrylic resin may be used to formulate a thermoplastic coating, as cross-linking reactions are absent or limited without additional reactive moieties in the monomers. Generally, a thermoplastic film produced from an acrylic resin-coating will possess a lower elongation, an increased hardness, an increased tensile strength, greater UV resistance (e.g., chalk resistance), color retention, a greater T_g, or a combination thereof, with increasing methacrylate ester monomer content in the acrylic resin. However, the ester of a monomer may comprise various alcohol moieties, and an alcohol moiety of larger size generally reduces the T_a. Examples a T_gvalue for a homopolymer strait acrylic resins with the include -100°C, poly(octadecyl methacrylate); -72^DC, poly(tetradecyl methacrylate); -65^DC, poly(laury) methacrylate); -60°C, poly(heptyl acrylate); -60°C, poly(n-decyl methacrylate); -55°C, poly(n-butyl acrylate); -50°C, poly(2-ethoxyethyl acrylate); -50°C, poly(2-ethylbutyl acrylate); -50°C, poly(2-ethylhexyl acrylate); -45°C, poly(propyl acrylate); -43°C, poly(isobutyl acrylate); -38°C, poly(2-heptyl acrylate); -24°C,

48sssss poly(ethyl acrylate); -20°C, poly(n-octyl methacrylate); -20°C, poly(sec-butyl acrylate); *20°C, poly(ethylthioethyl methacrylate); -10°C, poly(2-ethylhexyl methacrylate); -5°C, poly(n-hexyl methacrylate); -3°C, poly(isopropyl acrylate); 6°C, poiy(methyl acrylate); 11°C, poly(2-ethylbutyl methacrylate); 16°C, poly(cyclohexyl acrylate); 20°C, poly(n-butyl methacrylate); 35°C, poly(hexadecyl acrylate); 35°C, poly(n-propyl methacrylate); 43°C, poly(t-butyl acrylate); 53°C, poly(isobutyl methacrylate); 54°C, poly(benzyl methacrylate); 60 C, poly(sec-butyl methacrylate), 65°C, poly(ethyl methacrylate); 79°C, poly(3,3,5-trimethylcyclohexylmethacryiate); 81°C, poly(isopropyt methacrylate); 94°C, poly(isobornyl acrylate); 104°C, poly(cyclohexyl methacrylate); 105°C, poly(methyl methacrylate); 107°C, poly(t-butyl methacrylate); and 110°C, poly(phenyl methacrylate). Additionally, an estimated T_B of a copolymer comprising one or more monomers of an acrylate and/or methyacrylate monomer can be made by using the following equation: 1/T_a=W_l/T_a1 + W₂/T_a2, wherein W₁ and W₂ are the are the molecular weight ratios of the first and second monomer, respectively; and wherein T_B1 and T_g2 are glass transition temperatures of the first and second monomer, respectively (Fox, T. G., 1956). For many embodiments (e.g., solvent-borne coatings), it is contemplated that a T_B of 40°C to 60°C, including all intermediate ranges and combinations thereof, will be suitable.

The thermoplastic properties of an acrylic resin are also related to the molecular mass of the selected resin. Increasing the polymer size of an acrylic resin promotes physical polymer entanglement during film formation. Typically, a thermoplastic film produced from an acryliccoating will possess a lower flexibility, an increased exterior durability, an increased hardness, an increased solvent resistance, an increased tensile strength, a greater T_g, or a combination thereof, with increasing polymer size of the acrylic resin. However, increasing polymer size of an acrylic resin generally increases viscosity of a solution comprising a dissolved acrylic resin, which may make application to a surface more difficult, such as cobwebbing of coating during spray application and the changes of film properties generally will reach a plateau at 100 kDa. In most embodiments, it is contemplated that an acrylic resin will range in mass from 75 kDa to100 kDa, including all intermediate ranges and combinations thereof.

Examples of such a thermoplastic acrylic-coating include a lacquer. In specific facets, the lacquer possesses a good, high, or spectacular gloss. In specific aspects, such a thermoplastic acrylic-coating further comprises a pigment, in specific aspects, a wetting agent is less preferred in a coating comprising an acrylic resin and a pigment, due to the ease of dispersion of a pigment with an acrylic resin. In certain aspects, a thermoplastic acrylic-coating may be selected to coat a metal surface, a plastic surface, or a combination thereof. However, in particular aspects, a thermoplastic acrylic coating is an automotive coating. Such an automotive coating may comprise an acrylic binder with a high temperature T_B to produce a film of sufficient

48ttttt durability (e.g., hardness) for external use and contact with heated surfaces. In certain aspects, a thermoplastic acrylic coating comprises a binder with a T_H to 90°C to 110°C, including all intermediate ranges and combinations thereof. In additional aspects, an automotive coating comprises a plasticizer, a metallic pigment, or a combination thereof. In specific aspects, a binder for an automotive coating comprises a methylmethacrylate ester monomer. In specific facets, an automotive coating comprises poly(methyl methacrylate).

(2) Water-Borne Thermoplastic Acrylic Coatings

The thermoplastic acrylic coatings described above are solvent-borne coatings. In other embodiments, a thermoplastic acrylic resin may be a waterborne coating. A water-borne acrylic (acrylic latex) typically is an emulsion, wherein the acrylic binder is dispersed in the liquid component. In general embodiments, an emulsifier (e.g., a surfactant) promotes dispersion. In certain embodiments, an acrylic latex coating comprises 0% to 20% coalescent per weight of binder. In most embodiments, it is contemplated that a water-borne acrylic resin will range in mass from 100 kDa to1000 kDa, including all intermediate ranges and combinations thereof. In certain embodiments, a water-borne acrylic coating comprises an associative thickener (rheology modifier”), which may enhance flow, brushability, splatter resistance, film build, or a combination thereof. A water-borne acrylic may be selected as an architectural coating. An associative thickener forms a network with acrylic resin latex particles by hydrophobic interactions. Hydroxyethyl cellulose (HEC) changes the coating rheology by promoting flocculation, which tends to reduce gloss, flow, or a combination thereof. Selection of an acrylic resin with smaller size, greater hydrophobicity, or a combination thereof, and an associative thickener may produce higher gloss, better flow, lower roller splatter, or a combination thereof.

(I) Architectural Coatings

A flat interior coating typically comprises a vinyl acetate and a lesser amount of acrylate (e.g., butyl acrylate) monomers, which generally produces a film with suitable scrub resistance. A copolymer of acrylate and methacrylate may be selected for a semigioss or gloss coating, in certain embodiments, the acrylate resin has a T_g to 20°C to 50°C, including all intermediate ranges and combinations thereof. In some aspects, such a coating generally possesses good block resistance good print resistance, or a combination thereof. An acrylic resin that comprises a monomer that comprises a ureide moiety may be selected for enhanced film adhesion (e.g., to a coated surface), blistering resistance, or a combination thereof. An acrylic resin that comprises a styrene monomer may be selected for enhanced film water resistance.

An exterior latex coating typically produces a film with greater flexibility than an interior latex due to temperature changes and/or dimensional movement of a substrate (e.g., wood). In

48uuuuu certain embodiments, the acrylic resin has a T_g to 10°C to 35°C, including all intermediate ranges and combinations thereof. The selection of a T_g may be influences by the selection of the amount particulate material (e.g., pigment) in the coating to achieve a particular visual appearance. For example, a higher the pigment volume content (“PVC) that is typically selected to reduce gloss. However, to retain properties such as flexibility, a binder with a lower a T_g may be selected for combination with the higher PVC. For example, flat exterior latex a coating generally possesses a pigment volume content of 40% to 60% and a T_g of 10°C to 15°C including all intermediate ranges and combinations thereof, respectively. In another example, a semigloss or gloss exterior latex binder of a coating generally possesses a T_g of 20°C to 35°C, including all intermediate ranges and combinations thereof, respectively, in other embodiments^ the exterior latex binder particle size is selected to be relatively small such as 90 nm to 110 nm,' including all intermediate ranges and combinations thereof, in certain facets, a smaller latex particle size promotes adhesion of the coating and/or film, particularly to a surface that comprises a degraded (e.g., chalking) film. In certain other embodiments, a larger latex particle size may be selected to increase the coating and/or film’s build (e.g., thickness). In certain aspects, a larger latex particle size ranges from, for example 325 nm to 375 nm, including all intermediate ranges and combinations thereof.

(ii) Industrial Coatings

A water-borne thermoplastic acrylic latex industrial coating typically comprises a binder with a T_g of 30°C to 70°C, including all intermediate ranges and combinations thereof. Such a coating typically is applied to a metal surface, and thus often further comprises a surfactant, an additive, or a combination thereof to improve an anti-corrosion property. In specific aspects, the industrial coating comprises an anti-corrosion pigments, anti-corrosion pigment enhancers, or a combination thereof. In contrast, a water-borne acrylic latex industrial maintenance coating typically is similar to an exterior fiat architectural coating in selection of binders, though they preferably comprise anti-corrosion pigments, anti-corrosion pigment enhancers, and other anticorrosion components for use on a metal surface.

(3) Thermosetting Acrylic Resins

Unless otherwise noted, the following thermosetting acrylic resins and/or coatings are preferably solvent-borne coatings. In certain embodiments an acrylic coating comprises a thermosetting acrylic resin. A thermosetting acrylic coating typically possesses superior hardness, superior toughness, superior temperature resistance, superior resistance to a solvent, superior resistance to a stain, superior resistance to a detergent, higher application of solids, relative to a thermoplastic acrylic coating. The average size of a thermosetting acrylic resin is typically less than a thermoplastic acrylic resin, which promotes a relatively lower viscosity and/or

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17365S. 1 higher application of solids in a solution comprising a thermosetting acrylic resin. In certain embodiments, a thermosetting acrylic resin is from 10 kDa to 50 kDa, including all intermediate ranges and combinations thereof.

A thermosetting acrylic resin comprises a moiety capable of undergoing a cross-linking reaction. A monomer may comprise the moiety, and be incorporated into the polymer structure of an acrylic resin during resin synthesis (e.g., a styrene, a vinyltoluene), and/or the acrylic resin may be chemically modified after polymerization to comprise a chemical moiety. In additional embodiments, an acrylic resin may be selected to comprise chemical moieties, such as an amine, a carboxyl, an epoxy, a hydroxyl, an isocyanate, or a combination thereof, to confer a desirable property to the acrylic resin produced. Examples of such properties include the acrylic resin's chemical reactivity (e.g., crosslinkabiiity), acidity, alkalinity, hydrophobicity, hydrophilicity, glass transition temperature, or a combination thereof. In general embodiments, an acrylic resin comprising a carboxyl moiety, a hydroxyl moiety, or a combination thereof, promotes a crosslinking reaction with another binder. In other embodiments, an acrylic resin may be chemically modified to comprise a methylol and/or methylol ether group, which is a resin capable of self-crosslinking.

(I) Acrylic-Epoxy Combinations

In certain embodiments, a thermosetting acrylic resin may be combined with an epoxide resin, tn general embodiments, an acrylic resin comprising a carboxyl moiety may be selected for cross-linking with an epoxy resin. In specific aspects, an acrylic resin comprises 5% to 20% including all intermediate ranges and combinations thereof, of a monomer that comprises a carboxyl moiety, such as of an acrylic acid monomer, a methacrylic acid monomer, or a combination thereof. The carboxyl moiety may undergo a cross-linking reaction with an epoxide resin (e.g., a bisphenol A/epichlorohydrin epoxide resin) during film formation. In certain aspects, an epoxide resin cross-linked with an acrylic resin generally produces a film with good hardness, good alkali resistance, greater solvent resistance to a film, poorer UV resistance, or a combination thereof.

A thermosetting acrylic-epoxy coating may be selected for application to a metal surface. Examples of surfaces that an acrylic-epoxy coating is selected for use include an indoor surface, an indoor metal surface (e.g., an appliance), or a combination thereof. In certain aspects, an epoxide resin cross-linked with an acrylic resin generally produces a film with good hardness, good alkali resistance, greater solvent resistance to a film, poorer UV resistance, or a combination thereof, in some facets, an acrylic resin may be combined with an aliphatic epoxide resin to produce a film with relatively superior UV resistance than a bisphenol A/epichlorohydrin

8 www ww based epoxide resin. In another facet, an acrylic resin polymerized with an allyl glycidyl ether monomer, a glycidyl acrylate monomer, a glycidyl methacrylate monomer, or a combination thereof, may undergo a cross-linking reaction with an epoxide resin during film formation. In specific facets, a film produced from cross-linking an epoxide other than a bisphenol A/epichlorohydrin epoxide resin and an acrylic resin comprising an allyl glycidyl ether monomer, a glycidyl acrylate monomer, a glycidyl methacrylate monomer, or a combination thereof possesses a relatively superior UV resistance.

In certain embodiments, an acrylic epoxy coating comprises a catalyst to promote crosslinking during film formation. In specific aspects, the catalyst is a base such as a dodecyl trimethyl ammonium chloride, a tri(dimethylaminomethyl) phenol, a melamine-formaldehyde resin, or a combination thereof. In other embodiments, an acrylic epoxy coating is cured by baking at 150°C to 190°C, including all intermediate ranges and combinations thereof. In particular aspects, film formation time of an acrylic epoxy coating is from 15 minutes to 30 minutes, including all intermediate ranges and combinations thereof. In certain embodiments, a thermosetting coating comprises an acrylic epoxide melamine-formaldehyde coating, wherein an acrylic resin, an epoxide resin and a melamine-formaldehyde resin undergo cross-linking during film formation.

(il) Acryllc-Amlno Combinations

In other embodiments, a thermosetting acrylic resin may be combined with an amino resin. In general embodiments, an acrylic resin comprising an acid (e.g., carboxyl) moiety, a hydroxyl moiety, or a combination thereof, may be selected for cross-linking with an amino resin. An acrylic amino coating, wherein the acrylic resin comprises an acid moiety, may be cured by baking at, for example 150°C for 30 minutes. However, an acid moiety acrylic ammo coating is typically undergoes a greater degree of reactions between amino resins, which reduces properties such as toughness. In specific aspects, an acrylic resin comprises a monomer that comprises a hydroxyl moiety such as a hydroxyethyl acrylate (HEA), a hydroxyethyl methacrylate (“HEMA), or a combination thereof. An acrylic amino coating, wherein the acrylic resin comprises a hydroxyl moiety, typically comprises an acid catalyst to promote curing by baking at, for example 125°C for 30 minutes. An acrylic amino coating, wherein the amino resin was prepared from urea, generally produces a film with lower gloss, less chemical resistance, or a combination thereof, than an amino resin prepared from another nitrogen compound. Selection of a melamine and/or benzoguanamine based amino coating generally produces a film with excellent weathering resistance, excellent solvent resistance, good hardness, good mar resistance, or a combination thereof, and such an acrylic amino coating may be selected for an automotive topcoat.

48xxxxx (iii) Acryltc-Urethane Combinations

In other embodiments, a thermosetting acrylic resin may be combined with an urethane resin. In general embodiments, an acrylic resin comprising an acid moiety, a hydroxyl moiety, or a combination thereof, may be selected for crosslinking with an urethane resin. In specific embodiments, an acrylic resin comprises a hydroxyl moiety, such as, for example, a moiety provided by a HEA monomer, a HEMA monomer, or a combination thereof. Selection of an aliphatic isocyanate urethane (e.g., hexamethylene diisocyanate based) generally produces a film with superior color, weathering, or a combination thereof relative to other urethanes. An acrylic urethane coating may comprise a catalyst, such as, for example, triethylene diamine, zinc naphthenate, dibutyl tin-di-laurate, or a combination thereof. An acrylic urethane coating cures at ambient conditions. However, an acrylic urethane coating typically is a two-pack coating to separate the reactive binders until application. An acrylic urethane coating generally produces a film with good weathering, good hardness, good toughness, good chemical resistance, or a combination thereof. An acrylic urethane coating may be selected an aircraft coating, an automotive coating, an industrial coating (e.g., an industrial maintenance coating), or a combination thereof.

(iv) Water-Borne Thermosetting Acrylics

In other embodiments, a thermosetting acrylic coating may be a waterborne coating (e.g., a latex coating). Typically, such a thermosetting acrylic coating comprises an acrylic resin with a hydroxyl moiety, an acid moiety, or a combination thereof. An acrylic resin may further comprise an additional monomer such as a styrene, a vinyltoluene, or a combination thereof. The acrylic resin typically is combined in a coating with an amino resin, an epoxy resin, or a combination thereof as previously described. A film produced from a water-borne thermosetting acrylic coating is similar in properties as a solvent-borne counterpart. Such a coating may be selected for surfaces such as masonry, wood, metal, or a combination thereof.

k. Polyvinyl binders

A polyvinyl binder (polyvinyl, vinyl binder, “vinyl) is a binder comprising a polymer of a vinyl chloride monomer, a vinyl acetate monomer, or combination thereof. A solvent-borne polyvinyl coating may comprise a ketone, ester, chlorinated hydrocarbon, nitroparaffin, or a combination thereof, as a solvent. A solvent-borne polyvinyl coating may comprise a hydrocarbon (e.g., aromatic, aliphatic) as a diluent. A polyvinyl binder is generally insoluble in an alcohol, however, in embodiments wherein a solvent-borne polyvinyl coating that comprises an additional alcohol soluble binder, alcohol may comprise 0% to 20% of the liquid component. In embodiments wherein solvent-borne polyvinyi coating is cured by baking, a glycol ether and/or

48yyyyy glycol ester may be used in the liquid component to enhance a rheological property. In other embodiments, the liquid component of a polyvinyl coating may comprise a plasticizer (e.g., a phthalate, a phosphate, a glycol ester), wherein the plasticizer if 1 to 25 parts per hundred parts polyvinyl binder, including all intermediate ranges and combinations thereof, for a non-plastisol or non-organosol coating. A polyvinyl-coating may be used to prepare a thermoplastic coating, a thermosetting coating, or a combination thereof. In specific aspects, a thermoplastic polyvinyl binder coating possesses a T_B of 50°C to 85°C, including all intermediate ranges and combinations thereof. However, in some aspects, a polyvinyl-coating/film possesses moderate resistance to heat, UV irradiation, or a combination thereof. In specific aspects, a polyvinylcoating comprises a light stabilizer, a pigment, or a combination thereof. In particular facets, the light stabilizer, the pigment (e.g., titanium dioxide), or the combination thereof, improves the polyvinyl-coating and/or film's resistance to heat, UV irradiation, or a combination thereof.

In embodiments wherein a polyvinyl coating comprises a solvent-borne coating, it is contemplated that a polyvinyl resin will range in mass from 2 kDa to 45 kDa, including all intermediate ranges and combinations thereof. A typical solvent-borne polyvinyl coating comprises a polyvinyl resin, a liquid component wherein the liquid component comprises a solvent, and a plasticizer. A solvent-borne polyvinyl coating may additionally comprise a colorizing agent (e.g., a pigment), a light stabilizer, an additional binder, a cross-linker, or a combination thereof.

A polyvinyl binder typically possesses excellent adhesion for a plastic surface, an acrylic and/or acrylic coated surface, paper, or a combination thereof. A thermoplastic polyvinyl coating may be selected as a lacquer, a topcoat of a can coating (e.g., can interior surface), or a combination thereof. In some embodiments, an polyvinyl-coating may be selected to produce a film with such properties, for example, as excellent water resistance, excellent resistance to various solvents (e.g., an aliphatic hydrocarbon, an alcohol, an oil), excellent resistance to acid pH, excellent resistance to basic pH, inertness relative to food, or a combination thereof.

In many aspects, a polyvinyl resin is a copolymer that comprises a combination of a vinyl chloride monomer and vinyl acetate monomer. Often during resin synthesis (e.g., polymerization), a polyvinyl resin is prepared to further comprise monomers with specific chemical moieties to confer a property such as solubility in water, solubility in a solvent, compatibility with another coating component (e.g., a binder), or a combination thereof. In certain embodiments, a polyvinyl resin comprises a monomer comprising carboxyl moiety, a hydroxyl moiety (e.g., a hydroxyalkyl acrylate monomer), a monomer comprising an epoxy moiety, a monomer comprising a maleic acid, or a combination thereof. A carboxyl moiety may confer an

48zzzzz increased adhesion property (e.g., excellent adhesion to metal). However, a polyvinyl resin comprising a carboxyl moiety is generally not compatible with a basic pigment. A thermosetting polyvinyl coating comprising a polyvinyl binder that comprises a carboxyl moiety and a polyvinyl binder that comprises an epoxy moiety generally possesses one or more excellent physical properties (e.g., flexibility), and may be selected as a coil coating. A hydroxyl moiety may confer cross-iinkabiiity, compatibility with another coating component, an increased adhesion property (e.g. good adhesion to aluminum), or a combination thereof. Additionally, after polymer synthesis, a polyvinyl resin can be chemically modified to comprise such a specific chemical moiety. In some embodiments, a polyvinyl resin is chemically modified to comprise a secondary hydroxyl moiety, an epoxy moiety, a carboxyl moiety, or a combination thereof. A polyvinyl resin comprising a secondary hydroxyl moiety may be combined with another binder such as an alkyd, an urethane, an amino-formaldehyde, or a combination thereof. A thermosetting polyvinyl aminoformaldehyde coating comprising a polyvinyl binder that comprises a hydroxyl moiety generally possesses good corrosion resistance, water resistance, solvent resistance, chemical resistance, and may be selected as a can coating, a coating for an interior wood surface, or a combination thereof. Standards for physical properties, chemical properties, and/or procedures for testing the purity/properties of various polyvinyl monomers (e.g., vinyl acetate) and polyvinyl resins (e.g., polymer components, polymer mass, shear viscosity for a higher mass resin, chlorine content) are described, for example, in ASTM Book of Standards, Volume 06.04, Paint - Solvents; Aromatic Hydrocarbons,” D2190-97, D2086-02, D2191-97, and D2193-97, 2002; “ASTM Book of Standards, Volume 06.03, Paint - Pigments, Drying Oils, Polymers, Resins, Naval Stores, Cellulosic Esters, and Ink Vehicles, D4368-89, D3680-89, and D1396-92, 2002; and in “ASTM Book of Standards, Volume 06.01, Paint - Tests for Chemical, Physical, and Optical Properties;

Appearance, D2621-87, 2002.

In alternative embodiments, a polyvinyl resin temporary coating (e.g, a non-film forming coating) may be produced, for example, by selection of a polyvinyl resin that that comprises fewer or no crosslinkable moieties, selection of an addition binder that comprises fewer or no crosslinkable moieties, reducing the concentration of the a polyvinyl resin and/or additional binder, using a bake cured a polyvinyl resin coating at temperatures less than is needed for curing (e.g., ambient conditions), selection of size range for a plastisol or organisol polyvinyl resin coating that is less suitable for film formation (e.g1 ״ kDa to 60 kDa), selection of a polyvinyl resin with T_g that is lower than the temperature ranges herein and/or 20°C lower than the temperature range of use, or a combination thereof.

48aaaaa a

(1) Plastisols and Organlsols

A polyvinyl resin of 60 kDa to 110 kDa, Including all intermediate ranges and combinations thereof, may be selected for use as an organosol or a plastisol. A plastisol is coating comprising a vinyl homopolymer binder and a liquid component, wherein the liquid component comprises a plasticizer comprising a minimum of 55 parts or more of plasticizer per hundred parts of homopolymer binder in the coating. In certain embodiments, a plastisol comprises, by weight, 0% to 10% including all intermediate ranges and combinations thereof, of a thinner (e.g., an aliphatic hydrocarbon). A plastisol coating typically comprises an additional vinyl binder. A plastisol may comprise a pigment, however, a low oil absorption pigment is preferred to avoid undesirable increase in coating viscosity given the liquid component used for a plastisol.

An organosol is similar to a plastisol, except the less than 55 parts of plasticizer per hundred parts of homopolymer binder is used in the coating. In typical embodiments, the liquid component of comprises a weak solvent that may act as a dispersant and a thinner (e.g., a hydrocarbon). In typical aspects, the reduced content of plasticizer produced a film with a superior hardness property relative to a plastisol. In additional embodiments, the nonvolatile component of an organisol is 50% to 55%, including all intermediate ranges and combinations thereof. An organosol coating typically comprises a second binder. In specific aspects, the second binder is a vinyl copolymer, an acrylic, or a combination thereof. In certain aspects, the second binder comprises a carboxyl moiety, a hydroxyl moiety, or a combination thereof. In further aspects, an organisol may comprise a third binder. In specific facets, the third binder comprises an amino resin, a phenolic resin prepared from formaldehyde, or a combination thereof. In additional facets, a second binder that comprises a hydroxyl moiety may undergo a thermosetting cross-linking reaction with a third binder. An organisol may comprise a pigment suitable for general polyvinyl coatings.

A plastisol or organisol typically is cured by baking. In general embodiments, baking is at a temperature of 175°C to 180°C, including all intermediate ranges and combinations thereof. In general embodiments, a plastisol or organisol comprises a heat stabilizer. The heat stabilizer may protect a vinyl binder during baking. Examples of a suitable heat stabilizer include a combination of a metal salt of an organic acid and an epoxidized oil or a liquid epoxide binder. However, in an embodiment wherein the plastisol or organisol comprises a binder that comprises an carboxyl moiety, a metal salt is less preferred due to possible gellation of the coating, and may be substituted with a merapto tin and/or tin ester compound.

In embodiments wherein a plastisol or organisol comprise a binder with good adhesion properties for a surface such as a binder comprising carboxy moiety, the plastisol or organisol

48bbbbb b may be used as a single layer coating. For example, such an organisol may be selected to coat the end of a can. However, a plastisol or organisol typically is part of a multicoat system that comprises a primer to promote adhesion. In specific aspects, the primer comprises a vinyl resin comprising a carboxy moiety. In specific facets, the primer further comprises a thermosetting binder such as an amino-formaldehyde, phenolic, or a combination thereof, to enhance solvent resistance. In certain facets, it is preferred that a primer or other coat layer of a multicoat system possesses good solvent resistance to the plasticizers of the organosol and/or plastisol coat layer.

(2) Powder Coatings

A polyvinyl binder may be selected as a powder coating. Typically, coating components such as a polyvinyl binder and a plasticizer, colorizing agent, additive, or a combination thereof, admixed to prepare a powder coating. Such a powder coating is usually applied by a fluidized bed applicator, a spray applicator, or a combination thereof. In some aspects, the coating components are melted then ground into a powder. Such a powder coating is usually applied by an electrostatic spray applicator. The coating is cured by baking. A polyvinyl powder coating may be selected to coat a metal surface.

(3) Water-Borne Coatings

The previous discussions of polyvinyl coatings focused upon sotvent-borne and powder coatings. A polyvinyl binder with a T_g of 75°C to 85^DC, including all intermediate ranges and combinations thereof, may be selected for use in a dispersion waterborne coating. The liquid component may comprise a cosolvent such as a glycol ether, a plasticizer, or a combination thereof. Examples of a cosolvent include ethylene glycol monobutyl ether. The dispersion waterborne polyvinyl coating may be used as described for a solvent-borne polyvinyl coating. In another example, an organisol may be prepared with a plasticizer as a latex coating. Such a latex is suitable for selection as a primer coating. The latex coating is cured by baking.

I. Rubber Resins

In certain embodiments, a coating may comprise a rubber resin as a binder. A rubber may be either obtained from a biological source (natural rubber), synthesized from petroleum (synthetic rubber), or a combination thereof. Examples of synthetic rubber include polymers of styrene monomers, butadiene monomers, or a combination thereof. In alternative embodiments, a rubber temporary coating (e.g, a non-film forming coating) may be produced, for example, by selection of rubber resin that that comprises fewer or no crosslinkable moieties, selection of an addition binder that comprises fewer or no crosslinkable moieties, reducing the concentration of the a rubber resin and/or additional binder, or a combination thereof.

48ccccc (1) Chlorinated Rubber Resins

In general embodiments, a rubber resin comprises a chlorinated rubber resin, wherein a rubber isolated from a biological source has been chemically modified by reaction with chlorine to produce a resin comprising 65% to 68% chlorine by weight, including all intermediate ranges and combinations thereof. A chlorinated rubber resins generally are in a molecular weight range of

3.5 kDa to 20 kDa, including all intermediate ranges and combinations thereof. A chlorinated rubber coating may comprise another binder, such as, for example, an acrylic resin, an alkyd resin, a bituminous resin, or a combination thereof. In specific aspects, a chlorinated rubber resin comprises 10% to 50%, by weight, including all intermediate ranges and combinations thereof, of the binder when in combination with an acrylic resin, an alkyd resin, or a combination thereof. In general embodiments, a chlorinated rubber coating is a solvent-borne coating. In certain aspects, a chlorinated rubber coating comprises a liquid component, such as, for example, a solvent, a diluent, a thinner, a plasticizer, or a combination thereof. A chlorinated rubber coating may be a thermoplastic coating. To reduce the T_B of a film produced from a chlorinated rubber resin, the liquid component generally comprises a plasticizer. In certain aspects, a chlorinated rubber coating comprises 30% to 40%, by weight, including all intermediate ranges and combinations thereof, of plasticizer. In certain facets, a plasticizer is selected for water resistance (e.g., hydrolysis resistance) such as a bisphenoxyethylformal. In certain facets, a chlorinated rubber coating comprises light stabilizer, an epoxy resin, an epoxy plasticizer (e.g., epoxidized soybean oil), or combination thereof, to chemically stabilize a chlorinated resin, coating and/or film. In other embodiments, a chlorinated rubber coating comprises a pigment, an extender, or a combination thereof. In particular aspects, the pigment is a corrosion resistant pigment. A chlorinated rubber film are generally has good chemical resistance (e.g., acid resistance, alkali resistance), water resistance, Or a combination thereof. Coatings comprising chlorinated rubber resins may be used, for example, on surfaces that contact gaseous, liquid and/or solid external environments. Examples of such uses include a coating for an architectural coating (e.g., a masonry coating), a traffic marker coating, a marine coating (e.g., a marine vehicle, a swimming pool), a metal primer, a metal topcoat, or a combination thereof.

(2) Synthetic Rubber Resins

Examples of synthetic rubber include polymers comprising a styrene monomer, a methylstyrene (e.g., a-methylstyrene) monomer, or a combination thereof. A polystyrene and/or polymethylstyrene coating may be a solvent-borne coating. Examples of a solvent include an aliphatic hydrocarbon, an aromatic hydrocarbon, a ketone, an ester, or a combination thereof. A polystyrene and/or polymethylstyrene coating may possess good water resistance, good chemical resistance, or a combination thereof. A polystyrene and/or polymethylstyrene coating may be selected as a primer, a lacquer, a masonry coating, or a combination thereof. A

48ddddd d polystyrene homopolymer has a T_g of 100°C, and in certain embodiments, a polystyrene coating is bake cured. Standards for physical properties, chemical properties, and/or procedures for testing the purity/properties of a styrene monomer, a methylstyrene monomer, (e.g., amethylstyrene), a resin comprising a styrene and/or methylstyrene monomer, are described, for example, in ASTM Book of Standards, Volume 06.04, Paint - Solvents; Aromatic Hydrocarbons, D2827-00, D6367-99, D6144-97, D4590-00, D2119-96, D2121-00, and D234096, 2002.

Similar to the variability of T_g previously described for a thermoplastic acrylic resin, a styrene copolymer with a lower a T_B than polystyrene or other altered properties can be produced from polymerization with other monomers such as a butadiene monomer, an acrylic monomer, a maleate ester, an acrylonitrile, an allyl alcohol, a vinyltoluene, or a combination thereof. For example, a butadiene monomer decreases lightfastness, but confers setf-crossiinkability to the resin. In another example, an acrylic resin increases the resin's solubility in an alcohol. In a further example, an allyl alcohol monomer confers crosslinkability in combination with a polyol. In certain embodiments, a styrene-butadiene copolymer resin may be selected. In certain aspects, a styrene-butadiene resin comprises a carboxyl moiety to improve an adhesion property, dispersibility in a liquid component, or a combination thereof. In particular facets, a styrenebutadiene coating comprises an emulsifier to increase dispersion in a liquid component, a light stabilizer, or a combination thereof. A styrene-butadiene coating may be a thermosetting coating, due to oxidative crosslinking of a butadiene double bond moiety. However, styrenebutadiene film may have poor chalking resistance, poor color stability, poor UV resistance, or a combination thereof. A styrene-butadiene coating may be selected as a corrosion resistant primer, a wood primer, or a combination thereof. A styrene-vinnyltoluene-acrylate copolymer coating may be selected for an exterior coating, a traffic marker paint, a metal coating (e.g., a metal lacquer), a masonry coating, or a combination thereof.

m. Bituminous Binders

A bituminous binder (bituminous”) is a binder comprising a hydrocarbon soluble in carbon disulfide, is black or dark colored, and is obtained from a bitumen deposit and/or as a product of petroleum processing. A bituminous binder typically is used in asphalt, tar, and other construction materials. However, in certain embodiments, a bituminous binder may be used in a coating of the present invention, particularly in embodiments wherein good resistance to a chemical such as a petroleum based solvent, an oil, water, or a combination thereof, is desired. Examples of a bituminous binder include a coal tar, a petroleum asphalt, a pitch, an asphaltite, or a combination thereof. In certain embodiments, a coal tar and/or pitch is combined with an epoxy resin to form a thermosetting coating. Such as coating may be selected as a pipeline coating. In

48eeeee e other embodiments, an asphaltite and/or petroleum asphalt may be selected for use as an automotive coating (e.g., an underbody part coating). An asphaltite and/or petroleum asphalt coating may further comprise an additional binder such as an epoxy. In certain aspects, an asphaltite and/or petroleum asphalt coating is a solvent-borne coating. In specific aspects, an asphaltite and/or petroleum asphalt coating comprises a plasticizer. In further aspects, an asphaltite and/or petroleum asphalt coating comprises a wax to increase abrasion resistance.

In further embodiments, bituminous coating may be selected as a roof coating. Typically, a bituminous roof coating comprises an extender, a thixotrope, or a combination thereof. Examples of a thixotrope additive include asbestos, a silicon extender, a celluosic, a glass fiber, or a combination thereof. In some aspects, a bituminous roof coating comprises a solvent-borne coating or a water-borne coating. Examples of solvents that may be selected include a mineral spirit, an aliphatic hydrocarbon (e.g., a naphtha, a mineral spirit), an aromatic solvent (e.g., xylene, toluene) or a combination thereof. A bituminous roof coating may be selected as a primer, a topcoat, or a combination thereof. A bituminous roof topcoat typically further comprises a metallic pigment.

In certain aspects, a solvent-borne or water-borne bituminous coating is an emulsion comprising water and a bituminous binder. In specific facets, the emulsion further comprises a solvent, an extender (e.g., a silica), an emusifier (e.g., a surfactant), or a combination thereof. The extender typically functions to stabilize the emulsion. In particular facets, the emulsion bituminous coating is a roof coating, a road coating, a sealer, a primer, a topcoat, or a combination thereof. In facets wherein an emulsion bituminous coating is selected as a sealer, an additional binder may be added to increase solvent resistance.

In alternative embodiments, a bituminous temporary coating (e.g, a ηοπ-film forming coating) may be produced, for example, by selection of an addition binder that comprises fewer or no crosslinkable moieties, reducing the concentration of the a bituminous resin and/or additional binder, or a combination thereof.

n. Polysulfide Binders

A polysulfide binder is a polymer produced from a reaction of sodium polysufide, bls(2chlorethyl)formal and 1,2,3-trichloropropane. Typically, a polysulfide binder is 1 kDa to 8 kDa, including all intermediate ranges and combinations thereof. A polysulfide binder comprises a thiol (“mercaptan”) moiety capable of crosslinking with an additional binder. A poiysulfide may undergo crosslinking by an oxidative reaction with an additional binder comprising a peroxide (e.g., dicumen hydroperoxide), a manganese dioxide, p-quinonedioxime, or a combination

48ffffff thereof. A polysulfide binder may be crosslinked with a glycidyl epoxide, though a tertiary amine is preferably part of the coating to promote this reaction. A polysuifide may undergo crosslinking with a binder comprising an isocyanate moiety, though it is preferred that the binder comprise a plurality of isocyanates. A polysulfide film typically possesses excellent UV resistance, good general weatherabiiity properties, good chemical resistance, or a combination thereof.

In alternative embodiments, a polysuifide temporary coating (e.g, a non-film forming coating) may be produced, for example, by selection of an addition binder that comprises fewer or no crosslinkable moieties, reducing the concentration of the a bituminous resin and/or additional binder, or a combination thereof.

o. Silicone Binders

The previous described binders are molecules based on carbon, and are considered herein as “organic binders. A silicone binder (“silicone) is a binder molecule based on silicone. Examples of a silicone binder include a polydimethyllsiloxane and a methyltriacetoxy silane, a methyltrimethoxysilane, a methyltricyclorhexylaminosilane, a fluorosilicone, a trifluoropropyl methyl polysiloxane, or a combination thereof. In general embodiments, a silicone binder comprises a crossreactive silicon moiety, examples of which are described below. A silicone coating may be selected for excellent resistance to irradiation (e.g., UV, infrared, gamma), excellent weatherability, excellent biodegradation resistance, flame resistance, excellent dielectric property, which is poor electrical conductivity with little detrimental effect on an electrostatic field, or a combination thereof. In specific aspects, a silicon coating is an industrial coating. In particular facets, a silicon coating is applied to an appliance part, a furnace part, a jet engine part, an incinerator part, or a missile part. In other embodiments, a silicon coating comprises an organic binder. In particular aspects, a silicon organic binder coating possesses superior heat resistance to an organic binder coating. In other aspects, the greater the silicon binder to organic binder ratio, the greater the crosslinking reactions, greater film hardness, reduced flexibility, or a combination thereof.

In general embodiments, a silicone coating is a thermosetting coating. Often, a silicon coating is a multi-pack coating due to a limited pot life one the coating components are admixed. The crosslinking reaction depends upon the binder's specific silicon moiety. A plurality of binders may be used, each comprising one or more crosslinking moleties. A binder comprising crosslinking SiOH and HOSi moieties generally comprises a cure agent such as a lead octoate, a zinc octoate, or a combination thereof. In general aspects, the thermosetting SiOH and HOSi silicon coating is bake cured (e.g., 250°C for one hour). A binder comprising crosslinking SiOH and HSi moieties typically comprises a tin catalyst. A binder comprising crosslinking SiOH and

48ggggg g

ROS( moieties, wherein RO is an alkoxy moiety, also typically comprises a tin catalyst. A coating prepared using S1OH and ROSi silicon binder typically further comprises an iron oxide, a glass microballon, or a combination thereof to improve heat resistance. This type of silicon may be selected for rocket and jet engine parts. A binder comprising crosslinking SiOH and CHsCdOSi moieties is moisture cured, and typically comprises a tin catalyst (e.g., an organotin compound). A binder comprising crosslinking SiOH and R₂NOSi moieties, wherein R₂NO is an oxime moiety, is also moisture cured, and typically comprises a tin catalyst. The moisture cured silicon coatings may be selected for one-pack silicon coatings, though film formation is generally slower than other types of silicon thermosetting coatings. A binder comprising crosslinking SiCH=CH2 and R₂NOSi moieties, wherein R₂NO is an oxime moiety, typically comprises a platinum catalyst, and may be bake cured. A film produced by a SiCH=CH_z and RjNOSi silicon coating possesses excellent toughness, flame resistance, or a combination thereof. Such a coating may be selected for a rocket part. However, coating components such as a rubber, a tin compound (e.g., an organotin), or a combination thereof, may inhibit platinum catalyzed film formation in this silicon coating.

In certain embodiments, a silicone coating is a solvent-bome coating. Examples of liquid components that may function as a silicon solvent include a chlorinated hydrocarbon (e.g., 1,1,1trichloroethane), an aromatic hydrocarbon (e.g., a VMP naphtha, xylene), an aliphatic hydrocarbon, or a combination thereof. A silicone binder typically is insoluble or poorly soluble in an oxigenated compound such as an alcohol, a ketone, or a combination thereof, of relatively low molecular weight (e.g., ethanol, isopropanol, acetone). However, a fluorosilicone, which is a silicone binder that comprises a fluoride moiety, may be combined with a liquid component comprising a ketone such as methyl ethyl ketone, methyl isobutyl ketone, or a combination thereof. A fluorosilicone binder may be selected for producing a film with excellent solvent resistance. A silicon coating often comprises a pigment. In specific embodiments, a pigment comprises zinc oxide, titanium dioxide, zinc orthotitanate, or a combination thereof, which may improve a film's resistance to extreme temperature variations, such as those of outerspace. In specific embodiments, a silicon coating may comprise a silica extender (e.g., fumed silica), which often increases durability.

In certain embodiments, a silicon binder comprises a trifluoropropyl methyl polysiloxane binder. In certain aspects, a trifluoropropyi methyl polysiloxane binder may be selected for producing a film with excellent resistance to petroleum products (e.g. automotive fuel, aircraft fuel), but poor resistance to an acid or an alkali, particularly at baking conditions.

48hhhhh h

In alternative embodiments, a silicon temporary coating (e.g, a non-film forming coating) may be produced, for example, by selection of an addition binder that comprises fewer or no crosslinkable moieties, reducing the concentration of the a silicon resin and/or additional binder, using a bake-cured silicon coating at non-baking conditions, inclusion of a rubber, a tin compound (e.g., an organotin), or a combination thereof.

2. Liquid Components

A liquid component is a chemical composition that is in a liquid state while comprised in a coating and/or film. A liquid component is typically added to a coating composition, for example, to improve a rheological property for ease of application, alter the period of time that thermoplastic film formation occurs, alter an optical property (e.g., color, gloss) of a film, alter a physical property of a coating (e.g., reduce flammability) and/or film (e.g., increase flexibility), or a combination thereof.

As would be known to those of ordinary skill in the art, often a liquid component comprises a volatile liquid that is partly or fully removed (e.g., evaporated) from the coating during film formation. Examples of a volatile liquid include a volatile organic compound ( VOC ), water, or a combination thereof. In many embodiments, 0% to 100%, including all intermediate ranges and combinations thereof, of the liquid component is lost during film formation. Various environmental laws and regulations have encouraged the reduction of volatile organic compound use in coatings [see Paint and Coating Testing Manual, Fourteenth Edition of the Gardner-Sward Handbook, (Koleske, J. V. Ed.), pp. 3-12, 1995], As a consequence, a coating may comprise a solvent-borne coating, which typically comprises a VOC and was the coating usually selected prior to enactment of the environmental laws, a high solids coating, which is generally a solventborne coating formulated with a minimum amount of a VOC, a water-borne coating, which comprises water and typically even less VOC, or a powder coating, which comprises little or no VOC.

In many embodiments, a liquid component may comprise a liquid composition classified based upon function such as a solvent, a thinner, a diluent, a plasticizer, or a combination thereof. A solvent is a liquid component used to dissolve one or more coating components. A thinner is a liquid component used to reduce the viscosity of a coating, and often additionally confers one or more properties to the coating, such as, for example, dissolving a coating component (e.g., a binder), wetting a colorizing agent, acting as an antisettling agent, stabilizing a coating in storage, acting as an antifoaming agent, or a combination thereof. A diluent is a liquid component that does not dissolve a binder.

48iiiiii

Liquid components can be classified, based on their chemical composition, as an organic compound, an Inorganic compound, or a combination thereof. Preferred organic compounds include a hydrocarbon, an oxygenated compound, a chlorinated hydrocarbon, a nitrated hydrocarbon, a miscellaneous organic liquid component, or a combination thereof. A hydrocarbon consists of or consists essentially of one or more carbon and/or hydrogen atoms. Examples of a hydrocarbon include an aliphatic hydrocarbon, an aromatic hydrocarbon, a naphthene, a terpene, or a combination thereof. An oxygenated compound comprises of one or more carbon, hydrogen and/or oxygen atoms. Examples of an oxygenated compound include an alcohol, an ether, an ester, a glycol ester, a ketone, or a combination thereof. A chlorinated hydrocarbon comprises one or more carbon, hydrogen and/or chlorine atoms, but does not comprise an oxygen atom. A nitrated hydrocarbon comprises one or more carbon, hydrogen and/or nitrogen atoms, but does not comprise an oxygen atom. A miscellaneous organic liquid component is a liquid other than a chlorinated hydrocarbon and/or a nitrated hydrocarbon that comprises one or more carbon, hydrogen and/or other atoms. In certain aspects, a miscellaneous organic liquid component does not comprise an oxygen atom. Preferred inorganic compounds include ammonia, hydrogen cyanide, hydrogen fluoride, hydrogen cyanide, sulfur dioxide, or a combination thereof. However, an inorganic compound generally is used at temperatures less than room temperature, and at pressures greater than atmospheric pressure.

In certain embodiments, a liquid component may comprise an azeotrope. An azeotrope (”azeotropic mixture) is a solution of two or more liquid components at concentrations that produces a constant boiling point for the solution. An azeotrope BP (A-BP) is the boiling point of an azeotrope. Often, the boiling point (BP) of the majority component of an azeotrope is higher than the A-BP, and in some embodiments, such an azeotrope evaporates from a coating faster than a similar coating that does not comprise the azeotrope. However, in some aspects, a coating comprising an azeotrope with a superior evaporation property may possess a lower flash point temperature, a lower explosion limit, a reduced coating flow, greater surface defect formation, or a combination thereof, relative to a similar coating that does not comprise the azeotrope. Alternatively, an azeotrope may be selected for embodiments wherein a component's BP is increased. In specific aspects, a coating comprising such an azeotrope may have a relatively slower evaporation rate than a similar coating that does not comprise the azeotrope. It is contemplated that the greater the percentage of liquid component is an azeotrope, the greater the conference of an azeotrope’s property to a coating. Thus, a specific range of 50% to 100%, 90% to 100%, or 95% to 100%, including all intermediate ranges and combinations thereof, is sequentially more preferred in embodiments wherein an azeotrope's property is desired as a property of a coating.

48jjjjjj

In some embodiments, a chemically non-reactive (inert) liquid component may be selected. Typically, a liquid component is selected that Is inert relative to a particular chemical reaction to prevent an undesirable chemical reaction with other coating components. An example of such an undesirable chemical reaction is a binder-liquid component reaction that is inhibitory to a desired binder-binder film-formation reaction. Examples of a liquid component that are generally inert in an acetal formation reaction include benzene, hexane, or a combination thereof. An example of a liquid component that is generally inert in a decarboxylation reaction includes quinoline. Examples of a liquid component that are generally inert In a dehydration reaction include benzene, toluene, xylene, or a combination thereof. An example of a liquid component that is generally inert in a dehydrohalogenation reaction includes quinoline. Examples of a liquid component that are generally inert in a diazonium compound coupling reaction include ethanol, glacial acetic acid, methanol, pyridine, or a combination thereof. Examples of a liquid component that are generally inert in a diazotization reaction include benzene, dimethylformamide, ethanol, glacial acetic acid, or a combination thereof. Examples of a liquid component that are generally inert in an esterification reaction include benzene, dibutyl ether, toluene, xylene, or a combination thereof. Examples of a liquid component that are generally inert tn a Friedel-Crafts reaction include benzene, carbon disulfide, 1,2-dichloroethane, nitrobenzene, tetrachloroethane, tetrachloromethane, or a combination thereof. An example of a liquid component that is generally inert in a Grignard reaction includes diethyl ether. Examples of a liquid component that are generally inert in a halogenation reaction include dichlorobenzene, glacial acetic acid, nitrobenzene, tetrachloroethane, tetrachloromethane, trichlorobenzene, or a combination thereof. Examples of a liquid component that are generally inert in a hydrogenation reaction include an alcohol, dioxane, a hydrocarbon, glacial acetic acid, or a combination thereof. Examples of a liquid component that are generally inert in a ketene condensation reaction include acetone, benzene, diethyl ether, xylene, or a combination thereof. Examples of a liquid component that are generally inert in a nitration reaction include dichlorobenzene, glacial acetic acid, nitrobenzene, or a combination thereof. Examples of a liquid component that are generally inert in an oxidation reaction include glacial acetic acid, nitrobenzene, pyridine, or a combination thereof. Examples of a liquid component that are generally inert in a sulfonation reaction include dioxane, nitrobenzene, or a combination thereof.

A solvent-borne coating is a coating wherein 50% to 100%, the including all intermediate ranges and combinations thereof, of a coating’s liquid component is not water. Generally, the liquid component of a solvent-bome coating comprises an organic compound, an inorganic compound, or a combination thereof. The liquid component of a solvent-borne coating may function as a solvent, a thinner, a diluent, a plasticizer, or a combination thereof. In certain embodiments, a solvent-borne coating may comprise water. In specific aspects, the water may

48kkkkk k function as a solvent, a thinner, a diluent, or a combination thereof. The water component of a solvent-borne coating may comprise 0% to 49.999%, the including all intermediate ranges and combinations thereof, of the liquid component. In certain embodiments, the water component of a water-borne coating may be fully or partly miscible in the non-aqueous liquid component. Examples of the percent of water that is miscible, by weight at 20°C, in various liquids typically used in solvent-bome coatings include 0.01% water in tetrachloroethylene; 0.02% water in ethylbenzene; 0.02% water in p-xylene; 0.02% water in tricholorethylene; 0.05% water in 1,1,1tricholoroethane; 0.05% water in toluene; 0.1% water in hexane; 0.16% water in methylene chloride; 0.2% water in dibutyl ether; 0.2% water in tetrahydronaphthalene; 0.42% water in diisobutyl ketone; 0.5% water in cyclohexyl acetate; 0.5% water in nitropropane; 0.6% water in 2nitropropane; 0.62% water in butyl acetate; 0.72% water in dipentene; 0.9% water in nitroethane; 1.2% water in diethyl ether; 1.3% water in methyl tert-butyl ether; 1.4% water in trimethylcyclohexanone; 1.65% water in isobutyl acetate; 1.7% water in butyl glycol acetate; 1.9% water in isopropyl acetate; 2.4% water in methyl isobutyl ketone; 3.3% water In ethyl acetate; 3.6% water in cyclohexanol; 4.0% water in trimethylcyclohexanol; 4.3% water in isophorone; 5.8% water in methylbenzyl alcohol; 6.5% water in ethyl glycol acetate; 7.2% water in hexanol; 7.5% water in propylene carbonate; 8.0% water in methyl acetate; 8.0% water in cyclohexanone, 12.0% water in methyl ethyl ketone; 16.2% water in isobutanol; 19.7% water in butanol, 25.0/0 water in butyl glycolate; or 44.1% water in 2-butanol.

Various examples of such liquid components are described herein, including properties often used to select a chemical composition for use as a liquid component for a particular coating composition. Additionally, standards for physical properties, chemical properties, and/or procedures for testing purity/properties, are described for various types of liquid components (e.g., hydrocarbons, cycloaliphatic hydrocarbons, aromatic hydrocarbons, alcohols, ketones, esters, glycol ethers, mineral spirits, miscellaneous solvents, plasticizers) in, for example, “ASTM Book of Standards, Volume 06.04, Paint - Solvents; Aromatic Hydrocarbons, D4790-99, D26801, D3437-99, D1493-97, D235-02, D1836-02, D3735-02, D3054-98, D5309-02, D4734-98, D2359-02, D4492-98, D4077-00, D3760-02, D6526-00, D841-02, D843-97, D5211-01, D5471-97, D5871-98, D5713-00, D852-02, D1685-00, D4735-02, D3797-00, D3798-00, D5135-02, D513600, D5060-95, D3193-96, D3734-01, D1152-97, D770-95, D3622-95, D1007-00, D1719-95, D304-95, D319-95, D2635-01, D1969-01, D2306-00, D1612-95, D5008-01, D268-01, □1078-01, D329-02, D1363-94, D740-94, D2804-02, D1153-94, D3329-99, D2917-02, D3893-99, D4360-90, D2627-02, D2916-88, D2192-96, D4614-95, D3545-02, D3131-02, D3130-95, D1718-98, D461595, D3540-90, D1617-90, D2634-02, D5137-01, D3728-99, D4835-93, D4773-02, D3128-02, D331-95, D330-93, D4837-02, D4773-02, D4836-95, □5776-99, D5808-95, D5917-02, D6069-01, D6212-99, D6313-99, D6366-99, D6428-99, D6621-00, D6809-02, D5399-95, D6229-01, D656348111111

00, D6269-98, D3257-01, D847-96, D1613-02, D848-02, D1614-95, D4367-02, D4534-99,

D2360-00, D1353-02, D1492-02, D849-02, D3961-98, D1364-02, D3160-96, D1476-02 and

D1722-98, D853-97, D5194-96, D363-90, D1399-95, D1468-93, D3620-98, D3546-9O, and

D1721-97, 2002.

a. Solvents, Thinners, and Diluents

A coating may comprise a liquid component that may function as a solvent, a thinner, a diluent or a combination thereof. In one embodiment of a coating, a particular liquid component may function as a solvent, while in another coating composition comprising, for example, a different binder the same liquid component may function as a thinner and/or a diluent. Whether a liquid component functions primarily as a solvent, a thinner, or a diluent depends considerably upon the particular solvent and/or rheological property the liquid component confers to a specific coating composition. For example, the ability of the liquid component to function as a solvent, or lack thereof of such ability, relative to the other coating components generally differentiates a solvent from a diluent. A thinner is primarily included into a coating composition in combination with a solvent and/or diluent to alter a rheological property such as to reduce viscosity, enhance flow, enhance leveling, or a combination thereof. In addition to the ability of one of ordinary skill in the art to discern such differences of use for a specific liquid composition in a coating, examples of differing solubility properties for specific categories of liquid components, and empirical techniques for determining the solubility properties of a specific liquid component, relative to another coating component, are described herein.

A solute is a coating component dissolved by a solvent liquid component. A solute may be in solid, liquid or gas from prior to being dissolved. Solvency (solvent power) is the ability of a solvent to dissolve a solute, maintain a solute in solution upon addition of a diluent, and reduce the viscosity of a solution. A solvent is typically used to produce a solvent-bome coating, wherein the coating possesses desirable a rheological property for application to a surface and/or creation of a film of a desirable thickness. Additionally, a solvent may contribute to an appearance property, a physical property, a chemical property, or a combination thereof, of a coating and/or film. In most embodiments, a solvent is a volatile component of a coating, wherein 50% to 100%, including all intermediate ranges and combinations thereof, of the solvent is lost (e.g., evaporates) during film formation. In certain aspects, the rate of solvent loss slows during application and/or film formation. Such a change in solvent loss rate may promote a desirable Theologically related property during application and/or initial film formation, such as ease of application, minimum sag, reduce excessive flow, or a combination thereof, while still promoting a desirable Theologically related property post-application, such as a desirable leveling property, a desirable adhesion property, or a combination thereof.

48mmm mm tn

Depending upon the ability of a liquid component to dissolve, partly dissolve, or unsuccessfully dissolve a coating component, a coating may comprise, a real solution, a colloidal solution or a dispersion, respectively. Often the ability of a liquid component to dissolve a coating component is detrimentally affected by Increasing particulate matter size and/or molecular mass of the coating component. For example, a real solution comprises a clear and/or homogenous liquid solution. In typical embodiments, a real solution is produced when a potential solute of 1.0 nm or less in diameter is combined with a solvent. A colloidal solution comprises a physically non-homogenous solution, which may be a clear to opalescent in appearance. Often, a colloidal solution is produced when a potential solute of between 1.0 nm to 100 nm ( 0.1 pm ) in diameter is combined with a solvent. A dispersion is a composition comprising two liquid and/or solid phases, which is typically turbid to milky in appearance. Generally, a dispersion is produced when a potential solute of greater than 0.1 pm in diameter is combined with a solvent. In many aspects, a coating composition may comprise a combination of a real solution, a colloidal solution and/or a dispersion, depending upon the various solubility’s of coating components and liquid components. For example, a paint may comprise a real solution of a binder and a liquid component, and a dispersion of a pigment within the liquid component.

Depending upon other coating components, a liquid component may function as an active solvent or a latent solvent. An active solvent is capable of dissolving a solute. Additionally, an active solvent often reduces viscosity of a coating composition. In certain embodiments, an ester, a glycol ether, a ketone, or a combination thereof may be selected for use as an active solvent. A latent solvent, in pure form, does not demonstrate solute dissolving ability. However, the latent solvent may demonstrate the ability to dissolve a solute in a combination of an active solvent and the latent solvent; confer a synergistic improvement in the dissolving ability of an active solvent when combined with the active solvent, or a combination thereof. In certain embodiments, an alcohol may be selected for use as a latent solvent. In certain embodiments, a latent solvent is a thinner. A diluent, whether in pure form or in combination with an active solvent and/or a latent solvent, does not demonstrate solute dissolving ability, but may be combined with an active solvent and/or latent solvent to produce a liquid component with a suitable ability to dissolve a coating component. In certain embodiments, hydrocarbon may be selected for use as a diluent. In particular aspects, a hydrocarbon diluent comprises an aromatic hydrocarbon, an aliphatic hydrocarbon, or a combination thereof. In particular facets, an aromatic hydrocarbon diluent may be selected, due to a generally greater tolerance by a many solvents relative to an aliphatic hydrocarbon. In certain aspects, a diluent is used to alter a rheological property (e.g., reduce viscosity) of a coating composition, reduce cost of a coating composition, or a combination thereof.

48nnnnn

The ability of a solvent to dissolve a potential solute is related to the intermolecular interactions between the solvent molecules, between the potential solute molecules, between the solvent and the potential solute, as well as the molecular size of the potential solute. Examples of intermolecular interactions include, for example, ionic (“Coulomb), dipole-dipole (״directional״), ionic-dipole, induction (“permanent dipole/induced dipole), dispersion (nonpolar, atomic dipole,” London-Van der Walls”), hydrogen bond, or a combination thereof. As is known to those of ordinary skill in the art, the sum of intramolecular interactions for a compound, relevant for the preparation of a solution, is the solubility parameter (“δ). The solubility parameter Is a measure of the total energy needed to separate molecules of a liquid. Such a separation of molecules of a solvent occurs during the incorporation of the molecules of a solute during the dissolving process. The solubility parameter is the square root of the molar energy of vaporization of a liquid divided by the molar volume of a liquid, measured at 25°C. Additionally, the solubility parameter can also be expressed as the square root of the sum of the squares of the dispersion ( ), polar ( δ_ρ) and hydrogen bond (5_h”) solubility parameters.

Often, preparation of a coating composition may be aided by comparing the solubility parameter of a potential solvent and a potential solute {e.g., a binder) to ascertain the theoretical ability of a coating composition comprising a solution to be created. In many embodiments, coating components, wherein at least one coating component comprises a liquid, with a solubility parameter that is less than an absolute value of 6 are able to form a solution. The closer this value is to 0, the greater the general ability to form a solution. Additionally, the lower the individual absolute difference (e.g., six or less) between the dispersion solubility parameters of coating components, the polar solubility parameter of coating components, and/or the hydrogen bond solubility parameter of coating components, the generally greater ability to form a solution. The solubility parameter, dispersion solubility parameter, polar solubility parameter, and hydrogen bond solubility parameter, and methods for determining such values, and additional methods for determining the theoretical ability of coating components to form a solution have been described (see, for example, In “ASTM Book of Standards, Volume 06.03, Paint - Pigments, Drying Oils, Polymers, Resins, Nava! Stores, Cellulosic Esters, and Ink Vehicles, D3132-84, 2002).

However, due to exceptions to the ability of certain liquid components and potential solute coating components to form solutions, empirically determining the ability of a solute to dissolve in a solvent may be desirable in certain embodiments. Standard techniques for determining the ability of a liquid component comprising one or more liquids to function as an active solvent, a latent solvent, a diluent, or a combination thereof, relative to one or more potential solutes are known to those of ordinary skill in the art. For example, the solvency of a

4800000 liquid component comprising an active solvent (e.g., an oxygenated compound), a latent solvent, a diluent (e.g., a hydrocarbon), or a combination thereof, particularly for use tn a lacquer coating, may be determined as described in ASTM Book of Standards, Volume 06.04, Paint -- Solvents, Aromatic Hydrocarbons, D1720-96, 2002). In an additional example, the solvency for a liquid component that primarily comprises a hydrocarbon, and comprises little or lacks an oxygenated compound, may be determined as described in “ASTM Book of Standards, Volume 06.04, Paint -Solvents; Aromatic Hydrocarbons, D1133-02, 2002). In a further example, the solvency of a solution comprising liquid component and an additional coating component (e.g., a binder) may be used to determined, as described in “ASTM Book of Standards, Volume 06.03, Paint — Pigments, Drying Oils, Polymers, Resins, Naval Stores, Cellulosic Esters, and Ink Vehicles, D1545-98, D1725-62, D5661-95, D5180-93, D6038-96, D5165-93, and D5166-97, 2002. In a supplemental example, the dilutability of a solution comprising liquid component (e.g., a solvent and diluent) and an additional coating component (e.g., a binder) may be used to determined, as described in ASTM Book of Standards, Volume 06.03, Paint - Pigments, Drying Oils, Polymers, Resins, Naval Stores, Celluiosic Esters, and Ink Vehicles, D5062-96, 2002.

In certain embodiments, a liquid component may be selected on the basis of evaporation rate. The evaporation rate of a coating directly affects a physical aspect of film formation caused by loss of a liquid component, as well as the pot life of a coating, such as after a coating container is opened. Though the evaporation rate is known for various pure chemicals, one of ordinary skill in the art can empirically determine the evaporation rate of a liquid component and/or a coating, in ASTM Book of Standards, Volume 06.01, Paint - Tests for Chemical, Physical, and Optical Properties; Appearance, D3539-87, 2002. Additionally, the boiling point range of a liquid component often is useful in estimating whether the liquid component will evaporate faster or slower relative to another liquid component. Examples of methods for measuring a boiling point for a liquid component (e.g., a hydrocarbon, a chlorinated hydrocarbon) are described in ASTM Book of Standards, Volume 06.04, Paint - Solvents; Aromatic Hydrocarbons, D1078-01 and D850-02e1, 2002. The evaporation rate is also related to the flash point of a liquid component and/or coating. In certain embodiments, a liquid component may be selected on the basis of flash point and/or fire point, which is a measure of the danger of use of a flammable coating composition in, for example, storage, application in an indoor environment, etc. A flash point is the “lowest temperature at which the liquid gives off enough vapor to form an ignitable mixture with air to produce a flame when a source of ignition is brought close to the surface of the liquid under specified conditions of test at standard barometric pressure (760 mmHG, 101.3 kPa), and a fire point is the lowest temperature at which sustained burning of the sample takes place for at least 5 seconds [^,,Paint and Coating Testing Manual, Fourteenth Edition of the Gardner-Sward Handbook (Koleske, J. V. Ed.), pp. 140 and 142, 1995). Examples of methods for measuring the

48ppppp

P flash point and/or fire point for a liquid component and/or a coating are described in and “ASTM

Book of Standards, Volume 06.01, Paint - Tests for Chemical, Physical, and Optical Properties;

Appearance,” D1310-01, D3934-90, D3941-90, and D327896־e1, 2002.

Though it is contemplated that most or all liquid component will be lost from a coating composition during film formation, a liquid component may still contribute to the visual properties of a coating and/or film. In embodiments wherein a liquid component is selected as a colorizing agent, the color and/or darkness of the liquid may be empirically measured (see, for example, ASTM Book of Standards, Volume 06.04, Paint - Solvents; Aromatic Hydrocarbons, D1209-00, D1686-96, and D5386-93b, 2002); and ASTM Book of Standards, Volume 06.01, Paint - Tests for Chemical, Physical, and Optical Properties; Appearance,” D1544-98, 2002. in some embodiments, a liquid component and/or coating may be selected on the basis of odor (e.g., faint odor, pleasant odor, efc.). A coating or coating component can be evaluated for suitability in a particular application based on odor using, for example, techniques described tn “ASTM Book of Standards, Volume 06.04, Paint - Solvents; Aromatic Hydrocarbons, D1296-01, 2002; and “ASTM Book of Standards, Volume 06.01, Paint -- Tests for Chemical, Physical, and Optical Properties; Appearance,” D6165-97, 2002.

(1) hydrocarbons

A hydrocarbon is typically obtained as a petroleum product, a vegetable product, or a combination thereof. As a consequence of imperfect purification (e.g., distillation) from these sources, a hydrocarbon is often a mixture of chemical components. A hydrocarbon may be selected as an active solvent to dissolve an oil (e.g., a drying oil), an alkyd, an asphalt, a rosin, a petroleum product, or a combination thereof. A hydrocarbon is more suitable as a latent solvent or diluent in embodiments wherein an acrylic resin, an epoxide resin, a nitrocellulose resin, an urethane resin, or a combination thereof is to be dissolved. However, a hydrocarbon generally is immiscible in water.

(i) Aliphatic Hydrocarbons

In general embodiments, an aliphatic hydrocarbon may be selected as an active solvent for an alkyd, an oil, wax, a polyisobutene, a polyethylene, a poly(butyl acrylate), a poly(butyl methacrylate), a poly(vinyl ethers), or a combination thereof. In other embodiments, an aliphatic hydrocarbon may be selected as a diluent in combination with an additional liquid component. In alternative embodiments wherein an aliphatic hydrocarbon is selected as a non-solvent liquid component, a composition comprising a polar binder, a cellulose derivative, or a combination thereof, is usually insoluble. An aliphatic hydrocarbon is often selected as a liquid component in embodiments wherein a chemically inert liquid component is desired. Examples of an aliphatic

48qqqqq q hydrocarbon include, a petroleum ether, pentane (CAS No. 109-66-0), hexane (CAS No. 11054-3), heptane (CAS No. 142-82-5), isododecane (CAS No. 13475-82-6), a kerosene, a mineral spirit, a VMP naphthas or a combination thereof. A hexane, a heptane, or a combination thereof, may be selected for a coating wherein rapid evaporation of such a liquid component is desired (e.g., a fast drying lacquer). An example of an azeotrope comprising an aliphatic hydrocarbon includes an azeotrope comprising hexane. Examples of an azeotrope comprising a majority of hexane (BP 65°C to 70°C) include those comprising 2.5% isobutanol (azeotrope BP 68.3°C): 5.6% water (A-BP 61.6°C); 21% ethanol (A-BP 58.7°C); 22% isopropyl alcohol (A-BP 61.0°C); 26.9% methanol (A-BP 50.0°C); 37% methyl ethyl ketone (A-BP 64.2°C); or 42% ethyl acetate (ABP 65.0°C).

As would be known to one of ordinary skill in the art, an aliphatic hydrocarbon can comprise a petroleum distillation product of heterogeneous chemical composition. Such an aliphatic hydrocarbon may be classified by a physical and/or chemical property (e.g., boiling point range, flash point, evaporation rate) (see, for example, “ASTM Book of Standards, Volume 06.04, Paint - Solvents; Aromatic Hydrocarbons, D235-02 and D3735, 2002). In certain embodiments, such a petroleum distillation product aliphatic hydrocarbon may be classified, for example, as a mineral spirit, a VMP naphthas or a kerosene (e.g., deodorized kerosene). A mineral spirit (white spirit, petroleum spirit״) is a petroleum distillation fraction with a boiling point between 149°C to 204“C, including all intermediate ranges and combinations thereof, and a flash point of 38°C or greater. A mineral spirit may further be classified as a regular mineral spirit, which possesses the properties previously described for a mineral spirit; a high flash mineral spirit, which possesses a higher minimum flash point (e.g., 55°C or greater); a low dry point mineral spirit (Stoddard solvent), which typically evaporates 50% faster than a regular mineral spirit; or an odorless mineral spirit, which generally possesses less odor than a regular mineral spirit, but may also possess relatively weaker solvency property. A mineral spirit may be selected for embodiments wherein a solvent and/or diluent is desired for an alkyd coating, a chlorinated rubber coating, an oil-coating, a vinyl chloride copolymer coating, or a combination thereof. A VMP naphtha possess a similar solvency property as a mineral spirit, but evaporates faster with a BP of 121°C to 149°C, including all intermediate ranges and combinations thereof, and typically has a flash point of 4°C or greater. A VMP naphtha may further be classified as a regular VMP naphtha, which possesses the properties previously described for a VMP naphtha; a high flash VMP naphtha, which possesses a higher minimum flash point (e.g., 34°C or greater); or an odorless VMP naphtha, which generally possesses less odor than a regular mineral spirit. A VMP naphtha may be selected for a coating that is spray applied, an industrial coating, or a combination thereof. A petroleum ether is a petroleum distillation fraction with a boiling point

48πτπτ between 35°C to 80°C, including alt intermediate ranges and combinations thereof, with a low flash point (e.g., -46°C), and may be used in embodiments wherein rapid evaporation is desired.

(ii) Cycloaliphatic Hydrocarbons

In embodiments wherein a cycloaliphatic hydrocarbon is selected as a solvent, a composition comprising an oil, alkyd, bitumen, rubber, or a combination thereof, usually can be dissolved. In alternative embodiments wherein a cycloaliphatic hydrocarbon is selected as a nonsolvent liquid component, a composition comprising a polar binder such as a urea-formaidehyde binder, a melamine-formaldehyde binder, a phenol-formaldehyde binder; a cellulose derivative, such as, a cellulose ester binder; or a combination thereof, is usually insoluble. A cycloaliphatic hydrocarbon is generally soluble in other organic solvents, but not soluble in water. Examples of a cycloaliphatic hydrocarbon include cyclohexane (CAS No. 110-82-7); methylcyclohexane (CAS No. 108-87-2); ethylcyclohexane (CAS No. 1678-91-7); tetrahydronaphthalene (CAS No. 119-642); decahydronaphthalene (CAS No. 91-17-8); or a combination thereof. Tetrahydronaphthalene is often selected for coatings wherein oxidation of a binder is preferable during film formation; a high gloss is preferable in a film, smooth surface is preferable in a film, or a combination thereof. An example of an azeotrope comprising a cycloaliphatic hydrocarbon includes an azeotrope comprising cyclohexane. Examples of an azeotrope comprising a majority of cyclohexane (BP 80.5°C to 81.5°C) include those comprising 8.5% water (A-BP 69.8°C); 10% butanol (A-BP 79.8°C); 14% isobutanol (A-BP 78.1°C); 20% propanol (A-BP 74.3°C); 37% methanol (A-BP 54.2°C); or 40% methyl ethyl ketone (A-BP 72.0°C).

(iii) Terpene Hydrocarbons

A terpene typically possesses a superior solvency property, stronger odor, or a combination thereof, relative to an aliphatic hydrocarbon. Examples of a terpene include wood terpentine oil (CAS No. 8008-64-2); pine oil (CAS No. 8000-41-7); a-pinene (CAS No. 80-56-8); β-pinene; dipentene (CAS No. 138-86-3); D-iimonene (CAS No. 5989-27-5); or a combination thereof. Dipentene may be selected for embodiments wherein a superior solvency property, a slower evaporation rate, or a combination thereof, relative to a turpentine, is desired. Pine oil may be classified as an oxygenated compound, but is described under hydrocarbons due to convention by those of skill in the art. Pine oil generally comprises a terpene alcohol. Pine oil may be selected for embodiments wherein a greater range of solvency for solutes, a slow evaporation rate, or a combination thereof, is desired. An example of an azeotrope comprising a terpene includes an azeotrope comprising a-pinene. An example of an azeotrope comprising a majority of a-pinene (BP 154.0°C to 156.0°C) includes an azeotrope comprising 35.5% cyclohexanol (A-BP 149.9°C).

48ssssss

As would be known to one of ordinary skill in the art, a terpene hydrocarbon (terpene ) can comprise a by-product from pines tree and/or citrus processing of heterogeneous chemical composition. Such a terpene hydrocarbon (e.g., a terpentine) may be classified by a physical and/or chemical property (see, for example, “ASTM Book of Standards, Volume 06.03, Paint -Pigments, Drying Oils, Polymers, Resins, Naval Stores, Cellulosic Esters, and Ink Vehicles,” D804-02, D13-02, D233-02, D801-02, D802-02, and D6387-99, 2002. Examples of a terpentine include a gum turpentine, a steam-distilled wood turpentine, a sulfate wood turpentine, a destructively distilled wood turpentine, or a combination thereof. Both a gum turpentine and a sulfate wood turpentine generally comprise a combination of a-pinene and a lesser quantity of βpinene. A steam-distilled wood terpentine generally comprises a-pinene and a lesser component of dipentene and one or more other terpenes. Destructively distilled wood turpentine generally comprises various aromatic hydrocarbons and a lesser quantity of one or more terpenes.

(iv) Aromatic Hydrocarbons

An aromatic hydrocarbon typically possesses a greater solvency property and/or odor relative to other hydrocarbon types. Examples of an aromatic hydrocarbon include benzene (CAS No. 71-43-2); toluene (CAS No. 108-88-3; ״methylbenzene); ethylbenzene (CAS No. 10041-4); xylene (CAS No. 1330-20-7); cumene (isopropylbenzene; CAS No. 98-828־); a type I high flash aromatic naphthas; a type II high flash aromatic naphthas; mesitylene (CAS No. 108-67-8); pseudocumene (CAS No. 95-63-6); cymol (CAS No. 99-876־); styrene (CAS No. 100-42-5); or a combination thereof. Xylene typically comprises o-xylene (CAS No. 56004-61-6); m-xylene (CAS No. 108-38-3); p- xylene (CAS No. 41051-88-1); and trace ethylbenzene. Toluene may be selected for embodiments wherein rapid evaporation is desired. In specific aspects, toluene may be selected for a spray applied coating, an industrial coating, or a combination thereof. Xylene may be selected for embodiments wherein a moderate evaporation rate is desired. In specific aspects, xylene may be selected for an industrial coating. As would be known to one of ordinary skill in the art, an aromatic hydrocarbon may comprise a petroleum-processing product of heterogeneous chemical composition such as a high flash aromatic naphtha (e.g., type I, type II). A type I high flash aromatic naphtha and type II high flash aromatic naphtha possess a minimum flash point of 38°C and 60°C, respectively. Standards for the characteristic chemical an/or physical property of an aromatic naphtha are known to those of ordinary skill in the art (see, for example, “ASTM Book of Standards, Volume 06.04, Paint - Solvents; Aromatic Hydrocarbons, D3734, 2002). A high flash naphtha typically has a slow evaporation rate. In specific embodiments, a high flash aromatic naphtha may be used in an industrial coating, a coating that is baked, or a combination thereof. An example of a high flash aromatic is Solvesso 100 (CAS No. 64742-95-6). Examples of an azeotrope comprising an aromatic hydrocarbon include an azeotrope comprising toluene or m-xylene. Examples of an azeotrope comprising a majority of

48tttttt toluene (BP 110°C to 111°C) include those comprising 27% butanol (A-BP 105.6°C); or 44.5% isobutanol (A-BP 100.9°C). Examples of an azeotrope comprising a majority of m-xylene (BP

137.0°C to 142.0°C) include those comprising 14% cyclohexanol (A-BP 143.0°C); or 40% water (A-BP 94.5°C).

(2) Oxygenated Compounds

An oxygenated compound (oxygenated liquid compound”) is typically chemically synthesized by standard chemical manufacturing techniques. As a consequence, an individual oxygenated compound is typically an extremely homogenous chemical composition, with singular, rather than a range of, chemical and physical properties. The oxygen moiety of an oxygenated compound generally enhances the strength and breadth of solvency for potential solutes relative to a hydrocarbon. Additionally, an oxygenated compound typically has some or complete miscibility with water. Examples of an oxygenated compound include an alcohol, an ester, a glycol ether, a ketone, or a combination thereof. As would be known to one of ordinary skill in the art, a liquid component often comprises a combination of an alcohol, an ester, a glycol ether, a ketone and/or an addition liquid to produce suitable chemical and/or physical properties for a coating and/or film.

(ί) Alcohols

An alcohol comprises an alcohol moiety. However, a preferred “alcohol” comprises a single hydroxyl moiety. The alcohol moiety confers miscibility with water. Consequentially, increasing molecular size of an alcohol comprising a single alcohol moiety generally reduces miscibility with water. Alcohols typically possess a mild and/or pleasant odor. An alcohol is typically a poor primary solvent, though ethanol is an exception relative to a solute comprising a phenolic and/or polyvinyl resin. An alcohol may be selected as a latent solvent, co-solvent, a coupling solvent, a diluent, or combination thereof such as with solute comprising a nitrocellulose lacquer, melamine-formaldehyde, urea formaldehyde, alkyd, or combination thereof. Examples of an alcohol include methanol (CAS No. 67-56-1); ethanol (CAS No. 64-17-5); propanol (CAS No. 71-23-8); isopropanol (CAS No. 67-63-0); 1-butanol (CAS No. 71-36-3); isobutanol (CAS No. 7883-1); 2-butanol_(CAS No. 78-92-2); fert-butanol (CAS No. 75-650־); amyl alcohol (CAS No. 7141-0); isoamyl alcohol (123-51-3); hexanol (25917-35-5); methylisobutylcarbinol (CAS No. 10811-2); 2-ethylbutanol (CAS No. 97-95-0); isooctyl alcohol (CAS No. 26952-21-6); 2-ethylhexanol (CAS No. 104-76-7); isodecanol (CAS No. 25339-177־); cylcohexanol (CAS No. 1080־93־); methylcyciohexanol (CAS No. 5835־59־); trimethylcyclohexanol; benzyl alcohol (CAS No. 100-516); methylbenzyl alcohol (CAS No. 98-85-1); furfuryl alcohol (CAS No. 98-00-0); tetrahydrofurfuryl alcohol (CAS No. 97-99-4); diacetone alcohol (CAS No. 123-42-2); trimethylcyclohexanol (11602-9); or a combination thereof. Furfuryl alcohol and tetrahydrofurfuryl alcohol may be selected

48uuuuu as a primary solvent for a polyvinyl binder. Examples of an azeotrope comprising an alcohol include an azeotrope comprising butanol, ethanol, isobutanol, or methanol. Examples of an azeotrope comprising a majority of butanol (BP 117.7°C) include those comprising 97% butanol and 3% hexane (A-BP 67°C); 32% p-xylene (A-BP 115.7°C); 32.8% butyl acetate (A-BP 117.6°C); 44.5% water (A-BP 93°C); or 50% isobutyl acetate (A-BP 114.5°C). Examples of an azeotrope comprising a majority of ethanol (BP 78.3°C) include those comprising 4.4% water (ABP 78.2°C); or 32% toluene (A-BP 76.7°C). Examples of an azeotrope comprising a majority of isobutanol (BP 107.7°C) include those comprising 2.5% hexane (A-BP 68.3°C); 5% isobutyl acetate (A-BP 107.6°C): 17% p-xylene (A-BP 107.5°C); 33.2% water (A-BP 89.9°C); or 48% butyl acetate (A-BP 80.1 °C). An example of an azeotrope comprising a majority of methanol (BP 64.6°C) includes an azeotrope comprising 30% methyl ethyl ketone (A-BP 63.5°C).

(il) Ketones

A ketone comprises a ketone moiety. However, a preferred ketone comprises a single ketone moiety. A ketone generally possesses some miscibility with water, and a strong odor. In general embodiments, a ketone may be selected as a primary solvent, thinner, or combination thereof. Examples of a ketone include acetone (CAS No. 67-64-1); methyl ethyl ketone (CAS No. 78-93-3); methyl propyl ketone (CAS No. 107-87-9); methyl isopropyl ketone (CAS No. 563-80-4); methyl butyl ketone (CAS No. 591-78-6); methyl isobutyl ketone (CAS No. 108-10-1); methyl amyl ketone (CAS No. 110-43-0); methyl isoamyl ketone (CAS No. 110-12-3); diethyl ketone (CAS No. 9622-0־); ethyl amyl ketone (CAS No. 541-85-5); dipropyl ketone (CAS No. 110-43-0); diisopropyl ketone (CAS No. 565-80-0); cyclohexanone (CAS No. 108-94-1); methylcylcohexanone (CAS No. 1331-22-2); trimethylcyclohexanone (CAS No. 873-949־); mesityl oxide (CAS No. 141-79-7); diisobutyl ketone (CAS No. 108-83-8); isophorone (CAS No. 78-59-1); or a combination thereof. Acetone may be selected for complete miscibility in water, fast evaporation, or a combination thereof. In certain embodiments, acetone may be used as a liquid component in an aerosol, a spay-applied coating, or a combination thereof. In specific aspects, acetone may be used as a thinner. In other aspects, acetone may be used in a coating wherein nitrocellulose, an acrylic, or a combination thereof, is dissolved. Methyl ethyl ketone, methyl isobutyt ketone, and isophorone may be selected in embodiments wherein a fast evaporation rate, moderate evaporation rate, or slow evaporation rate, respectively, is desired. In specific facets, isophorone may be selected for a baked coating, an industrial coating, or a combination thereof. Examples of an azeotrope comprising a ketone include an azeotrope comprising acetone, methyl ethyl ketone or methyl isobutyl ketone. Examples of an azeotrope comprising a majority of acetone (BP 56.2°C) include those comprising 12% methanol (A-BP 55.7°C); or 41% hexane (A-BP 49.8°C). Examples of an azeotrope comprising a majority of methyl ethyl ketone (BP 79.6°C) include those comprising 11% water (A-BP 73.5°C); 32% isopropyl alcohol (A-BP 77.5°C); or 34% ethanol (A-BP 74.8°C).

48vww v

Examples of an azeotrope comprising a majority of methyl isobutyl ketone (BP 114°C to 117°C) include those comprising 24.3% water (A-BP 87.9°C); or 30% butanol (A-BP 114.35°C).

(ill) Esters

An ester may comprise an alkyl acetate, an alkyl propionate, a glycol ether acetate, or a combination thereof. An ester generally possesses a pleasant odor. In general embodiments, an ester possesses a solubility property that decreases with increasing molecular weight. A glycol ester acetate typically possesses a slow evaporation rate. In specific aspects, a glycol ester acetate may be selected as a retarder solvent, a coalescent, or a combination thereof. Examples of an ester include methyl formate (CAS No. 107-31-3); ethyl formate (CAS No. 109-94-4); butyl formate (CAS No. 592-84-7); isobutyl formate (CAS No. 542-55-2); methyl acetate (CAS No. 7920-9); ethyl acetate (CAS No. 141-78-6); propyl acetate (CAS No. 109-60-4); isopropyl acetate (CAS No. 108-21-4); butyl acetate (CAS No. CAS-No. 123-86-4); isobutyl acetate (CAS No. 11019-0); sec-butyl acetate (CAS No. 105-46-4); amyl acetate (CAS No. 628-63-7); isoamyl acetate (CAS No. 123-92-2); hexyl acetate (CAS No. 142-92-7); cyclohexyl acetate (CAS No. 622-45-7); benzyl acetate (CAS No. 140-11-4); methyl glycol acetate (CAS No. 110-49-6); ethyl glycol acetate (CAS No. 111-15-9); butyl glycol acetate (CAS No. 112-07-2); ethyl diglycol acetate (CAS No. 111-90-0); butyl diglycol acetate (CAS No. 124-17-4); 1-methoxypropyl acetate (CAS No. 108-65-6); ethoxypropyl acetate (CAS No. 54839-24-6); 3-methoxybutyl acetate (CAS No. 443553-4); ethyl 3-ethoxypropionate (CAS No. 763-69-9); isobutyl isobutyrate (CAS No. 97-85-8); ethyl lactate (CAS No. 97-64-3); butyl lactate (CAS No. 138-22-7); butyl glycolate (CAS No. 739762-8); dimethyl adipate (CAS No. 627-93-0); glutarate (CAS No. 119-40-0); succinate (CAS No. 106-65-0); ethylene carbonate (CAS No. 96-49-1); propylene carbonate (CAS No. 108-32-7); butyrolactone (CAS No. 96-48-0); or a combination thereof. Ethylene carbonate and propylene carbonate generally possess a high flash point, a slow evaporation rate, a weak odor, or a combination thereof. Ethylene carbonate is preferred for use in coatings at temperatures greater than 25°C. Examples of an azeotrope comprising an ester include an azeotrope comprising butyl acetate, ethyl acetate or methyl acetate. Examples of an azeotrope comprising a majority □f butyl acetate (BP 124°C to 128°C) include those comprising 27% water (A-BP 90.7°C) or 35.7% ethyl glycol (A-BP 125.8°C). Examples of an azeotrope comprising a majority of ethyl acetate (BP 76°C to 77°C) include those comprising 5% cyclohexanol (A-BP 153.8°C); 8.2% water (A-BP 70.4’C); 22% methyl ethyl ketone (A-BP 76.7°C); 23% isopropyl alcohol (A-BP 74.8°C); or 31% ethanol (A-BP 71.8°C). An example of an azeotrope comprising a majority of methyl acetate (BP 55.0°C57.0°־C) includes an azeotrope comprising 19% methanol (A-BP 54°C).

48www

WWW (iv) Glycol Ethers

A glycol ether comprises an alcohol moiety and an ether moiety. The glycol ether generally possesses good solvency, high flash point, slow evaporation rate, mild odor, miscibility with water, or a combination thereof. In some embodiments, a glycol ether may be selected as a coupling solvent, a thinner, or a combination thereof. In particular aspects, a glycol ether may be selected as a liquid component of a lacquer. Examples of a glycol ether include methyl glycol (CAS No. 109-86*4); ethyl glycol (CAS No. 110-80-5); propyl glycol (CAS No. 2807-30-9); isopropyl glycol (CAS No. 109-59-1); butyl glycol (CAS No. 111-76-2); methyl diglycol (111-77-3); ethyl diglycol (CAS No. 111-90-0); butyl diglycol (CAS No. 112-34-5); ethyl triglycol (CAS No. 112-50-5); butyl triglycol (CAS No. 143-22-6); diethylene glycol dimethyl ether (CAS No. 111-966); methoxypropanol (CAS No. 107-98-2); isobutoxypropanol (CAS No. 23436-193־); isobutyl glycol (CAS No. 4439-241־); propylene glycol monoethyl ether (CAS No. 52125-53-8); 1isopropoxy-2-propanol (CAS No. 3944-36-3); propylene glycol mono-n-propyl ether (CAS No. 30136-13-1); propylene glycol n-butyl ether (CAS No. 5131-66-8); methyl dipropylene glycol (CAS No. 34590-94-8); methoxybutanol (CAS No. 30677-36-2); or a combination thereof. An example of an azeotrope comprising a glycol ether includes an azeotrope comprising ethyl glycol. An example of an azeotrope comprising a majority of ethyl glycol (BP 134°C to 137°C) includes an azeotrope comprising 50% dibutyl ether (A-BP 127°C).

(v) Ethers

Examples of an ether include diethyl ether (CAS No. 60-29-7); diisopropyl ether (CAS No. 108-20-3); dibutyl ether (CAS No. 142-96-1); di-sec-butyl ether (CAS No. 6863-58-7); methyl tertbutyl ether (CAS No. 1634-04-4); tetrahydrofuran (CAS No. 109-99-9); 1,4-dioxane (CAS No. 123-91-1); metadioxane (CAS No. 505-22-6); or a combination thereof. Tetrahydrofuran may be selected as a primary solvent for a polyvinyl binder. An example of an azeotrope comprising an ether includes an azeotrope comprising tetra hydrofuran. An example of an azeotrope comprising a majority of tetrahydrofuran (BP 66°C) includes an azeotrope comprising 5.3% water (A-BP 64.0°C).

(3) Chlorinated Hydrocarbons

A chlorinated hydrocarbon generally comprises a hydrocarbon, wherein the hydrocarbon comprises a chloride atom moiety. A chlorinated hydrocarbon generally possesses a very high degree of non-flammability, and consequently lacks a flash point. A chlorinated hydrocarbon may be selected for embodiments where high flash point is desired. In particular facets, a chlorinated hydrocarbon may be added to a liquid component to reduce the liquid component’s flash point. In certain facets, it is less preferred that a chlorinated hydrocarbon be combined with a mineral spirit, methylene chloride, or a combination thereof, wherein reduction of the flash point is

48xxxxx x

desired. In particular aspects, a chlorinated hydrocarbon (e.g., methylene chloride, trichloroethylene) may be selected as a solvent for removal of hydrophobic material from a surface (e.g., grease, an undesired coating and/or film). However, a chlorinated hydrocarbon may be less preferred due to an environmental regulation or law. Examples of a chlorinated hydrocarbon include methylene chloride (CAS No. 75-09-2; dichloromethane); trichloromethane (CAS No. 67-66-3); tetrachloromethane (CAS No. 56-23-5); ethyl chloride (CAS No. 75-00-3); isopropyl chloride (CAS No. 75-29-6); 1,2-dichloroethane (CAS No. 107-06-2); 1,1,1trichloroethane (CAS No. 71-55-6; methylchloroform”); trichloroethylene (CAS No. 79-01-6);

1,1,2,2-tetrachlorethane (CAS No. 79-55-6); 1,2-dichloroethylene (CAS No. 75-35-4); perchloroethylene (CAS No. 127-18-4); 1,2-dichloropropane (CAS No. 78-87-5); chlorobenzene (CAS No. 108-90-7); or a combination thereof. Methylene chloride may be selected for embodiments wherein a fast evaporation rate is desired. 1,1,1-trichloroethane may be selected for embodiments wherein a photochemically inert liquid component is desired. Additionally, methylene chloride may be selected as a coating remover. Examples of an azeotrope comprising a chlorinated hydrocarbon include an azeotrope comprising methylene chloride, trichloroethylene or 1,1,1-trichloroethane. Examples of an azeotrope comprising a majority of methylene chloride (BP 40.2°C) include those comprising 1.5% water (A-BP 38.1 °C); 3.5% ethanol (A-BP 41.0°C); or 8% methanol (A-BP 39.2°C). Examples of an azeotrope comprising a majority of trichloroethylene (BP 86.7°C) include those comprising 6.6% water (A-BP 72.9°C); 27% ethanol (A-BP 70.9°C); or 36% methanol (A-BP 60.2°C). An example of an azeotrope comprising a majority of 1,1,1-trichloroethane (BP 74.0°C) includes an azeotrope comprising 4.3% water (A-BP 65.0°C).

(4) Nitrated Hydrocarbon

A nitrated hydrocarbon comprises a hydrocarbon, wherein the hydrocarbon comprises a nitrogen atom moiety. Examples of a nitrated hydrocarbon include a nitroparaffin, N-methyl-2pyrrolidone (NMP), or a combination thereof. Examples of a nitroparaffin include nitroethane, nitromethane, nitropropane, 2-nitropropane (2NP”), or a combination thereof. 2-nitropropane may be selected for embodiments as a substitute for butyl acetate relative to a solvent property, but wherein a greater evaporation rate is desired. N-methy!-2-pyrrolidone may be selected for embodiments wherein a strong solvent property, miscibility with water, high flash point, biodegradability, low toxicity, or a combination thereof is desired. In certain aspects, N-methyl-2pyrrolidone may be used in a water-borne coating, a coating remover, or a combination thereof.

(5) Miscellaneous Organic Liquids

A miscellaneous organic liquid is a liquid comprising carbon that are useful as a liquid component for a coating, but are not readily classified as a hydrocarbon, an oxygenated

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compound, a chlorinated hydrocarbon, a nitrated hydrocarbon, or a combination thereof. Examples of a miscellaneous organic liquid include carbon dioxide; acetic acid, methylal (CAS No. 109-87-5); dimethylacetal (CAS No. 534-15-6); Ν,Ν-dimethylformamide (CAS No. 68-12-2); A/./V-dimethylacetamide (CAS No. 127-19-5); dimethylsulfoxide (CAS No. 67-68-5); tetramethylene suflone (CAS No. 126-33-0); carbon disulfide (CAS No. 75-15-0); 2-nitropropane (CAS No. 79-46-9); W-methylpyrrolidone (CAS No. 872-50-4); hexamethylphosphoric triamide (CAS No. 680-31-9); 1,3-dimethyl-2-imidazolidinone (CAS No. 80-73-9); or a combination thereof. As would be known to one of ordinary skill in the art, carbon dioxide may function as a liquid component when prepared under pressure and temperature conditions to form a supercritical liquid. A supercritical liquid has properties between that of a liquid and a gas, and can be used in spray application of a coating wherein the appropriate pressure conditions can be maintained. Supercritical carbon dioxide may be formulated with a coating using the tradename technique Unicarb™ (Union Carbide Chemicals and Plastics Co., Inc.). Supercritical carbon dioxide may be selected as a substitute for a hydrocarbon diluent in embodiments wherein chemical inertness, non-flammability, rapid evaporation, or a combination thereof, is desirable. In certain aspects, 0% to 30%, including all intermediate ranges and combinations thereof, of a hydrocarbon liquid component may be replaced with supercritical carbon dioxide.

b. Plasticizers

In certain embodiments, a coating may comprise a plasticizer. A plasticizer may be selected for embodiments wherein the resin possesses an unsuitable brittleness and/or low flexibility property upon film formation. Properties a plasticizer typically confers to a coating and/or film include, for example, enhancing a flow property of a coating, lowering a film-forming temperature range, enhancing the adhesion property of a coating and/or film, enhancing the flexibility property of a film, lowering the T_a, improving film toughness, enhancing film heat resistance, enhancing film impact resistance, enhancing UV resistance, or a combination thereof. Since a function of a plasticizer typically is to alter a film’s properties, most plasticizer’s possess a high (e.g., baking temperature) boiling point, as such a compound is generally less volatile, with increasing boiling point temperature. In certain aspects, a plasticizer may function as a solvent, thinner, diluent, plasticizer, or combination thereof, for a coating composition and/or film at a temperature greater than ambient conditions.

A plasticizer is thought to interact with a binder by a polar Interaction, but is chemically inert relative to the binder. A plasticizer typically will lower the T_g of a binder below the temperature a coating comprising the binder will be applied to a surface. In many embodiments, a plasticizer have a vapor pressure less than 3 mm at 200°C, a mass of 200 Da to 800 Da, a specific gravity of 0.75 to 1.35, a viscosity of 50 cSt to 450 cSt, a flash point temperature greater

48zzzzz z than 120°C, or a combination thereof. Preferred plasticizers comprise an organic liquid (e.g., an ester). Standards for physical properties, chemical properties, and/or procedures for testing purity/properties, are described for plasticizers (e.g., undesired acidity, color, undesired copper corrosion, boiling point, ester content, odor, undesirable water contamination) in, for example, “ASTM Book of Standards, Volume 06.04, Paint - Solvents; Aromatic Hydrocarbons, □1613-02, D1209-00, D849-02, D1078-01, D1617-90, D1296-01, D608-90, and D1364-02, 2002; and “ASTM Book of Standards, Volume 06.01, Paint - Tests for Chemical, Physical, and Optical Properties; Appearance, D1544-98, 2002. Compatibility of a plasticizer with a binder and/or a solvent has been described (see, for example, Riley, Η. E., Plasticizers,” Paint Testing Manual, American Society for Testing Materials, 1972). Additionally, techniques previously described for estimating solubility for liquid and an additional coating component may be applied for a plasticizer.

Various plasticizers comprise an ester of a monoalcohol and an acid (e.g., a dicarboxylic acid). In many embodiments, the monoalcohol comprises 4 to 13 carbons. In specific aspects, the monoalcohol comprises butanol, 2-ethylhexanol, isononanol, isooctyl, isodecyl, or a combination thereof. Examples of an acid include an azelaic acid, a phthalic acid, a sebacic acid, a trimellitic acid, an adipic acid, or a combination thereof. Examples of such plasticizers include di(2-ethylhexyl) azelate (“DOZ); di(butyl) sebacate (DBS); di(2-ethylhexyl) phthalate (DOP); di(isononyl) phthalate (DINP); dibutyl phthalate (“DBP”); butyl benzyl phthalate (BBP’j; di(isooctyl) phthalate (“DIOP); di(idodecyl) phthalate (DIDP); tris(2־ethylhexyl) trimellitate (TOTM”); tris(isononyl) trimellitate (TINTM'j; di(2-ethylhexyl) adipate (DOA”); di(isononyl) adipate (“DINA); or a combination thereof.

A plasticizer may be classified by a moiety, such as, for example, as an adipate (e.g., DOA, DINA), an azelate (e.g., DOZ), a citrate, a chlorinated plasticizer, an epoxide, a phosphate, a sebacate (e.g., DBS), a phthalate (e.g., DOP, DINP, DIOP, DIDP), a polyester, or a trimellitate (e.g., TOTM, TINTM). An example of a citrate plasticizer includes acetyl tri-n-butyl citrate. Examples of an epoxide plasticizer include an epoxy modified soybean oil (ESO), 2-ethylhexyl epoxytallate (2EH tallate), or a combination thereof. Examples of a phosphate plasticizer include isodecyl diphenyl phosphate, tricresyl phosphate (“TPC), isodecyl diphenyl phosphate, tri-2-ethylhexyl phosphate (TOP), or a combination thereof. Tricresyl phosphate may function as a plastizer, confer flame resistance, confer fungi resistance, or a combination thereof to a coating. Examples of a polyester plasticizer include an adipic acid polyester, an azelaic acid polyester, or a combination thereof. In certain aspects, a plasticizer is selected for water resistance (e.g., hydrolysis resistance, inertness toward water) such as a bisphenoxyethylformal.

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c. Water-Borne Coatings

A water-borne coating (“water reducible coating) refers to a coating wherein components such as a pigment, a binder, an additive, or a combination thereof are dispersed in water. Often, an additional solvent, surfactant, emulsifier, wetting agent, dispersant, or a combination thereof promotes dispersion of a coating component. A latex coating refers to a water-borne coating wherein the binder is dispersed in water. Typically, a binder of a latex coating comprises a high molecular weight binder. Often a latex coating (e.g., a paint, a lacquer) is a thermoplastic coating. Film formation occurs by loss of the liquid component, typically through evaporation, and fusion of dispersed thermoplastic binder particles. Often, a latex coating further comprises a coalescing solvent (e.g., diethylene glycol monobutyl ether) that promotes fusion of the binder particles. In some embodiments, a film produced from a latex coating is more porous, possesses a lower moisture resistance property, is less compact (e.g., thicker), or a combination thereof, relative to a solvent-borne coating comprising similar non-volatile components. Specific procedures for determining the purity/properties of a latex coating, coating component (e.g., solids content, nonvolatile content, vehicles), and/or film have been described, for example, in “ASTM Book of Standards, Volume 06.01, Paint - Tests for Chemical, Physical, and Optical Properties; Appearance,” D4747-02 and D4827-93, 2002; “ASTM Book of Standards, Voiume 06.02, Paint - Products and Applications; Protective Coatings; Pipeline Coatings, D3793-00, 2002; and “ASTM Book of Standards, Volume 06.03, Paint - Pigments, Drying Oils, Polymers, Resins, Naval Stores, Cellulosic Esters, and Ink Vehicles,” D5097-90 D4758-92, and D4143-89, 2002.

In certain embodiments, a water-borne coating is a coating wherein 50% to 100%, the including all intermediate ranges and combinations thereof, of a coating s liquid component is water. In general embodiments, the water component of a water-borne coating may function as a solvent, a thinner, a diluent, or a combination thereof. In certain embodiments, a water-borne coating may comprise an additional non-aqueous liquid component. In specific aspects, such an additional liquid component may function as a solvent, a thinner, a diluent, a plasticizer, or a combination thereof. An additional liquid component of a water-borne coating may comprise 0% to 49.999%, the including all intermediate ranges and combinations thereof, of the liquid component. Examples of additional liquid components in a water-borne coating include a glycol ether, an alcohol, or a combination thereof.

In certain embodiments, an additional liquid component of a water-borne coating may be fully or partly miscible in water. Examples of a liquid that is completely miscible in water, and visa versa, include methanol, ethanol, propanol, isopropyl alcohol, tert-butanol, ethylene glycol, methyl glycol, ethyl glycol, propyl glycol, butyl glycol, ethyl diglycol, methoxypropanol, methyldipropylene

48bbbbb bb glycol, dioxane, tetrahydorfuran, acetone, diacetone alcohol, dimethylformamide or dimethyl sulfoxide. Examples of a liquid that is partly miscible in water, by weight at 20°C, include 0.02% ethylbenzene; 0.02% tetrachloroethylene; 0.02% p-xylene; 0.035% toluene; 0.04% diisobutyl ketone; 0.1% tricholorethylene; 0.19% trimethylcyclohexanol; 0.2% cyclohexyl acetate; 0.3% dibutyl ether; 0.3% trimethylcyclohexanone; 0.44% 1,1,1-trlcholoroethane; 0.53% hexane; 0.58% hexanol; 0.67% isobutyl acetate; 0.83% butyl acetate; 1.2% isophorone; 1.4% nitropropane; 1.5% butyl glycol acetate; 1.7% 2-nitropropane; 2.0% methylene chloride; 2.0% methyl isobutyl ketone; 2.3% cyclohexanone; 2.9% isopropyl acetate; 2.9% methylbenzyl alcohol; 3.6% cyclohexanol; 4.5% nitroethane; 4.8% methyl tert-butyl ether; 6.1% ethyl acetate; 6.9% diethyl ether; 7.5% butanol; 7.5% butyl glycolate; 8.4% isobutanol; 12.5% 2-butanol; 21.4% propylene carbonate; 23.5% ethyl glycol acetate; 24% methyl acetate; or 26.0% methyl ethyl ketone. Examples of an azeotrope comprising a majority of water (BP 100°C) include those comprising 16.1% isophorone (A-BP 99.5°C); 20% 2־ethylhexanol (A-BP 99.1°C); 20% cyclohexanol (A-BP 97.8°C); 20.8% butyl glycol (A-BP 98.8°C); or 28.8% ethyl glycol (A-BP 99.4°C).

3. Colorants

A colorant (colorizing agent”) is a composition that confers a desirable optical property to a coating. Examples of desirable optica! properties, depending upon the application of the present invention, include a reflection property, a light absorption property, a light scattering property, or a combination thereof. A colorant that increases the reflection of light may increase gloss. A colorant that increased light scattering may increase the opacity and/or confer a color to a coating and/or film. Light scattering of a broad spectrum of wavelengths can confer a white color to a coating and/or film. Scattering of a certain wavelength may confer a color associated with the wavelength to a coating and/or film. Light absorption also affects opacity and/or color. Light absorption over a broad spectrum confers a black color to a coating and/or film. Absorbance of a certain wavelength may eliminate the color associated with the wavelength from the appearance of a coating and/or film. Examples of colorants include pigments, dyes, extenders, or a combination thereof. Colorants (e.g., pigments, dyes) and procedures for determining the optical properties and physical properties (e.g., hiding power, transparency, light absorption, light scattering, tinting strength, color, particle size, particle dispersion, pigment content, color matching) of a colorant, coating component, coating and/or film are described in, for example, (in Industrial Color Testing, Fundamentals and Techniques, Second, Completely Revised Edition, 1995; Colorants for Non-Textile Applications, 2000). Various colorants are well known to those of ordinary skill in the art, and are often identified by their Colour Index (“Cl”) number (see, for example, Colour Index International,” 1971; and Colour Index International,״ 1997). In some cases, a common name for a colorant encompasses several related colorants, which can be differentiated by Cl number,

48ccccc cc

a. Pigments

A pigment is a composition that is insoluble in the other components of a coating, and further confers a desirable optical properties, confers a property affecting the application of the coating (e.g., a rheological property), confers a performance property to a coating, reduces the cost of the coating, or a combination thereof. In certain embodiment, a pigment confers a performance property to a coating such as a desirable corrosion resistance property, magnetic property, or a combination thereof. Examples of a pigment include an inorganic pigment, an organic pigment, or a combination thereof.

Pigments possess a variety of properties in addition to color that aid in the selection of a particular pigment for a specific application. Examples of such properties include a tinctorial property, an insolubility property, a corrosion resistance property, a durability property, a heat resistance property, an opacity property, a transparency property, or a combination thereof. A tinctorial property is the ability of a composition to produce a color, wherein a greater tinctorial strength indicating less of the composition is needed to achieve the color. A insolubility property is the ability of a composition to remain in a solid form upon contact with another coating component (e.g., a liquid component), even during a curing process involving chemical reactions (e.g., thermosetting, baking, irradiation). A corrosion resistance property is the ability of a composition to reduce the damage of a chemical (e.g., water, acid) that contacts metal.

Pigments (e.g., extenders, titanium pigments, inorganic pigments, surface modified pigments, bismuth vanadates, cadmium pigments, cerium pigment, complex inorganic color pigments, metallic pigments, be nzim id azoIone pigments, diketopyrroiopyrrole pigments, dioxazine violet pigments, disazocondensation pigments, isoindoline pigments, isoindolinone pigments, perylene pigments, phthalocyanine pigments, quinacridone pigments, quinophthalone pigments, thiazine pigments, oxazine pigments, zinc sulfide pigments, zinc oxide pigments, iron oxide pigments, chromium oxide pigments, cadmium pigments, cadmium sulfide, cadmium yellow, cadmium sulfoselenide, cadmium mercury sulfide, bismuth pigments, chromate pigments, chrome yellow, molybdate red, molybdate orange, chrome orange, chrome green, fast chrome green, ultramarine pigments, iron blue pigments, black pigments, carbon black, specialty pigments, magnetic pigments, cobalt-containing iron oxide pigments, chromium dioxide pigments, metallic iron pigments, barium ferrite pigments, anti-corrosive pigments, phosphate pigments, zinc phosphate, aluminum phosphate, chromium phosphate, metal phosphates, multiphase phosphate pigments, borosilicate pigments, borate pigments, chromate pigments, molybdate pigments, lead cyanamide pigments, zinc cyanamide pigments, iron-exchange pigments, metai oxide pigments, red lead pigment, red lead, calcium plumbate, zinc ferrite pigments, calcium ferrite pigments, zinc

48ddddd dd oxide pigments, powdered metal pigments, zinc dust, lead powder, flake pigments, nacreous pigments, interference pigments, natural pearl essence pigment, basic lead carbonate pigment, bismuth oxychloride pigment, metal oxide-mica pigments, metal effect pigments, transparent pigments, transparent iron oxide pigments, transparent iron blue pigment, transparent cobalt blue pigment, transparent cobalt green pigment, transparent iron oxide, transparent zinc oxide, luminescent pigments, inorganic phosphor pigments, sulfide pigments, selenide pigments, oxysulfide pigments, oxygen dominant phosphor pigments, halide phosphor pigments, azo pigments, monoazo yellow pigments, monoazo orange pigment, disazo pigments, β-naphtho! pigments, naphthol AS pigments, salt-type azo pigments, benzimidazolone pigments, disazo condensation pigments, metal complex pigments, isoindolinone pigments, isoindoline pigments, polycyclic pigments, phthalocyanine pigments, quinacrindone pigments, perylene pigments, perinone pigments, diketopyrrolo pyrrole pigments, thioindigo pigments, anthrapyrimidine pigments, flavanthrone pigments, pyranthrone pigments, anthanthrone pigments, dioxanzine pigments, triarylcarbonium pigments, quinophthalone pigments) and their chemical properties, physical properties and/or optical properties (e.g., color, tinting strength, lightening power, scattering power, hiding power, transparency, light stability, weathering resistance, heat stability, chemical fastness, interactions with a binder), in coating component, coating and/or film, and techniques for determining such properties, are known to one of ordinary skill in the art (see, for example, Solomon, D, H. and Hawthorne, D. G., Chemistry of Pigments and Fillers, 1983; “High Performance Pigments, 2002; Industrial Inorganic Pigments, 2002; ^,1Industrial Organic Pigments, Second, Completely Revised Edition,” 1993).

As would be known to one of ordinary skill in the art, specific standards for physical properties, chemical properties, purity, and/or procedures for testing the purity/properties of various pigments (e.g., lead chromate, chromium oxide, phthalocyanine green, a phthalocyanine blue, molybdate orange, white zinc, zinc oxide, calcium carbonate, barium sulfate, aluminum silicate, diatomaceous silica, magnesium silicate, mica, calcium borosilicate, zinc hydroxy phosphite, aluminum powder, micaceous iron oxide, zinc phosphate, basic lead silicochromate, strontium chromate, ochre, lampblack, orange shellac, raw umber, burnt umber, raw sienna, burnt sienna, bone black, carbon black, red iron oxide, brown iron oxide, basic carbonate, white lead, white titanium dioxide, iron blue, ultramarine blue, chrome yellow, chrome orange, hydrated yellow iron oxide, zinc chromate yellow, red lead, para red toner, toluidine red toner, chrome oxide green, zinc dust, cuprous oxide, mercuric oxide, iron oxide, anhydrous aluminum silicate, black synthetic iron oxide, gold bronze powder, aluminum powder, strontium chromate pigment, basic lead silicochromate) for use in a coating are described, for example in “ASTM Book of Standards, Volume 06.03, Paint - Pigments, Drying Oils, Polymers, Resins, Naval Stores, Cellulosic Esters, and Ink Vehicles, D280-01, D2448-85, D126-87, D305-84, D3021-01, D325648eeeee ee

86, D2218-67, D3280-85, D50-90, D79-86, D1199-86, D602-81, D715-86, D603-66, D718-86, D604-81, □719-91, D605-82, D717-86, D607-82, D716-86, D4288-02, D4487-90, D4462-02, D4450-85, D962-81, D5532-94, D6280-98, D1648-86, D1649-01, D85-87, D209-81, D237-57, D763-01, D765-87, D210-81, D561-82, D3722-82, D3724-01, D34-91, D81-87, D1301-91, D1394-76, D261-75, D262-81, D1135-86, D211-67, D768-01, D444-88, D3872-86, D478-02, D1208-96, D83-84, D49-83, D3926-80, D475-67, D656-87, □970-86, D3721-83, D263-75, D52000, D521-02, D283-84, D284-88, D3720-90, D3619-77, D769-01, D476-00, D267-82, D480-88, D1845-86, D1844-86, and D279-02, 2002; and in “ASTM Book of Standards, Volume 06.01, Paint - Tests for Chemical, Physical, and Optical Properties; Appearance,” D5381-93 and D6131-97 2002.

(1) Corrosion Resistance Pigments

Addition of certain pigments may improve the corrosion resistance of a coating and/or film, or specifically, the protection of a metal surface coated with a coating and/or film from corrosion. Often, a primer comprises such pigments. Examples of corrosion resistance pigments include aluminum flake, aluminum triphosphate, aluminum zinc phosphate, ammonium chromate, barium borosilicate, barium chromate, barium metaborate, basic calcium zinc molybdate, basic carbonate white lead, basic lead silicate, basic lead silicochromate, basic lead siiicosulfate, basic zinc molybdate, basic zinc molybdate-phosphate, basic zinc molybdenum phosphate, basic zinc phosphate hydrate, bronze flake, calcium barium phosphosilicate, calcium borosilicate, calcium chromate, calcium plumbate (Cl Pigment Brown 10), calcium strontium phosphosilicate, calcium strontium zinc phosphosilicate, dibasic lead phosphite, lead chromosilicate, lead cyanamide, lead suboxide, lead sulfate, mica, micaceous iron oxide, red lead (Cl Pigment Red 105), steel flake, strontium borosilicate, strontium chromate (Cl Pigment Yellow 32), tribasic lead phophosilicate, zinc borate, zinc borosilicate, zinc chromate (Cl Pigment Yellow 36), zinc dust (Cl Pigment Metal 6), zinc hydroxy phosphite, zinc molybdate, zinc oxide, zinc phosphate (Cl Pigment White 32), zinc potassium chromate, zinc silicophosphate hydrate, zinc tetraoxylchromate, or a combination thereof.

The selection of a corrosion resistant pigment may be made based on the mechanism of corrosion resistance it confers to a coating and/or film. Corrosion often occurs as a cathodic process wherein a metal surface acts as a cathode and passes electrons to an electron accepter moiety of a corrosive chemical, such as, for example, hydrogen, oxygen, or a combination thereof. Corrosion can also occur as an anodic process wherein ionized metal atoms then enter solution. Pigments such as, for example, mica, micaceous iron oxide, metallic flake pigments (e.g., aluminum, bronze, steel), or a combination thereof confer corrosion resistance to a coating and/or film by acting as a physical barrier between a metal surface and corrosive chemicals.

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However, a chemically reactive pigment such as a metal flake pigment be used in an environment at or near neutra! pH (e.g., pH 6 to pH 8), Micaceous iron oxide can be selected for a primer, a topcoat, or a combination thereof, and can also function as a UV absorber. Aluminum flake may be selected for an industrial coating, an automotive coating, an architectural coating, a primer, or a combination thereof. Aluminum flake may additionally confer heat resistance, moisture resistance, UV resistance, or a combination thereof to a coating and/or film. Aluminum flake may also be stearate modified for use in a topcoat. However, aluminum flake may produce gas in a coating comprising more than 0.15% water. A metallic zinc pigment (e.g., zinc flake, zinc dust) acts by functioning as an anode instead of the metal surface (e.g., steel). However, the effectiveness of a coating’s corrosion resistance fades as the zinc pigment is used up in protective reactions. A metallic zinc primer may be selected for a primer, particularly in combination with an epoxy topcoat, a urethane topcoat, or a combination thereof.

Red lead and/or basic lead silicochromate can confer an orange color, and may be selected for combination with an oil-based coating (e.g., a primer), as the pigment chemically reacts with an oil-based binder to produce a corrosion resistant lead soap in the coating and/or film. Red lead and/or basic lead is typically selected for a primer in an industrial steel coating.

A barium metaborate pigment acts by retarding an anodic process. A barium metaborate pigment is usual chemically modified by combination with silica to reduce solubility. A zinc borate combined with a zinc phosphate, a modified barium metaborate, or combination thereof demonstrates synergistic enhancement of corrosion resistance, as well as flame retardancy.

Zinc potassium chromate may confer a yellow color as well as an anticorrosive property. Zinc tetraoxylchromate can also confer a yellow color, and is typically selected for use in a two pack poly(viny butyryl) primer. Zinc oxide may be selected for an oleoresinous coating, a waterborne coating, a primer, or a combination thereof, and may be combined with a zinc chromate and/or calcium borosilicate, and additionally may improve thermosetting crosslinking density and/or act as a UV absorber. Strontium chromate may confer a yellow color, and may be selected for an aluminum surface, an aircraft primer,’ or a combination thereof. Strontium chromate may be combined with a zinc chromate in a water-borne coating, though it is preferred that total chromate content is less from 0.001% to 2%. Ammonium chromate, barium chromate and calcium chromate may be selected as a corrosion inhibitor, particularly as a flash rust inhibitor.

A zinc molybdate, zinc phosphate, zinc hydroxy phosphite, or a combination thereof may confer a white color. These zinc pigments function by reducing an anodic process, though zinc

48ggggg gg hydroxy phosphite may form corrosion resistant soap in an oleoresinous-coating. Basic zinc molybdate typically is selected for an alkyd-coating, an epoxide-coating, an epoxy ester-coating, a polyester-coating, a solvent-borne coating, or a combination thereof. Basic zinc molybdatephosphate is similar to basic zinc molybdate, though it may provide superior corrosion resistance for a rusted steel surface. Basic calcium zinc molybdate may be selected for a water-borne coating, a two-pack polyurethane coating, a two-pack epoxy coating, or a combination thereof. A combination of basic calcium zinc molybdate and zinc phosphate may confer a superior adhesion property to a surface comprising iron, and may be selected for a water-borne coating or a solventborne coating. A zinc phosphate may be selected for an alkyd coating, a water-reducible coating, a coating cured by an acid and baking, or a combination thereof. A zinc phosphate is less preferred for a marine coating for salt water embodiments. A modified zinc phosphate, such as, for example, aluminum zinc phosphate, basic zinc phosphate hydrate, zinc silicophosphate hydrate, basic zinc molybdenum phosphate, or a combination thereof may confer improved corrosion resistance for a salt water embodiment. Zinc hydroxy phosphite may be selected for a solvent-borne coating.

An aluminum triphosphate typically confers a white color, acts by chelating iron ions, and is preferred for a surface that comprises Iron. A grade I aluminum triphosphate is modified with zinc and silicate, and may be selected for an alkyd-coating, an epoxy coating, a solvent-borne coating, a primer, or a combination thereof. A grade II aluminum triphosphate is modified with zinc and silicate, and may be selected for a water-borne coating or a solvent-borne coating. A grade III aluminum triphosphate is modified with zinc, and may be selected for a water-borne coating or a solvent-borne coating.

A silicate pigment such as barium borosilicate, calcium borosilicate, strontium borosilicate, zinc borosilicate, a calcium barium phosphosilicate, a calcium strontium phosphosilicate, a calcium strontium zinc phosphosilicate, or a combination thereof, typically acts through inhibiting an anodic or cathodic process, as well as forming a corrosion resistant soap in an oleoresinous-coating. A grade I and/or 111 calcium borosilicate may be selected for a medium oil alkyd-coating, a long oil alkyd, an epoxy ester-coating, a solvent-borne coating, an architectural coating, an industrial coating, or a combination thereof, but is less preferred for a marine coating, an epoxide-coating, a water-borne coating, or a combination thereof. Calcium barium phosphosilicate grade I pigment may be selected for a solvent-borne epoxy-coating, to confer an antisettling property to a primer comprising zinc, or a combination thereof. Calcium barium phosphosilicate grade II pigment may be selected for a water-borne coating, an alkydcoating, or a combination thereof. Calcium strontium phosphosilicate may be selected for a water-borne acrylic lacquer, a water-borne sealant, or a combination thereof. In aspects wherein

48hhhhh hh a water-borne acrylic lacquer comprises calcium strontium phosphosilicate, it is preferred that a

1:1 ratio of zinc phosphate pigment is included. Calcium strontium zinc phosphosilicate may be selected for an alkyd-coating, an epoxide coating, a coating cured by a catalyst and baking, a water-borne coating, or a combination thereof.

(2) Camouflage Pigments

A camouflage pigment refers to a pigment typically selected to camouflage a surface (e.g., a military surface) from visual and, more preferred, infrared detection. Examples of a camouflage pigment include an anthraquinone black, a chromium oxide green, or a combination thereof. A chromium oxide green may be selected for embodiments wherein good chemical resistance, dull color, good heat stability, good infrared reflectance, good light fastness, good opacity, good solvent resistance, low tinctorial strength, or a combination thereof, is suitable. Anthraquinone black (Cl Pigment Black 20) may be selected for good light fastness and moderate solvent resistance, and is often selected for camouflage coatings, due to its Infrared absorption property.

(3) Color Property Pigments

A color property is the ability of a composition to confer a visual color and/or metallic appearance to a coating and/or a coated surface. Color pigments are often categorized by a common name recognized within the art, which often encompasses several specific color pigments, each identified by a Cl number.

(i) Black Pigments

A black pigment is a pigment that confers a black color to a coating. Examples of black pigments, identified by common name with examples of specific pigments in parentheses, include aniline, black; anthraquinone black; carbon black; copper carbonate; graphite; iron oxide; micaceous iron oxide; manganese dioxide; or a combination thereof.

Aniline black (e.g., Cl Pigment Black 1); may be selected for a deep black color (e.g., strong light absorption, low light scattering) and/or fastness. Coatings comprising aniline black typically comprise relatively higher concentrations of binder, and thus often possesses a matt property.

Anthraquinone black (e.g. Ci Pigment Black 20) may be selected for good light fastness and moderate solvent resistance.

48iiiiiii

Carbon black (e.g., Cl Pigment Black 6, Cl Pigment Black 7, Cl Pigment Black 8) generally possesses properties such as chemical stability, good light fastness, good solvent resistance, heat stability, or a combination thereof. Carbon black is often categorized into separate grades, based on the intensity of black color (jetness”). To reduce flocculation in preparing a coating comprising a carbon black pigment, such pigments may be incrementally added to a coating during preparation, chemically modified by surface oxidation, chemically modified by an organic compound (e.g., a carboxylic acid), or a combination thereof. Additionally, a carbon black pigment may absorb certain other coating components such as a metal soap drier. Typically, increasing the concentration of the susceptible component by, for example, two-fold will reduce this effect. A high jet channel black pigment is often selected for use in an automotive coating wherein a high jetness is desired. The other grades of carbon black pigments are often selected for architectural coatings.

Graphite (e.g., Cl Pigment Black 10) may be selected for properties such as relative chemically inertness, low in color intensity, low in tinctorial strength, an anti-corrosive property, an increase in coating spreading rate, or a combination thereof.

Iron oxide (e.g., Cl Pigment Black 11) may be selected for properties such as good chemical resistance, relative inertness, good solvent resistance, limited heat resistance, low tinctorial strength, or a combination thereof. Iron oxide possesses superior floating resistance than carbon black, particularly in combination with titanium dioxide.

Micaceous iron oxide may be selected for properties such as relative inertness, grayish appearance, shiny appearance, function as a UV absorber, function as an anti-corrosive pigment due to resistance to oxygen and moisture passage. However, over-dispersal of a micaceous iron oxide during coating preparation may damage the pigment.

(ii) Brown Pigments

A brown pigment is a pigment that confers a brown color to a coating. Examples of a brown pigment include azo condensation (Cl Pigment Brown 23, Cl Pigment Brown 41, Cl Pigment Brown 42); benzimidazolone (Cl Pigment Brown 25); iron oxide; metal complex brown; or a combination thereof. A synthetically produced iron oxide brown (Cl Pigment Brown 6, Cl Pigment Brown 7) may be selected for embodiments wherein a rich brown color, good lightfastness, or a combination thereof is suitable. A metal complex brown (Cl Pigment Brown 33) may be selected for embodiments wherein high heat stability, good fastness, or a combination thereof is suitable, A metal complex brown may be used, for example, in a coil coating, a coating for a ceramic surface, or a combination thereof.

48jjjjjij (ill) White Pigments

A white pigment is a pigment that confers a white color to a coating. Examples of a white pigment include antimony oxide; basic lead carbonate (Cl Pigment White 25); lithopone, titanium dioxide; white lead; zinc oxide; zinc sulphide (Cl Pigment White 7); or a combination thereof.

Antimony oxide (Cl Pigment White 11) is chemically inert, and used in fire resistant coatings. In some embodiments, antimony oxide may be combined with titanium dioxide, particularly in a coating where chalking is undesirable and a white color in the coating is desired.

Titanium dioxide (Cl Pigment White 6) is resistant to heat, many chemicals, and organic solvents, allowing use in many different applications where such properties are desirable. Titanium dioxide may be in the form of a crystal, such as an anatase crystal, a rutile crystal, or a combination thereof. Rutile is more opaque than anatase. Anatase has a greater ability to chalk and is whiter in color than rutile. In aspects wherein chalking is undesirable, a titanium dioxide crystal may be reacted with an inorganic oxide to enhance chalking resistance. Examples of such inorganic oxides include aluminum oxide, silicon oxide, zinc oxide, or a combination thereof.

White lead (Cl Pigment White 1) is chemically reactive with acidic binders to form strong films with elastic properties, but also chemically reacts with sulphur to become black in color. It is less preferred in certain coatings due to the toxic nature of lead.

Zinc oxide (Cl Pigment White 4) confers desirable properties such as resistance to mildew, as well as chemically reacting with oleoresin binders in film formation to enhance resistance to abrasion, to enhance resistance to moisture, to enhance hardness, and/or reduce chalking. However, these reactions may undesirably occur during storage. In some embodiments, it may be combined with titanium dioxide, particularly in a coating comprising an oleoresin binder when chalking is undesirable and a white color in the coating is desired.

Zinc sulfide (Cl Pigment White 7) is chemically inert, and confers a strong chalking property. In certain embodiments, a zinc sulfide comprises a lithopone. A lithopone (Cl Pigment White 5) comprises a mixture of ZnS and barium sulphate (BaSO&lt;)_t usually from 30% to 60% ZnS and 70% to 40% BaSO«.

48kkkkk kk

65-3T (iv) Pearlescent Pigments

A pearlescent pigment is a pigment that confers a pearl-like appearance to a coating.

Examples of a white pigment include titanium dioxide and ferric oxide covered mica, bismuth oxychloride crystal, ora combination thereof.

(v) Violet Pigments

A violet pigment is a pigment that confers a violet color to a coating. However, a violet pigment is often used in combination with a red pigment or a blue pigment to produce a desirable color of an intermediate hue between red and blue. Additionally, a violet pigment is often combined with titanium dioxide to balance the slight yellow color of that white pigment. An example of a violet pigment includes dioxanine violet (Cl Pigment Violet 23; Cl Pigment Violet 37). A dioxazine violet may be selected for embodiments wherein high heat stability, good light fastness, good solvent fastness, or a combination thereof is suitable. Cl Pigment Violet 23 (carbazole violet”) is relatively transparent and bluer than Cl Pigment 37, and is typically used in a metallic coating. A dioxazine violet is susceptible to flocculation, loss in a powder coating, or a combination thereof, due to small particle size.

(vl) Blue Pigments

A blue pigment is a pigment that confers a blue color to a coating. Examples of a blue pigment include carbazol Blue; carbazole Blue; cobalt blue; copper phthalocyanine; dioxanine Blue; indanthrone; phthalocyanin blue; Prussian blue; ultramarine; or a combination thereof.

A cobalt blue (Cl Pigment Blue 36) may be selected for embodiments wherein good chemical resistance, good iightfastness, good solvent fastness, or a combination thereof, is suitable. An indanthrone (Cl Pigment Blue 60) may be selected for embodiments wherein a redish-blue hue, good chemical resistance, good heat resistance, good solvent fastness, transparency, superior resistance to flocculation relative to a copper phthalocyanine, or a combination thereof, is suitable.

A copper phthalocyanine (Cl Pigment Blue 15, Ci Pigment Blue 15:1, Cl Pigment Blue 15:2, Ct Pigment Blue 15:3, Cl Pigment Blue 15:4, Cl Pigment Blue 15:6, Cl Pigment Blue 16) may be selected for embodiments wherein good color strength, good tinctorial strength, good heat stability, good lightfastness, good solvent resistance, transparency, or a combination thereof, is suitable. CI Pigment Blue 15 is redish in hue, but is chemically unstable upon contact with an aromatic hydrocarbon, and converts to a greenish blue compound. Cl Pigment Blue 15:1 is form of Cl Pigment Blue 15 chemically stabilized by chlorination, greener, and tinctorially weaker than Cl Pigment Blue 15. Cl Pigment Blue 15:2 is modified form of Cl Pigment Blue 15 that is

481111111 resistant to flocculation. Cl Pigment Blue 15:3 is greenish-blue, while Cl Pigment Blue 15:4 is modified form of Cl Pigment Blue 15:3 that is resistant to flocculation. Cl Pigment Blue 16 is relatively transparent. Examples of coatings wherein copper phthalocyanine are used include a metallic automotive coating. However, as described above, a copper phthalocyanine may be susceptible to flocculation due to small primary particle size, and various modified forms are known wherein flocculation is reduced. Examples of modifications used to reduce flocculation adding a sulfonic acid moiety; a sulfonic acid moiety and a long chain amine moiety; an aluminum benzoate; an acidic binder (e.g., a rosin); a chloromethyl moiety; or a combination thereof, to the phthalocyanine. A modified phthalocyanine may be selected for embodiments wherein superior color shade, dispersibility, gloss, or a combination thereof is suitable.

A Prussian blue (Cl Pigment Blue 27) may be selected for embodiments wherein a strong color, good heat stability, good solvent fastness, or a combination thereof is suitable. However, a Prussian blue is chemically unstable in alkali conditions. An ultramarine (Cl Pigment Blue 29) may be selected wherein a strong color, good heat stability, good light fastness, good solvent resistance, or a combination thereof is suitable. However, an ultramarine is chemically unstable in acidic conditions.

(vii) Green Pigments

A green pigment is a pigment that confers a green color to a coating. However, often a “green pigment comprises a mixture of a yellow pigment and a blue pigment, with the properties of each component pigment generally retained. Examples of a green pigment include chrome green; chromium oxide green; halogenated copper phthalocyanine; hydrated chromium oxide; phthalocyanine green; or a combination thereof.

A chrome green (Brunswick green, Cl Pigment Green 15) comprises a combination of a Prussian blue and/or a copper phthalocyanine blue and a chrome yellow. A coating comprising a chrome green may be susceptible to floating and flooding defects. A chromium oxide green (Cl Pigment Green 17) may be selected for embodiments wherein good chemical resistance, dull color, good heat stability, good infrared reflectance, good light fastness, good opacity, good solvent resistance, low tinctorial strength, or a combination thereof is suitable. A hydrated chromium oxide (Cl Pigment Green 18) is similar to chromium oxide, and may be selected for embodiments wherein good light fastness, relatively brighter appearance, relatively greater transparency, relatively less heat stability, relatively less acid stability, or a combination thereof, is suitable. A phthalocyanine green (Cl Pigment Green 7, Cl Pigment Green 36) may be selected for embodiments wherein good chemical resistance, good heat stability, good light fastness, good solvent resistance, good tinctorial strength, color transparency, or a combination thereof Is

48mmm mmmm suitable. Cl Pigment Green 7 may be selected for a bluish green color, while Cl Pigment Green may be selected for a yellower-greenish color. A phthalocyanine green is often selected for an automotive coating (e.g., a metallic coating), an industrial coating, an architectural coating, a powder coating, or a combination thereof.

(vtli) Yellow Pigments

In certain embodiments, a coating may comprise a yellow pigment. A ״yellow pigment is a pigment that confers a yellow color to a coating. Examples of a yellow pigment include anthrapyrimidine; arylamide yellow; barium chromate; benzimidazolone yellow; bismuth vanadate (Cl Pigment Yellow 184); cadmium sulfide yellow (Cl Pigment Yellow 37); complex inorganic color pigment; diarylide yellow; disazo condensation; flavanthrone; isoindoline; isoindolinone; lead chromate; nickel azo yellow; organic metal complex; quinophthalone; yellow iron oxide; yellow oxide; zinc chromate; or a combination thereof.

An anthrapyrimidine pigment (Cl Pigment Yellow 108) may be selected for embodiments wherein, moderate light fastness, moderate solvent resistance, a dull color, transparency, or a combination thereof is suitable.

An arylamide yellow (Hansa® yellow,” Cl Pigment Yellow 1, Cl Pigment Yellow 3, Cl Pigment Yellow 65, Cl Pigment Yellow 73. Cl Pigment Yellow 74, Cl Pigment Yellow 75, Cl Pigment Yellow 97, Cl Pigment Yellow 111) may be selected for embodiments wherein, poor heat stability, good light fastness, poor solvent resistance, moderate tinctorial strength, or a combination thereof is suitable. Cl Pigment 1 and Cl Pigment 74 are mid-yellow in hue. Cl Pigment Yellow 3 is greenish in hue. Cl Pigment Yellow 73 is mid-yellow in hue, and resistant to recrystalization during dispersion. Cl Pigment 97 possesses superior solvent fastness than other arylamide yellow pigments, and has been used in a stoving enamel, an automotive coating, or a combination thereof. Other arylamide yellow pigments may be used in a water-borne coating, a coating comprising a white spirit liquid component, or a combination thereof.

A benzimidiazoIone yellow (Cl Pigment Yellow 120, Cl Pigment Yellow 151, Cl Pigment Yellow 154, Cl Pigment Yellow 175, Cl Pigment Yellow 181, Cl Pigment Yellow 194) may be selected for embodiments wherein, good chemical resistance, good heat stability, good light fastness, good solvent resistance, or a combination thereof is suitable. A benzimidiazolone with larger particle size been used in an automotive coating, a powder coating, or a combination thereof.

48nnnnn nn

A cadmium sulfide yellow (Cl Pigment Yellow 37) may be selected for embodiments wherein good stability in basic pH, good heat stability, good light fastness, good opacity, good solvent fastness, or a combination thereof is suitable. However, a cadmium yellow comprises cadmium, which may limit suitability relative to an environmental law or regulation,

A complex inorganic color pigment (“mixed phase metal oxide, Cl Pigment Yellow 53, Cl Pigment Yellow 119, Cl Pigment Yellow 164); may be selected for embodiments wherein, good chemical stability, good heat resistance, good light fastness, good opacity, good solvent fastness, or a combination thereof is suitable. However, a complex inorganic color pigment generally produces a pale color, and is often combined with an additional pigment (e.g., an organic pigment). A complex inorganic color pigment is often selected for an automotive coating, a coil coating, or a combination thereof. A bismuth vanadate is similar to a complex inorganic pigment, but possesses superior color of green-yellow hue, poorer light fastness, and greater use in a powder coating. A bismuth vanadate is often combined with a light stabilizer.

A diarylide yellow (Cl Pigment Yellow 12, Cl Pigment Yellow 13, Cl Pigment Yellow 14, Cl Pigment Yellow 17, Cl Pigment Yellow 81, Cl Pigment Yellow 83) may be selected for embodiments wherein, good chemical resistance, poor light fastness, good solvent resistance, good tinctorial strength, or a combination thereof is suitable. A diarylide yellow is not stable at a temperature of 200°C or greater. Cl Pigment Yellow 83 has superior light fastness than other diarylide yellow pigments, and has been used in an industrial coating, a powder coating, or a combination thereof.

A diazo condensation pigment (Cl Pigment Yellow 93, Cl Pigment Yellow 94, Cl Pigment Yellow 95, Cl Pigment Yellow 128, Cl Pigment Yellow 166) may be selected for embodiments wherein, good chemical resistance, good heat stability, good solvent resistance, good tinctorial strength, or a combination thereof is suitable. A diazo condensation pigment typically is used in plastics, though Cl Pigment Yellow 128 has been used in a coating such as an automotive coating.

A flavanthrone pigment (Cl Pigment Yellow 24) may be selected for embodiments wherein, good heat stability, moderate light fastness, a reddish yellow hue superior to an anthrapyrimidine, transparency, or a combination thereof is suitable.

An isoindoline yellow pigment (Cl Pigment Yellow 139, Cl Pigment Yellow 185) may be selected for embodiments wherein, good chemical resistance, good heat stability, good light fastness, good solvent resistance, moderate tinctorial strength, or a combination thereof is

4800000 00 suitable. An isoindolinone yellow pigment (Cl Pigment Yellow 109, Cl Pigment Yellow 110, Cl Pigment Yellow 173) typically has been used in an automotive coating or an architectural coating. An isoindoline yellow pigment may be selected for embodiments wherein, good light fastness, good tinctorial strength, or a combination thereof is suitable. However, an isoindoline pigment is not stable in a basic pH. An isoindoline yellow pigment typically has been used in an industrial coating.

A lead chromate (Cl Pigment Yellow 34) may be selected for embodiments wherein moderate heat stability, low oil absorption, good opacity, good solvent resistance, or a combination thereof is suitable. However, a lead chromate is susceptible to an acidic or a basic pH, and a lower light fastness so that the pigment darkens upon Irradiation by light. The pH and lightfastness properties of commercially produced lead chromate are often improved by treatment of a lead chromate with siiica, antimony, alumina, metal, or a combination thereof. Additionally, a lead chromate comprises lead and/or chromium, which may limit suitability relative to an environmental law or regulation. A lead chromate may comprise a lead sulfate, which is used to modify color. Examples of lead chromates include a iemon chrome, which comprises from 20% to 40% lead sulfate and is greenish yellow in color; a middle chrome, which comprises little lead sulfate and is reddish yellow in color; orange chrome, which comprises no detectable lead sulfate; and primrose chrome, which comprises from 45% to 55% lead chrome and is greenish yellow in color.

An organic metal complex (Cl Pigment Yellow 129, Cl Pigment Yellow 153) may be selected for embodiments wherein good solvent resistance is suitable. An organic meta! complex typically is transparent and dull in color.

A quinophthalone pigment (Cl Pigment Yellow 138) may be selected for embodiments wherein, good heat stability, good light fastness, good solvent resistance, a reddish yellow hue, or a combination thereof is suitable. A quinophthalone can be either highly opaque or transparent, A quinophthalone pigment has been used as a substitute for chrome as a pigment.

A yellow iron oxide (Cl Pigment Yellow 42, Cl Pigment Yellow 43) may be selected for embodiments wherein good covering power, good disperability, good resistance to chemicals, good light fastness, good solvent resistance, a yellow with greenish hue is desired, or a combination thereof is suitable. A yellow iron oxide can function as a U.V. absorber. However, a yellow iron oxide is generally of duller color relative to other pigments, and is susceptible to temperatures of 105°C or greater. Additionally, a yellow iron oxide may comprise a a-crystal, a βcrystal, a γ-crystal, or a combination thereof. Overdispersion may damage the needle-shape

48ρρρρρ

PP crystal structure, which can reduce the color intensity. Additionally, a transparent yellow iron oxide can be prepared by selecting particles with minimum size, and such a pigment is used, for example, in an automotive coating or a wood coating.

(ix) Orange Pigments

In certain embodiments, a coating may comprise an orange pigment. An “orange pigment is a pigment that confers an orange color to a coating. Examples of an orange pigment include perinone orange; pyrazolone orange; or a combination thereof.

A perinone orange pigment (Cl Pigment Orange 43) may be selected for embodiments wherein very good resistance to heat, good light fastness, good solvent resistance, high tinctorial strength, or a combination thereof is suitable.

A pyrazolone orange pigment (Ci Pigment Orange 13, Cl Pigment Orange 34) is similar to a diarylide yellow pigment, and may be selected for embodiments wherein moderate resistance to heat, poor light fastness, moderate solvent resistance, high tinctorial strength, or a combination thereof is suitable. However, Cl Pigment Orange 34 possesses greater lightfastness relative to Cl Pigment Orange 13, and has been used in an industrial coating and/or a replacement for chrome.

(x) Red Pigments

In certain embodiments, a coating may comprise a red pigment. A “red pigment is a pigment that confers a red color to a coating. Examples of an red pigment include anthraquinone; benzimidazolone; BON arylamide; cadmium red; cadmium selenide; chrome red; dibromanthrone; diketopyrrolo-pyrrole pigment (Cl Pigment Red 254, Cl Pigment Red 255, Cl Pigment Red 264, Cl Pigment Red 270, Cl Pigment Red 272); disazo condensation pigment (Cl Pigment Red 144, Cl Pigment Red 166, Cl Pigment Red 214, Cl Pigment Red 220, Cl Pigment Red 221, Cl Pigment Red 242); lead molybdate; perylene; pyranthrone; quinacridone; quinophthalone; red iron oxide; red lead; toluidine red; tonor pigment (Cl Pigment Red 48, Cl Pigment Red 57, Cl Pigment Red 60, Cl Pigment Red 68); β-naphthol red; or a combination thereof.

A lead molybdate red pigment (Cl Pigment Red 104) may be selected for embodiments wherein good resistance to heat, moderate resistance to basic pH, good opacity, excellent solvent resistance, or a combination thereof is suitable. A molybdate red is bright in color, and is often combined with an organic pigment to extend a color range. However, a molybdate is easy to disperse, and overdispersion may damage this pigment. Additionally, a molybdate red

48qqqqq qq comprising lead and/or chromium may have limited suitability relative to an environmental law or regulation.

A cadmium red pigment (Cl Pigment Red 108) may be selected for embodiments wherein excellent resistance to heat, good lightfastness, poor resistance to acidic pH, good opacity, excellent solvent resistance, or a combination thereof is suitable. However, a cadmium red comprises cadmium, and may have limited suitability relative to an environmental law or regulation.

A red iron oxide pigment (Cl Pigment Red 101, Cl Pigment Red 102) may be selected for embodiments wherein excellent resistance to heat, good lightfastness, poor resistance to acidic pH, good opacity, excellent solvent resistance, or a combination thereof is suitable. However, a cadmium red comprises cadmium, and may have limited suitability relative to an environmental law or regulation.

β-naphthol red (Cl Pigment Red 3) may be selected for embodiments wherein modest heat resistance, good lightfastness, modest solvent resistance, or a combination thereof is suitable.

BON arylamide (Cl Pigment Red 2, Cl Pigment Red 5, Cl Pigment Red 12, Cl Pigment Red 23, Cl Pigment Red 112, Cl Pigment Red 146, Cl Pigment Red 170) comprises various pigments that generally have good lightfastness, good solvent resistance, or a combmation thereof.

Tonor pigment (Cl Pigment Red 48, Cl Pigment Red 57, Cl Pigment Red 60, Cl Pigment Red 68) comprises various pigments that generally have good solvent resistance, but often have poor acid resistance, poor alkali resistance, or a combination thereof.

Benzimidazolone (Cl Pigment Red 171, Cl Pigment Red 175, Cl Pigment Red 176, Cl Pigment Red 185, Cl Pigment Red 208) comprises various pigments that generally have good heat stability, excellent solvent resistance, or a combination thereof.

Disazo condensation pigment (Cl Pigment Red 144, Cl Pigment Red 166, Cl Pigment Red 214, Cl Pigment Red 220, Cl Pigment Red 221, Cl Pigment Red 242) comprises various pigments that generally have excellent heat stability, good solvent resistance, or a combination thereof.

׳481111111

Quinacridone (Cl Pigment Red 122, Cl Pigment Red 192, Cl Pigment Red 202, Cl

Pigment Red 207, Cl Pigment Red 209) comprises a various pigments that generally have bright color, excellent heat stability, excellent solvent resistance, excellent chemical resistance, good lightfastness, or a combination thereof.

Perylene (Cl Pigment Red 123, Cl Pigment Red 149, Cl Pigment Red 178, Cl Pigment Red 179, Cl Pigment Red 190, Cl Pigment Red 224) comprises a various pigments that generally have excellent heat stability, excellent solvent resistance, excellent lightfastness, or a combination thereof.

Anthraquinone (Cl Pigment Red 177) has a bright color, good heat stability, good solvent resistance, good lightfastness, or a combination thereof.

Dibromanthrone (Cl Pigment Red 168) has a bright color, moderate heat stability, good solvent resistance, excellent lightfastness, or a combination thereof.

Pyranthrone (Cl Pigment Red 216, Cl Pigment Red 226) has a dull color, moderate heat stability, good solvent resistance, poor lightfastness in combination with titanium dioxide, or a combination thereof.

Diketopyrrolo-pyrrole pigment (Ct Pigment Red 254, Cl Pigment Red 255, Cl Pigment Red 264, Cl Pigment Red 270, Cl Pigment Red 272) comprises a various pigments that generally have a bright color, good opacity, excellent heat stability, excellent solvent resistance, or a combination thereof.

(xl) Metallic Pigments

In certain embodiments, a coating may comprise a metallic pigment. A metallic pigment is a pigment that confers a metallic appearance to a coating, and as previously described, is often a corrosion resistance pigment. A metallic pigment may be selected for a topcoat, particularly to confer a metallic appearance, a primer, particularly to confer a corrosion resistance property, an automotive coating, an industrial coating, or a combination thereof. Metallic flake pigments are preferred for embodiments wherein UV and/or infrared resistance is to be conferred to a coating. Additionally, as some enzymes comprise a metal atom in the active site, inclusion of a metallic pigment and/or other composition comprising a metal during coating preparation, or addition fater (e.g., a multipack coating) may stimulate a desired enzyme activity. Examples of a metallic pigment include aluminum flake (Cl Pigment Metat 1); aluminum non-leafing, gold bronze flake, zinc dust, stainless steel flake, nickel (e.g., flake, powder), or a combination thereof.

48ssssss s (4) Extender Pigments

An extender pigment (“inert pigment,״ “extender, inert, filler”) is a substance that is insoluble in the other components of a coating, and further confers a desirable optical property (e.g., opacity, gloss), a rheological property, physical property, an antisettling property, or a combination thereof, to the coating and/or film. An extender pigment is often white or near white in color, and typically are used to provide a cheap partial substitute for a more expensive white pigment (e.g., titanium dioxide). Often an extender has a refractive index below 1.7. In some aspects, an extenders refractive index is 1.30 to 1.70, including all intermediate ranges and combinations thereof. Examples of an inorganic extender include a barium sulphate (Cl Pigment White 21, Cl Pigment White 22); 1); a calcium carbonate (Cl Pigment White 18); a calcium sulphate; a silicate (Cl Pigment White 19, Cl Pigment White 26); a silica (Cl Pigment White 27); or a combination thereof.

Calcium carbonate (“calcite, “whiting, “limestone.” Cl Pigment White 18) is generally chemically inert with the exception of reactions with an acid. Calcium carbonate may be used in a water-borne coating or a solvent-borne coating. Properties specifically associated with calcium carbonate include conferring settling resistance, sag resistance, or a combination thereof. Precipitated calcium carbonate obtained from processing of limestone, and may have superior opacity.

Kaolin (“china clay”) is typically selected for a latex coating, an alkyd coating, an architectural coating, or a combination thereof. In addition to the typical properties of an extender (e.g., opacity), kaolin can confer scrub resistance to a coating.

Talc is a hydrated magnesium aluminum silicate, and is soluble in water. Talc may be selected for an architectural coating (e.g., interior, exterior), a primer, a traffic marker coating, an industrial coating, or a combination thereof. Talc comprising a platy particle shape can confer chemical resistance, water resistance, improved flow property, or a combination thereof.

Silica is silicon dioxide, and may be classified as crystalline silica, diatomaceous silica or synthetic silica. Crystalline silica is produced from crushed and ground quartz, and may be selected for an architectural coating, an industrial coating, a primer, a latex coating, a powder coating, or a combination thereof. Crystalline silica may confer burnish resistance to a coating and/or film. Diatomaceous silica (“diatomaceous earth, diatomite) is the mineral fossil of diatoms which were single celled aquatic plants. Diatomaceous silica may be selected for an architectural coating, a latex coating, or a combination thereof. Diatomaceous silica may also function as a flattening agent. Synthetic silica is produced from chemical reactions, and includes, for example, precipitated silica, fumed silica, or a combination thereof. Precipitated silica may be selected for an industrial coating, a solvent-borne coating, or a combination thereof. Precipitated silica may also function as a flattening agent. Fumed silica may be selected for an industrial coating. Fumed silica may also function as a flattening agent, a rheology modifier, or a combination thereof.

Mica is a hydrous silica aluminum potassium silicate, and typically comprises plate shaped particles. Mica may be selected for an architectural coating, an exterior coating, a traffic marker coating, a primer, or a combination thereof. Mica may also confer durability, moisture resistance, corrosion resistance, heat resistance, chemical resistance, cracking resistance, sagging resistance, or a combination thereof, to a coating and/or film.

Barium sulfate may be classified as baryte or a blanc fixe. Baryte may be selected for an automotive coating, an industrial coating, a primer, an undercoat, or a combination thereof. Blanc fixe has good opacity for an extender, and may be selected for an automotive coating, an industrial coating, ora combination thereof.

Wollastonite is a calcium metasiiicate, and may be selected for a latex coating. Wollasonite may also function as an alkali pH buffer. Surface modified wollasonite may be selected for an industrial coating.

Nepheline syenite is an anhydrous sodium potassium aluminum silicate, and may be selected for an architectural coating, a latex coating, an interior coating, an exterior coating, or a combination thereof. Nepheline syenite may function may confer cracking resistance, scrub resistance, or a combination thereof.

Sodium aluminosilicate may be selected for a latex coating, an architectural coating, or a combination thereof. Sodium aluminosilicate may also function as a flattening agent.

Alumina trihydrate may be selected for an architectural coating, a thermoplastic coating, a thermosetting coating, or a combination thereof. Alumina trihydrate may confer flame retardancy to a film.

b. Dyes

A dye is a composition that is soluble in the other components of a coating, and further confers a desirable color property to the coating. It is contemplated that many of the compounds

48uuuuu uu that give a microorganism derived particulate material of the present invention colorsuch as photosynthetic pigment and/or carotenoid pigment, will be partly or fully soluble in many nonaqueous liquids described herein. It is further contemplated that a microorganism derived particulate material of the present invention is added to a coating comprising such a liquid component, the material may act as a dye, as well as a pigment and/or extender, due to the dissolving of colored compounds into the liquid component.

4. Coating Additives

A coating additive is any material which is added to a coating to confer a desirable property other than that described for a binder, a liquid component, a colorizing agent, or a combination thereof. It is contemplated that, in addition to the examples of additives described herein, any additive known to one of ordinary skill in the art, in light of the present disclosures, may be included in a composition of the present invention.

Examples of coating additives include a microorganism based particulate material of the present invention, as well as an antifloating agent, an antiflooding agent, an antifoaming agent, an antisettling agent, an antiskinning agent, a catalyst, a corrosion inhibitor, a film-formation promoter, a leveling agent, a matting agent, a neutralizing agent, a preservative, a thickening agent, a wetting agent, or a combination thereof. The content for an individual coating additive in a coating generally is 0.001% to 20.0%, including all intermediate ranges and combinations thereof. However, in most embodiments, it is contemplated the concentration of a single additive in a coating will comprise between 0.001% and 10,0%, including all intermediate ranges and combinations thereof.

a. Preservatives

A coating may comprise a preservative to reduce or prevent the deterioration of a coating and/or film by a microorganism. As would be known to one of ordinary skill in the art, a microorganism is generally considered a contaminant capable damaging a film and/or coating the point of suitable usefulness in a given embodiment. A surprising and unexpected aspect of the present invention is the discovery of the suitability of a microorganism based particulate material of the present invention for use as a purposefully added coating component. However, it is preferred that a coating comprising a microorganism based particulate material of the present invention also comprises a preservative. It is contemplated that continued growth of a microorganism from the microorganism base particulate material of the present invention would be detrimental to a coating and/or film, and a preservative may reduce or prevent such growth. It is further contemplated that a contaminating microorganism could use the microorganism based particulate material of the present Invention as a readily available source of nutrients for growth,

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and a preservative may reduce or prevent such growth. It is also contemplated that the amount of preservative added to a coating comprising a microorganism based particulate material of the present invention may be increased relative to a preservative content of a similar coating lacking such an added microorganism based particulate material. In certain aspects, it is contemplated that the amount of preservative may be increased 1.1 to 10-fold or more, including all intermediate ranges and combinations thereof, the amount of an example of a preservative content described herein or as would be known to one of ordinary skill in the art in light of the present disclosures.

Examples of preservatives include a biocide, which kills an organism, a biostatic, which reduces or prevents the growth of an organism, or a combination thereof. Examples of a biocide include, for example, a bactericide, a fungicide, an algaecide, or a combination thereof. Examples of bacteria commonly found to contaminate a coating and/or film include Pseudomonas spp., Aerobacter spp., Enterobacter spp., Flavobacterium spp. (e.g., Flavobacterium marinum). Bacillus spp., or a combination thereof. Examples of fungi commonly found to contaminate a coating and/or film include Aureobasidium pullulans, Alternaria dianthicola, Phoma pigmentivora, or a combination thereof. Examples of algae commonly found to contaminate a coating and/or film include Oscillotoria sp., Scytonema sp., Protoccoccus sp., or a combination thereof. Techniques are known to those of ordinary skill in the art for determining microbial contamination of a coating and/or coating component (see, for example, ASTM Book of Standards, Volume 06.01, Paint - Tests for Chemical, Physical, and Optical Properties; Appearance, D5588-97, 2002).

In addition to the disclosures herein, a preservative and use of a preservative in a coating is known to those of skill in the art, and all such materials and techniques for using a preservative in a coating may be applied in the practice of the present invention (see, for example, Flick, E. W. “Handbook of Paint Raw Materials, Second Edition, 263-285 and 879-998, 1989; in “Paint and Coating Testing Manual, Fourteenth Edition of the Gardner-Sward Handbook, (Koleske, J. V. Ed.), pp 261-267 and 6541995 ,661־; in “Paint and Surface Coatings, Theory and Practice, Second Edition, (Lambourne, R. and Strivens, T, A., Eds.), pp. 193-194, 371-382 and 543-547, 1999; Wicks, Jr., Z. W., Jones, F. N., Pappas, S. P. Organic Coatings, Science and Technology, Volume 1; Film Formation, Components, and Appearance,” pp. 318-320,1992; Wicks, Jr., Z. W., Jones, F. N., Pappas, S. P. Organic Coatings, Science and Technology, Volume 2: Applications, Properties and Performance,” pp. 145, 309, 319323־ and 340-341, 1992; and in “Paints, Coatings and Solvents, Second, Completely Revised Edition, (Stoye, D. and Freitag, W., Eds.) pp 6, 127 and 165, 1998.

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A coating, film, surface, or a combination thereof may be detrimentally affected by the presence of a living microorganism. For example, a living microorganism can alter viscosity due to damage to a cellulosic viscosifier; alter a rheological property by increasing the gelling of a coating; produce an undesirable color alteration (discoloration) by production of a colorizing agent; produce undesirable gas and increase foam; produce an undesirable odor; lower pH; damage a preservative; produce slime; reduce adhesion by a film; increase corrosion of a metal surface by moisture production by an organism; increase corrosion of a metal surface by film damage; damage a wooden surface by colonization (e.g., fungal colonization); or a combination thereof. These changes can lead to the coating and/or film becoming unsuitable for use. The undesirable growth of a microorganism is generally more prevalent in a water-borne coating, as the solvent component of a solvent borne-coating usually acts as a preservative. However, a film is generally susceptible to such damage by growth of a microorganism after loss of a solvent (e.g., evaporation) during film formation. Additionally, various bacteria (e.g., Bacillus spp.) and fungi produce spores, which are cells that are relatively durable to unfavorable conditions (e.g., cold, heat, dehydration, a biocide), and may persist in a coating and/or film for months or years prior to germinating into a damaging colony of cells.

In certain embodiments, a preservative may comprise an in-can preservative, an in-film preservative, or a combination thereof. An in-can preservative is a composition that reduces or prevents the growth of a microorganism prior to film formation. Addition of an in-can preservative during a water-borne coating production typically occurs with the introduction of water to a coating composition. Typically, an in-can preservative is added to a coating composition for function during coating preparation, storage, or a combination thereof. An in-film preservative is a composition that reduces or prevents the growth of a microorganism after film formation. In many embodiments, an in-film preservative is the same chemical as an in-can preservative, but added to a coating composition at a higher (e.g., two-fold) concentration for continuing activity after film formation.

Examples of preservatives that have been used in coatings include a metal compound (e.g., an organo-metal compound) biocide, an organic biocide, or a combination thereof. Examples of a metal compound biocide include barium metaborate, which is a fungicide and bactericide; copper(ll) 8-quinolinolate, which is a fungicide; phenylmercuric acetate, tributyltin oxide, which is less preferred for use against Gram-negative bacteria; tributyltin benzoate, which is a fungicide and bactericide; tributyltin salicylate, which is a fungicide; zinc 2-pyridinethiol-Noxide, which is a fungicide; zinc oxide, which is a fungistatic/fungicide and aigaecide; a combination of zinc-dimethyldithiocarbamate and zinc 2-mercaptobenzothiazole, which acts as a fungicide; zinc 2-pyridinethiol-N-oxide, which is a fungicide; a metal soap; or a combination

48xxxxx xx thereof. Examples of metals comprised in a metal soap biocide include copper, mercury, tin, zinc, or a combination thereof. Examples of an organic acid comprised in a metal soap biocide include a butyl oxide, a laurate, a naphthenate, an octoate, a phenyl acetate, a phenyi oleate, or a combination thereof.

An example of an organic biocide that acts as an algaecide includes 2-methylthio-4-tertbutylamino-6-cyclopropylamino-s-triazine. Examples of an organic biocide that acts as a bactericide include a combination of 4,4-dimethyl-oxazolidine and 3,4,4־trimethyloxazolidine; 5hydroxy-methyl-1-aza-3,7-dioxabicylco (3.3.0.) octane; 2(hydroxymethyl)-aminoethanol; 2(hydroxymethyl)-amino-2-methyl-1-propanol; hexyhydro-1,3,5-tri-ethyl-5-triazine; 1-(3-chloroallyl)3,5,7-triaza-1-azonia-adamantane chloride; 1-methyl-3,5,7-triaza-1-azonia-adamantane chloride; p-chloro-m-cresol; an alkylamine hydrochloride; 6-acetoxy-2,4-dimethyl-1,3-dioxane; 5-chloro-2_methy|-4-isothiazolin-3-one; 2-methyl-4-isothiazolin-3-one; 1,3-bis(hydroxymethyl)-5,5dimethylhydantoin; hydroxymethyl-5,5-dimethylhydantoin; or a combination thereof. Examples of an organic biocide that acts as a fungicide include a parabens; 2-(4-thiazolyl)benzimidazole; Ntrichloromethyl-thio-4-cyclohexene-1,2-dicarboximide; 2-n-octyl-4-isothiazoline-3־one; 2,4,5,6tetrachloro-isophthalonitrile; 3-iodo-2-propynyl butyl carbamate; N-(trichloromethyithio)phthalimide; tetrachloroisophthalonitrile; potassium N-hydroxy-methyl-N-methyldithiocarbamate; sodium 2-pyridinethiol-1-oxide; or a combination thereof. Examples of a parbens include butyl parahydroxybenzoate; ethyl parahydroxybenzoate; methyl parahydroxybenzoate; propyl parahydroxybenzoate; or a combination thereof. Examples of an organic biocide that acts as an bactericide and fungicide include 2-mercaptobenzo-thiazole; a combination of 5-chloro-2-methyl-3(2H)-isothiazoline and 2-methyl-3(2H)-isothiazolone; a combination of 4-(2-nitrobutyl)-morpholine and 4,4^l-(2-ethylnitrotrimethylene dimorpholine; tetrahydro-3,5-di-methyl-2H-1,3,5-thiadiazine-2-thione; potassium dimethyldithiocarbamate; or a combination thereof. An example of an organic biocide that acts as an algaecide and fungicide includes diiodomethyl-p-tolysulfone. Examples of an organic biocide that acts as an algaecide, bactericide and fungicide include glutaraldehyde; methylenebis(thiocyanate); 1,2-dibromo-2,4dicyanobutane; 1,2-benzisothiazoline-3-one; 2-(thiocyanomethyl-thio)benzothiazole; or a combination thereof. An example of an organic biocide that acts as an algaecide, bactericide, fungicide and molluskicide includes 2-(thiocyanomethy!-thio)benzothiozole and methylene bis(thiocyanate).

In certain embodiments an environmental law or regulation may encourage the selection of an organic biocide such as a benzisothiazolinone derivative. An example of a benzisothiazolinone derivative is Busan™ 1264 (Buckman Laboratories, Inc.), Proxel™ GXL (Avecia Inc.), or Preventol® VP OC 3068 (Bayer Corporation), which comprises 1,248yyyyy yy

־ . י 1 benzisothiazolinone (CAS No. 2634-33-5). In the case of Busan™ 1264, the primary use Is a bactericide and/or fungicide at 0.03% to 0.5% in a water-borne coating.

Often, a preservative is a proprietary commercial formulation and/or a compound sold under a tradename. Examples include organic biocides under the tradename Nuosept® (International Specialty Products), which are typically used in a water-borne coating. Specific examples of a Nuosept® biocide includes. Nuosept® 95, which comprises a mixture of bicyclic oxazolidines, and is typically added to 0.2% to 0.3% concentration to a coating composition; Nuosept® 145, which comprises an amine reaction product, and is typically added to 0.2% to 0.3% concentration to a coating composition; Nuosept® 166, which comprises 4,4dimethyloxazolidine, and is typically added to 0.2% to 0.3% concentration to a basic pH waterborne coating composition; or a combination thereof. A further example is Nuocide® (International Specialty Products) biocides, which are typically used fungicides and/or algaecides. Examples of a Nuocide® biocide is Nuocide® 960, which comprises 96% tetrachlorisophthalonitrile (CAS No. 1897-45-6), and is typically used at 0.5% to 1.2% in a waterborne or solvent-borne coating as a fungicide; Nuocide® 2010, which comprises chlorothalonil and IPBC at 30%, and is typically used at 0.5% to 2.5% in a coating as a fungicide and algaecide; Nuocide® 1051 and Nuocide® 1071, each which comprises 96% N-cyclopropyl-N-(1dimethylethyl)-6-(methylthio)-1,3,5-triazine-2,4-diamine, and is typically used as an algaecide in antifouling coatings at 1.0% to 6.0% or water-based coatings at 0.05% to 0.2%, respectively; and Nuocide® 2002, which comprises chlorothalonil and a triazine compound at 30%, and is typically used at 0.5% to 2.5% in a coating and/or a film as a fungicide and algaecide.

An additional example of a tradename biocide for coatings includes Vancide® (R. T. Vanderbilt Company, Inc,), Examples of a Vancide® biocide include Vancide® TH, which comprises hexahydro-1,3,5-trlethyl-s-triazine, and is generally used in a water-borne coating; Vancide® 89, which comprises N-trichloromethylthio-4-cyclohexene-1,2-dicarboximide and related compounds such as captan, and is used as a fungicide in a coating composition; or a combination thereof. A bactericide and/or fungicide for coatings, particularly a water-borne coating, is a Dowicil™ (Dow Chemical Company). Examples of a Dowicil™ biocide include Dowicil™ QK-20, which comprises 2,2-dibromo-3-nitrilopropionamide (CAS No. 10222-01-2), and is used as a bactericide at 100 ppm to 2000 ppm in a coating; Dowicil™ 75, which comprises 1(3-chloroallyl)-3,5,7-triaza-1-azoniaadamantane chloride (CAS No. 51229-78-8), and is used as a bactericide at 500 ppm to 1500 ppm in a coating; Dowicil™ 96, which comprises 7-ethyl bicyclooxazolidine (CAS No. 7747-35-5), and is used as a bactericide at 1000 ppm to 2500 ppm in a coating; Bioban™ CS-1135, which comprises 4,4-dimethyloxazolidine (CAS No. 51200-87-4), and is used as a bactericide at 100 ppm to 500 ppm in a coating; or a combination thereof. An

48zzzzz zz additional example of a tradename biocide for coatings includes Kathon® (Rohm and Haas Company). An example of a Kathon® biocide includes Kathon® LX, which typically comprises 5chloro-2-methyl-4־isothiazoiin-3-one (CAS no 2617255-4־) and 2-methyl-4-isothiazo!in-3-one (CAS no 2682-20-4) at 1.5%, and is added from 0.05% to 0.15% in a coating. Examples of tradename fungicides and algaecides include those described for Fungitrol® (International Specialty Products), which are often formulated for solvent-borne and water-borne coatings, and in-can and film preservation. An example is Fungitrol® 158, which comprises 15% tributyltin benzoate (15%) and 21.2% alkylamine hydrochlorides, and is typically used at 0.35% to 0.75% in a water-borne coating for in-can and film preservation. An additional example is Fungitrol® 11, which comprises N-(trichloromethylthio) phthalimide, and is typically used at 0.5% to.1.0% as a fungicide for solvent-borne coating. A further example is Fungitrol® 400, which comprises 98% 3-iodo-2-propynl N-butyl carbamate (IPBC) (Cas No. 55406-53-6), and Is typically used at 0.15% to 0.45% as a fungicide for a water-borne or a solvent-borne coating.

As would be known to one of ordinary skill in the art, determination of whether damage to a coating and/or film is due to microorganisms (e.g., film algal defacement, film fungal defacement), as well as the efficacy of addition of a preservative to a coating and/or film composition in reducing microbial damage to a coating and/or film, may be empirically determined by techniques such as those that are described in ASTM Book of Standards, Volume 06.01, Paint - Tests for Chemical, Physical, and Optical Properties: Appearance, D3274-95, D4610-98, D2574-00, D3273-00, D3456-86, D5589-97, and D5590-00, 2002; and in Paint and Coating Testing Manual, Fourteenth Edition of the Gardner-Sward Handbook, (Koleske, J. V. Ed.), pp. 654-661, 1995. Examples of microorganisms typically selected in such procedures as positive controls of a coating and/or film damaging microorganism include, for example, Aspergillus oryzae (ATCC 10196), Aspergillus flavus (ATCC 9643), Aspergillus niger (ATCC 9642), Pseudomonas aeruginosa (ATCC 10145), Aureobasidium pullulans (ATCC 9348), Peniciliium citrinum (ATCC 9849), Peniciliium funiculosum (ATCC 9644), or a combination thereof.

b. Wetting Additives and Dispersants

It is contemplated that one or more types of particulate matter may be incorporated into a coating composition of the present invention. As is known to those of ordinary skill in the art, physical force and/or chemical additives are used to promote a desirable level of dispersion of particulate matter in a coating composition, for purposes such as coating homogeneity and ease of application. Depending upon whether such an additive is admixed earlier or latter in a coating composition, such an additive is known as a wetting agent or a dispersant, respectively, though it is common that an additive has dual classification. A wetting agent and/or a dispersant often can be used to reduce the particulate matter grinding time during coating preparation, improve wetting

48aaaaa aaa of particulate matter, improve dispersion of particulate matter, improve gloss, improve leveling, reduce flooding, reduce floating, reduce viscosity, reduce thixotropy, or a combination thereof.

(1) Wetting Additives

As is known to those of ordinary skill in the art, preparation of a coating comprising particulate material often comprises a step wherein the particulate material is dispersed in an additional coating component. An example of this type of dispersion step is the dispersion of a pigment into a combination of a liquid component and a binder to form a material known as a millbase. A wetting additive (“wetting agent) is a composition added to promote dispersion of particulate material during coating preparation.

In certain embodiments, a wetting agent is a molecule that comprises a polar region and a nonpolar region. An example is an ethylene oxide molecule comprising a hydrophobic moiety. Such a wetting agent is thought to act by reducing interfacial tension between a liquid component and particulate matter. In specific aspects, a wetting agent comprises a surfactant. Examples of such a wetting agent include pine oil, which is typically added at 1% to 5% of the total coating liquid component, including all intermediate ranges and combinations thereof. Other examples of wetting agents include a metal soap, such as, for example, calcium octoate, zinc octoate, aluminum stearate, zinc stearate, or a combination thereof. An additional example of a wetting agent is bis(2-ethylhexyl)sulfosuccinate (Aerosol OT) (Gas No. 577-11-7); (octylphenoxy)polyethoxyethanoi octylphenyl-polyethyiene glycol (“lgepal-630”) (Cas no. 903619-5); nonyl phenoxy poly (ethylene oxy) ethanol (Tergitol NP-14) (Cas No. 9016-45-9); ethylene glycol octyl phenyl ether (“Triton X-100) (CAS No. 9002-93-1); or a combination thereof.

Often a wetting agent and/or dispersant Is a proprietary formulation and/or commonly available under a trade name. Examples include an Anti-Terra® or Disperbyk® (BYK-Chemie GmbH) and EnviroGem® or Surfynol® (Air Products and Chemicals, Inc.) wetting agents and/or dispersants. An example is Anti-Terra®-U, which comprises a 50% solution of an unsaturated polyamine amide salt and a lower molecular weight acid, dissolved in xylene and isobutanol, and preferred for used in a solvent-borne coating. Anti-Terra®-U is typically added from 1% to 2% to an inorganic pigment, 1% to 5% to an organic pigment, and at 0.5% to 1.0% to titanium dioxide, and 30% to 50% to a bentonite. An example of a Disperbyk® is Disperbyk®, which comprises a polycarboxylic acid polymer alkylolammonium salt and water, and is added to 0.3% to 1.5% to the solvent-borne or water-borne coating composition. A further example is Disperbyk®-101, which comprises a 52% solution of a long chain polyamine amide salt and a polar acidic ester, dissolved in a mineral spirit and butylglycol, and preferred for used in a solvent-borne coating. The ranges for addition to particulate material for Disperbyk®-101 is similar to Anti-Terra®-U. An additional

48bbbbb bbb example Is Disperbyk®-108, which comprises over 97% of a hydroxyfunctional carboxylic acid ester that includes moieties with pigment affinity, and is typically added from 3% to 5 % to an inorganic pigment, 5% to 8% to an organic pigment. However, Disperbyk®-108 is typically added at 0.8% to 1.5% to titanium dioxide, or 8% to 10% to a carbon black, and is preferred for coatings lacking a non-aqueous solvent. A supplemental example is EnviroGem® AD01, which comprises a non-ionic wetting agent with a defoaming property, and is added to 0.1% to 2% to a water borne coating composition. An additional example is Surfynol® TG (Air Products and Chemicals, Inc.), which comprises a non-ionic wetting agent, and is added to 0.5% to 5% to a water-borne coating composition. A further example is Surfynol® 104 (Air Products and Chemicals, Inc.), which comprises a non-ionic wetting agent, dispersant, and defoamer, and is added to 0.05% to 3% to a water-borne coating composition.

(2) Dispersants

As is known to those of ordinary skill in the art, maintenance of the dispersal of particulate matter comprised within a coating composition is often promoted by the addition of a dispersant. A dispersant (“dispersing additive,” “deflocculant, antisettling agent”) is a composition that is added to promote continuing dispersal of particulate matter. In specific aspects, a dispersant is added to a coating composition to reduce or prevent flocculation. Flocculation is the process wherein a plurality of primary particles that have been previously dispersed form an agglomerate. In other aspects, a dispersant is added to a coating composition to prevent sedimentation of particulate matter. Standard procedures to determining the degree of settling by particulate matter in a coating (e.g., paint) are described, for example, in “ASTM Book of Standards, Volume 06.02, Paint - Products and Applications; Protective Coatings; Pipeline Coatings, D869-85, 2002.

Often a dispersant is a compound comprising phosphate, such as, for example, tetrapotassium pyrophosphate or TKPP (CAS No. 7320-34-5). Examples of a tradename/ proprietary phosphate compounds are those known as a Strodex™ (Dexter Chemical L.L.C.), including Strodex™ PK-90, Strodex™ PK-OVOC, and/or Strodex™ MOK-70, which comprise a phosphate ester surfactant.

In some aspects, a dispersant may be a particulate material. Examples include Winnofil® SPT Premium, Winnofil® S, Winnofil® SPM, and Winnofil® SPT (Solvay Advanced Functional Minerals), which comprise 97.4% calcium carbonate (CAS No. 471-34-1) coated with 2.6% fatty acid (CAS No. 64755-01-7) and generally used at 2% to 3%.

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Various preparations of modified montmorillonite clay are known in the art as a dispersant. Examples include those under the name Bentone® (Elementis Specialties, Inc). Bentone® 34 (Elementis Specialties, Inc), which comprises tetraallkyl ammonium bentonite, and is prepared with 33% or more polar solvent prior to addition to a coating composition. M-P-A® 14 (Elementis Specialties, Inc.), which comprises a montmorillonite clay modified by and organic chemical, and is prepared with 33% or more polar solvent prior to addition to a solvent-borne coating composition. Bentone® SD-1 (Elementis Specialties, Inc.), which comprises a montmorillonite clay modified by and organic chemical, and typically added from 0.2% to 2% by weight to a solvent-borne coating composition, particularly those comprising an aliphatic liquid component.

A further example of a dispersant Is a castor wax formulation under the trade names Crayvallac® SF, Crayvallac® MT, and Crayvallac® AntiSettle CVP (Cray Valley Limited), each of which are typically added from 0.2% to 1.5% as a dispersant, thixotropy additive, anti-sagging agent, or a combination thereof. Crayvallac® AntiSettle CVP comprises caster wax (“hydrogenated caster oil), and is suitable for a solvent free epoxy-coating and a mineral spirit liquid component. Crayvallac® SF and Crayvallac® MT each comprise amide modified caster wax, and may be used in an epoxy-coating, an acrylic-coating, a chlorinated rubber-coating, or a combination thereof. Crayvallac® SF and Crayvallac® MT are preferred for use with a liquid component comprising an aromatic hydrocarbon, an alcohol, a glycol ether, or a combination thereof with Crayvallac® MT being also preferred for use with a mineral spirit.

c, Buffers

In certain embodiments, it is preferred to maintain a coating's pH within a certain range. A coating may be acidic, which is a pH between 0 and 7, including all intermediate ranges and combinations thereof, or basic, which is a pH between 7 and 14, including all intermediate ranges and combinations thereof. A neutral pH is pH 7.0, and it is contemplated that a coating may have a neutral pH, or a pH that is near neutral, which is a pH between 6.5 and 7.5, including all intermediate ranges and combinations thereof. A buffer may be added to maintain a coating’s pH as acidic, basic, neutral, or near neutral. In certain aspects, a basic pH is preferred to optimize the function of a preferred enzyme, such as, for example, OPH. Examples of buffers include a bicarbonate (e.g., an ammonium bicarbonate), a monobasic phosphate buffer, a dibasic phosphate buffer, Trizma base, a 5 zwitterionic buffer, triethanolamine, or a combination thereof. In particular facets, a buffer such as a bicarbonate, may provide a ligand or co-substrate (e.g., water) on activator (e.g., carbon dioxide) to an enzyme to promote an enzymatic reaction.

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d. Rheology Modifiers

A rheology modifier (rheology control agent,” rheology additive, “thickener and rheology modifier, TRM, rheological and viscosity control agent, viscosifier, viscosity control agent, thickener) is a composition that alters (e.g., increases, decreases, maintains) a rheological property of a coating. A thickener (“thickening agent”) increases and/or maintains viscosity. A rheological property is a property of flow and/or deformation. Examples of a rheological property include viscosity, brushability, leveling, sagging, or a combination thereof. Viscosity is a measure of a fluid's resistance to flow (e.g., a shear force). Brushability is the ease a coating can be applied using an applicator (e.g., a brush). Leveling is the ability of a coating to flow into and fill uneven areas of coating thickness (e.g., brush marks) after application to a surface and before sufficient film formation to end such flow. Sagging is the gravitationally induced downward flow of a coating after application to a surface and before sufficient film formation to end such flow. It is specifically contemplated that a microorganism based particulate material of the present invention may be added to a coating as a rheology modifier.

A rheology modifier that alters viscosity (e.g., increases, decreases, maintains) is known as a viscosifier.” During application, a coating is usually subjected to a shear force 10³ s’¹ to 10⁴s¹־ by techniques such as brush application, and a shear force up to or greater than 10⁶ s'¹ by techniques including, for example, blade application, high-speed roller application, spray application, or a combination thereof. As would be known to one of ordinary skill in the art, a coating typically is formulated to possess a viscosity upon the shear force of application that promotes the ease of application. An example of a coating viscosity during application is between 0.5 P (50 mPa s) to 2.5 P (250 mPa s”), including all intermediate ranges and combinations thereof. In certain aspects, a coating may possess a viscosity greater or lower than this range, however, it is contemplated such a viscosity may make the coating more difficult to apply using the above application techniques. Post-preparation and/or post-application, a coating is usually subjected to a shear force of 10 s¹־ to 10’³ s'¹ produced, for example, by forces such as gravity, capillary pressure, or a combination thereof. In embodiments wherein a coating’s viscosity is too high at these levels of shear force, leveling during and/or after application may be undesirably low. In embodiments wherein a viscosity is to low at these levels of shear force, a coating may suffer in-can settling, sagging during or after application, or a combination thereof. A preferred viscosity of a coating post-preparation and/or application is between 100 P (10 Pa s) to 1000 P (“100 Pa s”), the including all intermediate ranges and combinations thereof. Of course, the viscosity of a coating will change post-application in embodiments wherein film formation occurs; however, the post-application viscosity refers to the viscosity prior to completion of film formation, and may be determined immediately post-application (e.g., within seconds, within minutes) as appropriate to the coating, as would be known to one of ordinary skill in the art.

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In certain aspects, a coating may possess a viscosity greater or lower than this range, however, it is contemplated such a viscosity may make the coating more prone to sagging and/or settling defects.

A rheology modifier is typically added to alter and/or maintain a rheology property within a desired range post-formulation, during application, post-application, or a combination thereof. In specific embodiments, a rheology modifier alters viscosity at or above 10³ s’¹ and/or at or below 10 s’¹. Viscosity, including non-Newtonian (e.g., shear-thinning) viscosity for coatings and/or coating components (e.g., binders, binder solutions, vehicles) upon formulation with or without a viscosity modifier can be empirically determined, particularly for shear rates comparable to application techniques (e.g., blade, brush, roller, spray) by standard techniques such as in ASTM Book of Standards, Volume 06.01, Paint - Tests for Chemical, Physical, and Optical Properties; Appearance, D562-01, D2196-99, D4287-00, D4212-99, D1200-94, D5125-97, and D5478-98, 2002; “ASTM Book of Standards, Volume 06.02, Paint -- Products and Applications; Protective Coatings; Pipeline Coatings, D4958-97, 2002; and ASTM Book of Standards, Volume 06.03, Paint - Pigments, Drying Oils, Polymers, Resins, Naval Stores, Cellulosic Esters, and Ink Vehicles, D1545-98, D1725-62, D6606-00 and D6267-98, 2002. Additionally, other rheological properties can be determined to aid formulation of a coating of the present invention using techniques known to those of ordinary skill in the art. For example, brush drag, which is the resistance during coating (e.g., a latex) application using a brush, can be determined by standard techniques, such as, for example, in ASTM Book of Standards, Volume 06.02, Paint - Products and Applications; Protective Coatings; Pipeline Coatings, D4040-99, 2002. In an additional example, leveling and sagging can be empirically determined for a coating by standard techniques such as in ASTM Book of Standards, Volume 06.02, Paint - Products and Applications; Protective Coatings; Pipeline Coatings, D4062-99 and D4400-99, 2002.

As would be known to one of ordinary skill in the art, the addition of a coating component to a coating composition typically alters a rheological property, and many coating components have multiple classifications to include function as a rheology modifier. Examples of coating components more commonly added for function as a rheology modifier includes an inorganic rheology modifier, an organometallic rheology modifier, an organic rheology modifier, or a combination thereof. An example of an inorganic rheology modifier includes a silicate such as a montmorillonite silicate. An example of a montomori I Ionite silicate includes aluminum silicate, a bentonite, magnesium silicate, or a combination thereof. A silicate rheology modifier typically confers a superior washfastness property, a superior abrasion resistance property, or a combination thereof, to a coating relative to an organic rheology modifier. An example of an organic rheology modifier includes a cellulose ether, a hydrogenated oil, a polyacrylate, a polyvinylpyrrolidone, 8 urethane, or a combination thereof. Organic rheology modifiers of 8 polymeric nature (e.g., a cellulose ether, a urethane, a polyacrylate, etc.) are sometimes used as an associative thickener, and are preferred for a latex coating. An organic rheology modifier typically confers a greater water retention capacity property (“open time) to a coating relative to a silicate rheology modifier. A common example of a cellulose ether is a methyl cellulose, a hydroxyethyl cellulose, or a combination thereof. An example of a hydroxyethyl cellulose includes Natrosol® (Hercules Incorporated); Cellosize™ (Dow Chemical Company); or a combination thereof. An example of hydrogenated oil includes hydrogenated castor oil. An example of a urethane rheology modifier (associative thickener) includes a hydrophobically modified ethylene oxide urethane (“HEUR), which comprises a polyethylene glycol block covalently linked by urethane, and has both a hydrophilic and hydrophobic regions capable of use in an aqueous environment. An example of a HEUR includes a block of polyethylene oxide linked by an urethane and modified with a nonyl phenol hydrophobe (Rohm and Haas Company). Often a urethane rheology modifier confers a superior leveling property over another type of organic rheology modifier. An example of an organometallic rheology modifier includes a titanium chelate, a zirconium chelate, or a combination thereof.

In addition to the disclosures herein, a rheology modifier and use of a rheology modifier in a coating is known to those of skill in the art, and such compositions and techniques may be included in the practice of the present invention (see, for example, Flick, E. W. “Handbook of Paint Raw Materials, Second Edition,” 808-843 and 879-998, 1989; in ״Paint and Coating Testing Manual, Fourteenth Edition of the Gardner-Sward Handbook, (Koleske, J. V. Ed.), pp 268-285 and 3481995 ,349־; in Paint and Surface Coatings, Theory and Practice, Second Edition, (Lambourne, R. and Strivens, T. A., Eds.), pp. 73, 218, 227, 352, 558-559 and 718, 1999; Wicks, Jr., Z. W., Jones, F. N., Pappas, S. P. Organic Coatings, Science and Technology, Volume 2: Applications, Properties and Performance, pp. 42, 215, 293, 315, 320 and 323-328, 1992; and in “Paints, Coatings and Solvents, Second, Completely Revised Edition, (Stoye, D. and Freitag, W״ Eds.) pp 6, 128 and 166-167, 1998.

e. Defoamers

A coating sometimes comprises a gas capable of forming a bubble (foam”) that can undesirably alter a physical and/or aesthetic property. Undesirable gas incorporation into a coating composition is often a side affect of coating preparation processes, and a particular bane of latex coatings. Often, a wetting agent and/or a dispersant used in a coating may promote creation or retention of foam. Additionally, microorganisms can produce gas, and in certain embodiments, a coating comprising a microorganism based particulate material of the present invention may also comprise a defoamer. A defoamer (“antifoaming agent, “antifoaming

48ggggg ggg additive) is a composition that releases gas (e.g., air) and/or reduces foaming in a coating during production, application, film formation, or a combination thereof. A defoamer often acts by lowering the surface tension around a bubble, allowing merging of a bubble with a second bubble, which produces a larger and less stable bubble that collapses.

Examples of a defoamer Include an oil (e.g., a mineral oil, a silicon oil), a fatty acid ester, dibutyl phosphate, a metallic soap, a siloxane, a wax, an alcohol comprising between six to ten carbons, or a combination thereof. An example of an oil defoamer is pine oil. In some aspects, an antifoaming agent is combined with an emulsifier, a hydrophobic silica, or a combination thereof. Examples of a tradename defoamer is a TEGO® Foamex 8050 (Goldschmidt Chemical Corp.), which comprises a polyether siloxane copolymer and fumed silica, and typically is used at 0.1% to 0.5% during coating preparation; and BYK®-31 (BYK-Chemie), which comprises a paraffin mineral oil and hydrophobic compounds, and typically is used at 0.1% to 0.5% in a coating.

f. Catalysts

A catalyst is an additive that promotes film formation by catalyzing a cross-linking reaction in a thermosetting coating. Examples of a catalyst include a drier, an acid or a base, and the selection of the type of catalyst is specific to the chemistry of the film formation reaction.

(1) Driers

A drier (siccative”) catalyzes is an oxidative film formation reaction, such as those that occur in an oil-based coating. In addition to the disclosures herein, an drier and use of an drier in a coating is known to those of skill in the art, and such materials and techniques for using an drier in a coating may be applied in the practice of the present invention (see, for example, Flick, E. W. Handbook of Paint Raw Materials, Second Edition, pp. 73-93 and 879-998, 1989; in Paint and Coating Testing Manual, Fourteenth Edition of the Gardner-Sward Handbook, (Koleske, J. V. Ed.), pp 30-35, 1995; in “Paint and Surface Coatings, Theory and Practice, Second Edition, (Lambourne, R. and Strivens, T. A., Eds.), pp. 190-192, 1999; Wicks, Jr., Z. W., Jones, F. N., Pappas, S. P. Organic Coatings, Science and Technology, Volume 1; Film Formation, Components, and Appearance,” pp. 138, 317-318, 1992; Wicks, Jr., Z. W., Jones, F. N., Pappas,

S. P. Organic Coatings, Science and Technology, Volume 2: Applications, Properties and Performance pp. 138, 197-198, 330, 344, 1992; and in Paints, Coatings and Solvents, Second, Completely Revised Edition,” (Stoye, D. and Freitag, W., Eds.) pp. 11, 48, 165, 1998.

A drier may comprise a metal drier, an alternative drier, a feeder drier, or a combination thereof. Usually a drier comprising a metal (a metal drier) catalyzes the oxidative reaction.

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Examples of a metal typically used in a drier includes aluminum, barium, bismuth, calcium, cerium, cobalt, iron, lanthanum, lead, manganese, neodymium, potassium, vanadium, zinc, zirconium, or a combination thereof. Examples of types of metal driers include an inorganic metal salt, a metal-organic acid salt (“soap), or a combination thereof. A “salt is the composition formed between the anion of an acid and the cation of a base. Typically, the acid and base of a salt interact by an ionic bond. Examples of organic acids used in such a soap include a monocarboxyiic acid of 7 to 22 carbon atoms. Examples of such a monocarboxyiic acid include a linoleate, a naphthenate, a neodecanoate, an octoate, a rosin, a synthetic acid, a tallate, or a combination thereof. Examples of a drier comprising a synthetic acid include those under the tradenames Troymax™ (Troy Corporation). Though most driers are water insoluble, water dispersible driers can be prepared by combining a surfactant with a naphthenate drier and/or a synthetic acid drier. However, water dispersible driers are typically obtained under a tradename such as, for example, Troykyd® Calcium WD, Troykyd® Cobalt WD, Troykyd® Manganese WD Troykyd® Zirconium WD (Troy Corporation). Additionally, a potassium soap, lithium soap, or a combination thereof, has limited aqueous solubility.

A primary drier (“surface drier,” “active drier, “top drier”) acts at the coating-external environment interface. A secondary drier (“auxiliary drier,”״through drier”) acts throughout the coating. Examples of primary driers include metal driers comprising cobalt, manganese, vanadium, or a combination thereof. Examples of secondary driers include metal driers comprising aluminum, barium, calcium, cerium, iron, lanthanum, lead, manganese, neodymium, zinc, zirconium, or a combination thereof. A rare earth drier comprises lanthanum, neodymium, cerium, or a combination thereof.

In many embodiments, it is contemplated that a coating will comprise from 0.01% to 0.1%, including all intermediate ranges and combinations thereof, of an individual metal of a primary drier, by weight of the non-volatile components of a coating composition. In many embodiments, it is contemplated that a coating will comprise from 0.1% to 1.0%, including all intermediate ranges and combinations thereof, of an individual metal of a secondary drier, by weight of the non-volatile components of a coating composition. Standard physical and/or chemical properties for various driers comprising a metal (e.g., calcium, cerium, cobalt, iron, lead, manganese, nickel, rare earth, zinc, zirconium), and procedures for determining various metals’ content for a driers are described in, for example, “ASTM Book of Standards, Volume 06.04, Paint -- Solvents; Aromatic Hydrocarbons, D600-90, 2002; and “Volume 06.01, Paint — Tests for Chemical, Physical, and Optical Properties; Appearance,” D2373-85, D2374-85, D2375-85, D2613-01, D3804-02, D3969-01, D3970-80, D3988-85, and D3989-01, 2002; and ASTM Book of

48iiiiiiii

Standards, Volume 06.01, Paint - Tests for Chemical, Physical, and Optical Properties;

Appearance, D564-87, 2002.

It is particularly preferred that in embodiments wherein a secondary drier is used, it is combined with a primary drier, as the activity of most secondary driers are often very limited when acting without the presence of a primary drier. Skinning is film-formation disproportionately at the coating-external environment interface. Skinning often results in undesirable wrinkle formation (“wrinkling”) in the film. A primary drier undesirably promotes skinning when acting without the presence of a secondary drier. In certain aspects, zinc may be selected for reducing wrinkling in thick films. In other aspects, calcium and/or zirconium may be selected instead of lead, which may be limited due to an environmental law or regulation. In some facets, an iron drier, rare earth drier, or combination thereof, may be selected for use during film formation by baking. However, an Iron drier may darken a coating. In further aspects, an aluminum drier may be selected for an alkyd-coating.

An alternative drier is a type of drier developed for use in a high solid and/or water-borne coating, due to the inefficiency of a metal-soap drier in these types of coatings. Often, an alternative drier is combined with a metal-soap drier. An example of a metal soap drier include a 1, 10-phenanthronine, 2,2’-dipyridyl. A feeder drier is a type of drier designed to prolong the pot life of a coating in embodiments wherein a metal soap drier is absorbed by a coating component such as a carbon black pigment, an organic red pigment, or a combination thereof. A feeder drier dissolves over time into the coating, thereby providing a continual supply of drier. An example a feeder drier include a tradename composition such as Troykyd® Perma Dry (Troy Corporation).

(2) Acids

An acid catalyzes amino resin cross-linking between a plurality of amino resins and/or an amino resin and an addition resin, though an acid is more effective in promoting cross-iinking between the additional resin and an amino resin. A coating may comprise a strong acid, a weak acid, or a combination thereof. Examples of an acid include a strong acid or a weak acid. The rate of curing is typically accelerated by selection of a strong acid over a weak acid. Examples of a strong acid include, p-toluenesulfonic acid (“PTSA), dodecylbenzenesulfonic acid (DDBSA), or a combination thereof. Examples of a weak acid include phenyl acid phosphate (PAP), butyl acid phosphate (“BAP”), or a combination thereof.

(3) Bases

A base catalyzes cross-linking between an acrylic resin and an epoxy resin in film formation. In specific aspects, the base comprises, for example, a dodecyl trimethyl ammonium

48jjjjjjjj chloride, a tri(dimethylaminomethyl) phenol, a melamine-formaldehyde resin, or a combination thereof.

(iv) Urethane Catalysts

In specific aspects, a urethane coating comprises a catalyst to accelerate the reaction between an isocyanate moiety and a reactive hydrogen moiety. Examples of such a urethane catalyst include a tin compound, a zinc compound, a tertiary amine, or a combination thereof. Examples of a zinc compound include zinc octoate, zinc naphthenate, or a combination thereof. Examples of a tin compound include dibutyltin dilaurate, stannous octoate, or a combination thereof. An example of a tertiary amine includes a triethylene diamine.

g. Antiskinning Agent

An antiskinning agent is a composition, other than a drier, that reduces film-formation at the coating-external environment interface, reduce shrinkage (wrinkling”), or a combination thereof. Such antiskinning agents are often used to protect coatings from undesired filmformation after a container of coating has been opened, during normal film-formation, or a combination thereof. Examples of antiskinning agents, with commonly used coating concentrations in parentheses, include butyraloxime (0.2%), cyclohexanone oxime, dipentene, exkin 1, exkin 2, exkin 3, guaiacol (0.001% to 0.1%), methyl ethyl ketoxime (0.2%), pine oil (1% to 2%), or a combination thereof. Generally, an antiskinning agent acts by reducing the rate of filmformation and/or promotes even film-formation throughout a coating by slowing an oxidative reaction that occurs as part of film formation. Examples of antioxidant antiskinning agents include a phenolic antioxidant, an oxime, or a combination thereof. Example of a phenolic antioxidant includes guaiacol, 4-tert-butylphenol, or a combination thereof. Oximes tend to evaporate such as during film formation, are colorless, do not affect a coating's color property, and generally do not significantly alter the time of film-formation. Examples of an oxime include, butyraldoxime, methyl ethyl ketoxime, cyclohexanone oxime, or a combination thereof. In certain facets, an oxime is used to slow skinning promoted by a copper drier.

h. Light Stabilizers

A coating, a film and/or a surface may be undesirably altered by contact with an environmental agent such as, for example, oxygen, pollution, water (e.g., moisture), and/or irradiation with light (e.g., UV light). To reduce such damaging alterations to a coating and/or film, it is contemplated that a coating composition may comprise a light stabilizer. A light stabilizer (stabilizer) is a composition that reduces or prevents damage to a coating, film and/or surface by an environmental agent. Such agents may alter the color, cause a separation between two layers of film (“delamination), promote chalking, promote crack formation, reduce gloss, or a

48kkkkk kkk combination thereof. This is a particular problem for a film in an exterior environment, such as, for example, an automotive film. Additionally, wood surfaces are susceptible to damage by environmental agents, particularly UV light.

Typically, a light stabilizer may comprise a UV absorber, a radical scavenger, or a combination thereof. A UV absorber is a composition that absorbs UV light. Examples of UV absorbers include a hydroxybenzophenone, a hydroxyphenylbenzotriazole, a hydrozyphenyl-Striazine, an oxalic anilide, yellow iron oxide, or a combination thereof. A hydroxyphenyibenzotriazole generally demonstrates the broadest range of UV wavelength absorption, and converts the absorbed UV light into heat. Additionally, a hydroxyphenyibenzotriazole and/or a hydrozyphenyl-S-triazine usually have the longest effective use in a film due to a higher resistance to photochemical reactions, relative to a hydroxybenzophenone or an oxalic anilide.

A radical scavenger light stabilizer (e.g., a stericaily hindered amine) is a composition that chemically reacts with a radical (free radical). Examples of a stericaily hindered amine (“hindered amine light stabilizer, “HALS) include the ester derivatives of decanedioic acid, such as HALS I [ייbis{1,2,2,6,6,-pentamethyl-4-poperidinyl) ester], which is used in non-acid catalyzed coatings; HALS II [“bis(2,2,6,6,-tetramethyl-1-isooctyloxy-4-piperidinyl) ester], which is typically used in an acid catalyzed coating.

For embodiments wherein a coating, film, and/or surface is primarily located in-doors, a range of 1% to 3%, including all intermediate ranges and combinations thereof, of a light stabilizer relative to binder content is contemplated. A range of 1% to 5%, including all intermediate ranges and combinations thereof, of a light stabilizer relative to binder content is contemplated for exterior uses. Additionally, a combination of a UV absorber and a radical scavenger light stabilizer are contemplated in some embodiments, as the heat released by a UV absorber may promote radical formation. Light stabilizers are often commercially produced, and examples of UV absorber and/or a radical scavenger light stabilizer sold under a tradename include Tinuvin® (Ciba Specialty Chemicals) or Sanduvor® [Clariant LSM (America) Inc.].

I. Corrosion Inhibitors

A coating comprising a liquid component that comprises water, particularly a water-borne coating, may promote corrosion in a container comprising iron, particularly at the lining, seams, handle, efc. A corrosion inhibitor reduces corrosion by water or another chemical. Examples of a corrosion inhibitor includes a chromate, a phosphate, a molybdate, a wollastonite, a calcium ion4811111111 exchanged silica gel, a zinc compound, a borosilicate, a phosphosilicate, a hydrotalcite, or a combination thereof.

In certain embodiments, a corrosion inhibitor is an in-can corrosion inhibitor, a flash corrosion inhibitor, or a combination thereof. An in-can corrosion inhibitor (“can-corrosion inhibitor) is a composition that that reduces or prevents such corrosion. Examples of an in-can corrosion inhibitor are sodium nitrate, sodium benzoate, or a combination thereof. These compounds are typically used at a concentration of 1% each in a coating composition. In-can corrosion inhibitor are often commercially produced, and an example includes SER-AD® FA179 (Condea Servo LLC.), typically used at 0.3% in a coating composition. A flash corrosion inhibitor (flash rust inhibitor) is a composition that reduces or prevents corrosion produced by application of a coating comprising water to a metal surface (e.g., an iron surface). Often, in-can corrosion inhibitors at increased concentrations are added to a coating composition to act as a flash corrosion inhibitor. An example of a flash corrosion inhibitor includes sodium nitrite, ammonium benzoate, 2-amino-2-methyl-propan-1-ol (“AMP), SER-AD® FA179 (Condea Servo LLC.), or a combination thereof. Standard procedures to determining the effectiveness of corrosion inhibition by a coating comprising a flash rust inhibitor are described, for example, in “ASTM Book of Standards, Volume 06.02, Paint - Products and Applications; Protective Coatings; Pipeline Coatings,” D5367-00, 2002.

j. Dehydrators

In some embodiments, preventing moisture from contacting coating component such as a binder, solvent, pigment, or a combination thereof, may be desired. For example, certain urethane coatings undergo film-formation in the presence of moisture, as well as produce a film with increased yellowing, increased hazing and/or decreased gloss. A dehydrator may be added during coating production and/or storage to minimalize contact with moisture. Examples of a dehydrator include Additive TI (Bayer Corporation), Additive OF (Bayer Corporation), or a combination thereof. Additive TI comprises a compound with one reactive isocyanate moiety, and it is capable of reacting with compounds with a chemically reactive hydrogen such as water, an alcohol, a phenol, or an amide. However, in a preferred reaction with water, the reaction products are carbon dioxide and toluenesulfonamide. The toluenesulfonamide is generally inert relative to a urethane binder, and soluble in many non-aqueous liquid components. In certain embodiments, a urethane coating may comprise 0.5% to 4% Additive TI. Additive OF Is a dehydrator generally used in a urethane coating. In certain embodiments, a urethane coating may comprise 1% to 3% Additive OF.

48mmm mmmm m

k. Electrical Additives

In some embodiments, it is desirable to include an additive to alter an electrical property of a coating (e.g., electrical conductivity, electrical resistance). Examples of an additive to alter an electrical property of a coating and/or coating component include an anti-static additive, an electrical resistance additive, or a combination thereof. An anti-static additive may be included in a coating composition comprising a flammable component to reduce the chance of an electrostatic spark occurring and igniting the coating. An anti-static additive is a composition that increases the electrical conductivity of a coating. An example of a flammable component is a hydrocarbon solvent. Examples of an anti-static additive include Stadis® 425 (Octel-Starreon LLC USA), Stadis® 450 (Octel-Starreon LLC USA), or a combination thereof. An electrical resistance additive is a composition that reduces the resistance to electricity by a coating. An electrical resistance additive may be included in a coating to improve the ability of a coating to be applied to a surface using an electrostatic spray applicator. For example, an oxygenated compound (e.g., a glycol ether) often possesses a high electrical conductivity, which can make use of an electrostatic spray applicator to apply a coating comprising an oxigenated compound relatively more difficult than a similar coating lacking an oxigenated compound. Examples of an electrical resistance additive include Ramsprep, Byk-ES 80 (BYK-Chemie GmbH), or a combination thereof. Byk-ES 80 comprises, for example, an unsaturated acidic carboxylic acid ester alkylolammonium salt, and typically is added between 0.2% and 2% to a coating composition. Additionally, techniques for determining an electrical property (e.g., electrical resistance) of a coating comprising an electrical additive are known to those of ordinary skill in the art (see, for example, ASTM Book of Standards, Volume 06.01, Paint - Tests for Chemical, Physical, and Optical Properties; Appearance, D5682-95, 2002).

l. Anti-Insect Additives

Certain coatings may serve a protective role for a surface or surrounding environment against insects, and thus may comprise an anti-insect agent. An example of a surface where a coating comprising an anti-insect agent may be desirable is a wooden surface. Examples of an area where coating comprising an anti-insect agent may be desirable would be a storage facility, such as a cargo hold of a ship or railcar. An anti-insect agent is a composition that, upon contact, is detrimental to the well-being (e.g., life, reproduction) of an invertebrate pest (e.g., an insect, an arachnid, efc). Examples of anti-insect additives that have been used in coatings include copper naphthenate, tributyl tin oxide, zinc oxide, 6-chloro epoxy hydroxy naphthalene, 1-dichloro 2,2'bis(p-chlorophenyl)ethane, or a combination thereof.

5. Coating Preparation

As would be known to one of ordinary skill in the art, a coating may comprise insoluble

48nnnnn nnn particulate material. Particulate material may comprise a primary particle, an agglomerate, an aggregate, or a combination thereof. A primary particle is a single particle not in contact with a second particle. An agglomerate is two or more particles in contact with each other, and generally can be separated by a dispersion technique, a wetting agent, a dispersant, or a combination thereof. An aggregate is two or more particles in contact with each other, which are generally difficult to separate by a dispersion technique, a wetting agent, a dispersant, or a combination thereof.

Usually, a pigment, an extender, certain types of rheology modifiers, certain types of dispersants, or a combination thereof are the major sources of particulate material In a coating. In the present invention, microorganism derived particulate material will also be a source of particulate material in a coating, in certain embodiments, a micoorganism-based particulate matter of the present invention may be used in combination with and/or as a substitute for a pigment, an extender, a rheology modifier, a dispersant, or a combination thereof. In specific facets, a micoorganism-based particulate matter of the present invention may substitute for 0.001% to 100%, including ail intermediate ranges and combinations thereof, of a pigment, an extender, a rheology modifier, a dispersant, or a combination thereof. It is contemplated that any technique used in the preparation of a coating that comprises a pigment, extender or any other form of particulate material described herein or would be known to one of ordinary skill in the art may be applied in the preparation of a coating comprising the microorganism derived particulate material of the present invention. Incorporation of particulate materials (e.g., pigments), assays for determining a rheological property and/or a related property (e.g., viscosity, flow, molecular weight, component concentration, particle size, particle shape, particle surface area, particle spread, dispersion, flocculation, solubility, oil absorption values, CPVC, hiding power, corrosion resistance, wet abrasion resistance, stain resistance, optical properties, porosity, surface tension, volatility, settling, leveling, sagging, slumping, draining, floating, flooding, cratering, foaming, splattering, ) of a coating component and/or a coating (e.g., pigment, binder, vehicle, surfactant, dispersant, paint) and procedures for determining such properties, as well as procedures for large scale (e.g., industrial) coating preparation (e.g., wetting, pigment dispersion into a vehicle, milling, letdown) are described in, for example, in Patton, T. C. Paint Flow and Pigment Dispersion, A Rheological Approach to Coating and Ink Technology,” 1979.

In many embodiments, dispersion of the particulate material is promoted by application of physical force (e.g., impact, shear) to the composition. Techniques such as grinding and/or milling are typically used to apply physical force for dispersion of particulate matter. Though it is contemplated that such application of physical force may be used in the dispersal of the microorganism based particulate material of the present invention, such force may damage the

4800000 structural integrity of the cell wall and/or cell membrane that confers size and shape to the material. The average particle size and shape will be altered by the degree of damage to the cell wall and/or cell membrane, which may alter a physical property, a chemical property, an optical property, or a combination thereof, of a microorganism based particulate material of the present invention. Examples of a physical property that may be altered by cell fragmentation include a rheological property, such as the contribution to viscosity, flow, etc., the tendency to form a primary particle, an agglomerate, an aggregate, efc. An example of a chemical property that may be altered includes allowing greater contact between amine and hydroxyl moieties of internally located biomolecules (e.g., a proteinaceous molecule) with a coating component, which may undergo a chemical reaction (e.g., crosslinking) with a binder. An example of an optical property that may be altered includes an alteration in the gloss characteristic of a coating and/or film by a reduction in particle size due to cell fragmentation.

A preferred embodiment of the microorganism based particulate material is the material in the form of a “whole cell material, which refers to particulate material resembling an intact living cell upon microscopic examination, in contrast to cell fragments of varying shape and size. It is contemplated that such whole ceil particulate material of the present invention will encapsulate an expressed biomolecule (e.g., an enzyme) located in and/or internal to a cell wall and/or cell membrane. In certain aspects, the encapsulation of a biomolecule by a whole cell particle may provide greater protection of the biomolecule from a coating component (e.g., a solvent, a binder, an additive), a coating related chemical reaction (e.g., thermosetting film formation), a potentially damaging agent a coating and/or film may contact (e.g., a chemical, a solvent, a detergent, etc.), or a combination thereof, relative to a biomolecule located on the external surface of a cell or otherwise not comprised within and/or encapsulated by a cell wall and/or cell membrane. As would be known to those of ordinary skill in the art, any preparation of a microorganism will comprise a certain percentage of cell fragments, which comprise pieces of a cell wall, cell membrane, and other cell components (e.g., an expressed biomolecule). The whole cell microorganism based particulate material of the present invention will comprise 50% to 100%, including all intermediate ranges and combinations thereof of whole cell material. The percentage of whole cell material and cell fragments may be determined by any applicable technique known to one of ordinary skill in the art such as microscopic examination, centrifugation, etc, as well as any technique described herein for determining the properties of a pigment, extender, or other particulate material either alone or comprises in a coating. It is contemplated that in some aspects, cell fragments may be used as microorganism based particulate material. The cell fragment microorganism based particulate material of the present invention will comprise 50% to 100%, including all intermediate ranges and combinations thereof of cell fragment material.

48ppppp

PPP

For example, during typical preparation of a water-borne and/or solvent-borne coating comprising particulate material such as a pigment and/or extender, the particulate material is dispersed into a paste known as a “grind or millbase.״ A combination of a binder and a liquid component know as a vehicle is used to disperse the particulate material into the grind. Often, a wetting additive is included to promoted dispersion of the particulate material. Additional vehicle and/or additives are admixed with the grind in a stage refered to as the “letdown to produce a coating of a desired composition and/or properties. These techniques and others for coating preparation are well known to those of ordinary skill in the art [see, for example, in ASTM Book of Standards, Volume 06.01, Paint - Tests for Chemical, Physical, and Optical Properties; Appearance, D6619-00, 2002; in “Paint and Surface Coatings, Theory and Practice, Second Edition, (Lambourne, R. and Strivens, T. A., Eds.), pp. 286-329, 1999; and in “Paints, Coatings and Solvents, Second, Completely Revised Edition, (Stoye, D. and Freitag, W״ Eds.) pp. 178193, 1998.] It is specifically contemplated that these techniques may be used in preparing a coating comprising the microorganism based particulate matter of the present invention, wherein the particulate matter of the present invention is treated as a pigment, extender, or other such particulate material dispersed into a coating.

In another example, the effectiveness of the convertion of agglomerates and/or an aggregates into primary particles in the grind (e.g., pigments, pigment-vehicle combinations, pastes), and latter stages (e.g., lacquer, paint) are typically measured to insure quality, using techniques such as, for example, those described in “ASTM Book of Standards, Volume 06.01, Paint - Tests for Chemical, Physical, and Optical Properties; Appearance, D1210-96, 2002; ASTM Book of Standards, Volume 06.02, Paint - Products and Applications; Protective Coatings; Pipeline Coatings, D2338-02, D1316-93, and D2067-97, 2002; and in “ASTM Book of Standards, Volume 06.03, Paint - Pigments, Drying Oils, Polymers, Resins, Naval Stores, Cellulosic Esters, and Ink Vehicles,” D185-84, 2002. It is specifically contemplated that these techniques for the preparation of coatings comprising a pigment, exender, or other particulate material may be used in the practice of the present invention in the preparation of a coating comprising a microorganism based particulate material of the present invention.

In a further example, a microorganism based particulate material of the present invention may be adapted for use in standard coating formulation techniques to optimize a coating composition for desired properties. As is known to those of ordinary skill in the art, the pigment volume concentration is the volume of pigment in the total volume solids of a dry film. The volume solids is the fractional volume of binder and pigment in the total volume of a coating. It is contemplated that in calculating the PVC, the content of a microorganism based particulate

48qqqqq qqq material of the present invention would be included in this or related calculations as a pigment or extender. A related calculation to the PVC that is specifically contemplated is the critical pigment volume concentration (“CPVC) is the formulation of pigment and binder wherein the coating comprises the minimum amount of binder to fill the voids between the pigment particles. A pigment to binder concentration that exceeds the CVPC threshold produces a coating with empty spaces wherein gas (e.g., air, evaporated liquid component), may be trapped. Various properties rapidly change above the CPVC. For example, corrosion resistance, scrub resistance, stain resistance, opacity, moisture resisitance, rigidity, gloss, or a combination thereof, are more rapidly reduced above the CPVC, while reflectance is often increased. However, in certain embodiments, coating may be formulated above the CPVC and still preduce a film suitable for given use upon a surface. Standard procedures for determining CPVC are known to those of ordinary skill in the art [see, for example, in “ASTM Book of Standards, Volume 06.01, Paint Tests for Chemical, Physical, and Optical Properties; Appearance, D1483-95, D281-95, and D6336-98, 2002; and in Paint and Coating Testing Manual, Fourteenth Edition of the GardnerSward Handbook, (Koleske, J. V. Ed.), pp. 252-258, 1995].

The physical and/or optical properties of a coating are affected by the size of particulate material comprised within the coating. For example, inclusion of a physically hard particulate material, such as a silica extender, may increase the abrasion resistance of a film. In another example, gloss is reduced when particulate material of a larger average particle size increases the roughness of the surface of a coating and/or film. Standard procedures for determining particle properties (e.g., size, shape) are known to those of ordinary skill in the art (see, for example, ASTM Book of Standards, Volume 06.03, Paint - Pigments, Drying Oils, Polymers, Resins, Naval Stores, Cellulosic Esters, and Ink Vehicles, D1366-86 and D3360-96, 2002; and in Paint and Coating Testing Manual, Fourteenth Edition of the Gardner-Sward Handbook, (Koleske, J. V. Ed.), pp. 305-332,1995).

It is also contemplated that a biomolecule composition, particularly one prepared as a particulate or powder material, may be incorporated into a powder coating. Specific procedures for determining the properties (e.g., particle size, surface coverage, optical properties) of a powder coating and/or film have been described, for example, in “ASTM Book of Standards, Volume 06.02, Paint - Products and Applications; Protective Coatings; Pipeline Coatings, D3451-01, D2967-02a, D4242-02, D5382-02 and D5861-95, 2002.

In some embodiments, the dispersion of particulate material (“fineness of grind) in a coating is, in Hegman units (Hu), 0.0 Hu to 8.0 Hu, including all intermediate ranges and combinations thereof. The dispersion of particulate material content of a coating can be

481τ1τπτ r empirically determined, for example, as described in ASTM Book of Standards, Volume 06.01, Paint -- Tests for Chemical, Physical, and Optical Properties; Appearance, D1210-96, 2002. The size of particulate matter in a coating can affect gloss, with smaller particle size generally more conducive for a higher gloss property of a coating and/or film. It is contemplated that a whole cell particulate material of the present invention will possess similar size and shape as the organism from which it was derived. For example, E. coli is about 2 pm in length and 0.8 pm in diameter, maize cells vary more in size, but a size of about 65 pm in diameter may be found in some cell types, and Saccaromyces cerivsia is about 10 pm in diameter. Of course, processing and purifying techniques may reduce the particle size by fragmentation of the cell wall and membrane, and it is contemplated that a biomolecule composition of the present invention may be prepared to an average particle size for a specific purpose (e.g., gloss). In certain facets, a visibly coarse and/or low gloss coating (e.g., a low gloss finish, a flat latex paint) has a dispersion of particulate material of 2.0 Hu to 4.0 Hu. A particle size of 100 pm to 50 pm is associated with a dispersion of 0.0 Hu to 4.0 Hu. In some aspects, a semi-gloss or gloss coating has a dispersion of particulate material of 5.0 Hu to 7.5 Hu. A particle size of 50 pm to 40 pm, 40 pm to 26 pm, 26 pm to 13 pm, and 13 pm to 6 pm is associated with a dispersion of 4.0 Hu to 5.0 Hu, 5.0 Hu to 6.0 Hu, 6.0 Hu to 7.0 Hu, and 7.0 Hu to 7.5 Hu, respectively. In other aspects, a high gloss coating has a dispersion of particulate material of 7.5 Hu to 8.0 Hu. A particle size of 6 pm to 3 pm and 3 pm to 0.1 pm is associated with a dispersion of 7.5 Hu to 7.75 Hu and 7.75 Hu to 8.0 Hu, respectively. In embodiments wherein a coating comprises a combination of particulate materials, wherein the different particulate materials such as a combination of a microorganism derived particulate material of the present invention and one or more of different pigments, with each type of particulate material possessing a different average particle size, it is contemplated that the gloss will be affected most by the particle size of the largest type of particulate material added. However, gloss can also be empirically determined for a coating and/or film, as described herein or as would be known to one of ordinary skill in the art in light of the present disclosures.

6. Empirically Determining the Properties of Biomolecule, Coatings and/or Film

A coating with a desired set of properties for a particular use may be prepared by varying the ranges and/or combinations of coating components, and such coating selection and preparation is within the ability of one of ordinary skill in the art in light of the present disclosures. For example, as would be known to those of ordinary skill in the art, a variety of assays are available to measure various properties of a coating, coating application, and/or a film to determine the degree of suitability of a coating composition for use in a particular application.

48ssssss ss

It is contemplated that in general embodiments, a coating comprising a microorganism derived particulate material of the present invention may be subjected to one or more of such assays. Additionally, a microorganism derived particulate material may further comprise a desired biomolecule (e.g., a colorant, an enzyme), whether endogenously or recombinantly produced, that may confer a desired property to a coating and/or film of the present invention. As used herein, bioactivity refers to desired property such as color, enzymatic activity, efc, conferred to a coating by a biomolecule of a microorganism derived particulate material of the present invention. As used herein, bioactivity resistance refers to the ability of a biomolecule to confer a desired property during and/or after contact with a stress condition normally assayed for in a standard coating and/or film assay procedure. Examples of such a stress condition includes, for example, a temperature (e.g., a baking condition), contact with a coating component (e.g., an organic liquid component), contact with a chemical reaction (e.g., thermosetting film formation), contact with coating and/or film damaging agent (e.g., weathering, detergents, solvents), etc. In specific facets, wherein a microorganism derived particulate material of the present invention that comprises a desired biomolecule, a biomolecule may possess a greater bioactivity resistance such as as determined with standard assay procedure, than a purified or partly purified-like biomolecule.

It is contemplated that such bioactivity resistance may be determined using a standard procedure for a coating and/or film described herein or as would be known to one of ordinary skill in the art in light of the present disclosures. In one example, it is contemplated that a microorganism derived particulate material may comprise a desired colorant such as a chlorophyll, a caroteinoid, etc, which may undergo a desired or undesired change in its optical characteristics (e.g., color, opacity) upon baking at a particular temperature. Various procedures for measuring the visual properties of a coating and/or film are described herein or would be known to those of ordinary skill in the art in light of the present disclosures may be used to determine the properties and/or tolerances of any such colorant.

In an additional example, any assay described herein or would be known to one of ordinary skill in the art in light of the present disclosures may be used to determine the bioactivity resistance wherein an enzyme retains detectable enzymatic activity upon contact with a condition typically encounter in a standard assay. Additionally, in certain aspects, it is contemplated that a coating and/or film comprising an enzyme may lose part of all of a detectable, desirable bioactivity during the period of time of contact with standard assay condition, but regain part or all of the enzymatic bioactivity after return to non-assay conditions. An example of this process is the thermal denaturation of an enzyme at an elevated temperature range into a configuration with lowered or absent bioactivity, followed by refolding of an enzyme, upon return to a preferred

48tttttttt temperature range for the enzyme, into a configuration possessing part or all of the enzymatic bioactivity detectable prior to contact with the elevated temperature. In another example, an enzyme may demonstrate such an increase in bioactivity upon removal of a solvent, chemical, etc.

In some embodiments, an enzyme identified as having a desirable enzymatic property for one or more target substrates may be selected for incorporation into a composition of the present invention. The determination of an enzymatic property may be conducted using any technique described herein or known to those of ordinary skill in the art, in light of the present disclosures. For example, the determination of the rate of cleavage of a substrate, with or without a competitive or non-competitive enzyme inhibitor, can be utilized in determining the enzymatic properties of an enzyme, such as V_max, K_m, Kca/Kn, and the like, using analytical techniques such as Lineweaver-Burke analysis, Bronsted plots, etc (Dumas, D. P. et al., 1989a; Dumas, D. P. et al., 1989b; Dumas, D. P. et al., 1990; Caldwell, S. R. and Raushel, F. M1991 ״c; Donarski, W. J. et al., 1989; Raveh, Let al., 1992; Shim, H. et al., 1998; Watkins, L. M. et al., 1997a; diSioudi, B. et al., 1999; Hill, C. M., 2000; Hartleib, J. and Ruterjans, H., 2001b; Lineweaver, H. and Burke, D1934 ״; Segel, I. H., 1975). It is contemplated that any such analysis may be used to identify an enzyme with a specifically desirable enzymatic property for one or more substrates.

In a specific example, phosphoric triester hydrolases have demonstrated the ability to degrade a wide variety of OP compounds. Methods for measuring the ability of an enzyme to degrade an OP compound are known to those of ordinary skill in the art. It is contemplated that any such technique may be utilized to determine enzymatic activity of a composition of the present invention for a particular OP compound.

Techniques for measuring the kinetics of enzymatic detoxification for various OPcompounds comprising a P-S bond at the phosphorous center (e.g., an OP-phosphonothiolate) such as VX [ΈΑ 1701, TX60, 'O-ethyl-S-(diisopropylaminoethyl) methylphosphonothioate]; Russian VX (R-VX, O-isobutyl-S-(diisopropylaminoethyl) methylphosphonothioate], tetriso [ O, O-diisopropyl S-(2-diisoprpylaminoethyl) phosphorothiolate״], echothiophate (phospholine, 0,0diethyl-phosphorothiocholine”), malathion [“phosphothion, S-(1,2-dicarbethoxyethyl)-0.0dimethyl dithiophosphate), dimethoate (“Cygon®,״ “Dimetate®,״ O,O-dimethyl-S-(Nmethylcarbomoyl-methyl)phosphorodithioate], EA 5533 (“OSDMP, O,S-diethyl methyiphosphonothioate״]. IBP (Kitazin P,” O.O-dilsopropyl-S-benzylphosphothioate), acephate (O,S-dimethyl acetyl phosphoroamidothioate), azinophos-ethyl [S-(3,4-dihydro-4-oxobenzo(d)-

1,2,3-triazin-3-yl methyl-O,O-diethyl) phosphorothioate], demeton S (VX analogue,” 0,0diethyl-S-2-ethylthiolethyl phosphorothioate], malathion [“Phosphothion,” S-(1,2-

48uuuuu uuu dicarbethoxyethyl)-O,O-dimethyl dithiophosphate], and phosalone [“O,O-diethyl-S-(6-chloro-2oxobenzoxazolin-3-yl-methyl) phosphorodithioate], have been described (see, for example, diSioudi, B. D. et al., 1999; Hoskin. F. C. G. et al., 1995; Watkins, L. M. et al., 1997a; Kolakowski,

J. E. et al., 1997; Gopal, S. et aL, 2000; and Rastogi, V. K. et a!., 1997).

Techniques for measuring the kinetics of enzymatic detoxification for various OPcompounds comprising a P-F bond at the phosphorous center (e.g., an OP-phosphonofluoridate) such as soman (“l,2,2-trimethylpropyl־methylphosphonofluoridate), sarin (isopropylmethylphosphonofluoridate), DFP (“Ο,Ο-diisopropyl phosphorofluoridate), alpha (“1ethylpropylmethylphosphonofluoridate), and mipafox (N,N diisopropylphosphorofluorodiamidate) have been described (see, for example Dumas, D. P. et al., 1990; Li, W.-S. et al., 2001; diSioudi, B. D. et al., 1999; Hoskin, F. C. G. et al., 1995; Gopal, S. et al., 2000; and DeFrank, J. and Cheng, T., 1991).

A technique for measuring the kinetics of enzymatic detoxification for an OP-compound comprising a P-CN bond at the phosphorous center (e.g., an OP-phosphonocyanate) such as tabun (ethyl N,W-demethylamidophosphorocyanidate) has been described (see, for example, Raveh, L. et al., 1992).

Techniques for measuring the kinetics of enzymatic detoxification for various OPcompounds comprising a P-0 bond at the phosphorous center (e.g., an OP-triester) such as paraoxon (diethyl p-nitrophenylphosphate), the soman analogue O-pinacolyl p-nitrophenyl methylphosphonate, the sarin analogue O-isopropyl p-nitrophenyl methylphosphonate, NPPMP (p-nitrophenyl-o-pinacolyl methylphosphonate), coumaphos [‘Ό,Ο-diethyl 0-(3-chloro-4-methyl-

2-oxo-2H-1benzyran-7-yl)phosphorothioate], cyanophos [‘Ό,Ο-dimethyl p-cyanophenyl phosphorothioate}, diazinon (Ο,Ο-diethyl O-2-is0-propyl-4-methyl-6-pyrimidyl phosphorothiate”), dursban (Ο,Ο-diethyl O-3,5,6-trichloro-2-pyridyl phosphorothioate), fensulfothion {0,O-diethyt [p-(methyisulfinyl)phenyl] phosphorothioate}, parathion ( Ο,Ο-diethyl O-p-nitrophenyl phosphorothioate”), methyl parathion (“Ο,Ο-dimethyl p-nitrophenyl phosphorothioate), ethyl parathion [0,0-diethyl-0-(4-nitrophenyl)phosphorothioate”], EPN (O-ethyl O-(4-nitrophenyl) phenylphosphonothioate), DEPP (diethylphenylphosphate”), NPEPP (pnitrophenylethylphenylphosphinate”) have been described (see, for example, Dumas, D. P. etal., 1990; Li, W.-S. et al., 2001; diSioudi, B. D. et al., 1999; Watkins, L. M. et al., 1997a; Gopal, S. et at., 2000; Mulbry, W. and Karns, J., 1989; Hong, S.-B. and Raushel, F. M., 1996; and Dumas, D. P. et al., 1989b).

48wvw wv

In one example, the cleavage rate of a phosphonothiolate OP substrate comprising a PS bond can be measured using a method known as the Ellman reaction. All such substrates produce a P-S bond cleavage product comprising a free thiol group, which can chemically react with the Ellman's reagent, 5,5’-dithio-bis-2-nitrobenzoic acid (“DTNB). This reaction produces 5’thiol-2-nitrobenzoate anions with a maximum absorbency at 412 nm. P-S cleavage can be determined by the appearance of the free thiol group, measured using a spectrophotometer (Rastogi, V. H. et al., 1997; Gopal, S. et al., 2000; diSioudi, B. et ah, 1999; Watkins, L.

M. et al., 1997a; Hoskin, F. C. G. et al., 1995; Chae, Μ. Y. et aL, 1994; Ellman, G. L. et aL, 1961).

In an additional example, the cleavage of an OP substrate can be measured by detecting the production of a cleavage product that comprises a released ion. In a further example, the cleavage of a phosphonofluoridate can be measured by the release of cleavage product comprising a fluoride ion (F) using a fluoride ion specific electrode and a pH/mV meter (Hartleib, J. and Ruterjans, H., 2001a; Gopal, S. et al., 2000; diSioudi, B. et al., 1999; Watkins, L. M. etal., 1997a; DeFrank, J. and Cheng, T., 1991; Dumas, D. P. etal1990 ״; Dumas, D. P. et al., 1989a). In another example, the cleavage of a phosphonocyanate can be measured by the release of a cleavage product comprising a cyanide ion (CN) using a cyanide selective electrode with a pH meter (Raveh, L. et al., 1992).

In another example, cleavage of an OP substrate can be measured, for example, by P NMR spectroscopy. For example, the disappearance of VX and the formation of the cleavage product ethyl methylphosphonic acid (EMPA), has been measured using this technique (Kolakowski, J. E. etal., 1997; Lai, K. etal., 1995). In another example, the disappearance of tabun and the appearance of the Ν,Ν-dimethylamindophosphosphoric acid cleavage product has been measured by ³¹P NMR spectroscopy (Raveh, L. etal., 1992). In a further example, the disappearance of DFP and appearance of a F cleavage product has been determined using ¹⁹F־ and ³¹P NMR spectroscopy (Dumas, D. P. et al., 1989a).

The cleavage of many OP compounds’ such as paraoxon, coumaphos, cyanophos, diazinon, dursban, fensulfothion, parathion, methyl parathion, DEPP, and various phosphodiesters, can be determined by measuring the production of a cleavage product spectrophotometrically at visible or UV wavelengths (Dumas, D. P. et al., 1989b). For example, the cleavage of DEPP can be measured at 280 nm, using a spectrophotometer to detect a phenol cleavage product (Watkins, L. M. et al., 1997a; Hong, S.־B. and Raushel, F. M., 1996). In a further example, various phosphodiesters (e.g., ethyl-4-nitrophenyl phosphate) have been made to evaluate OPH cleavage rates, and their cleavage measured at 280 nm by the production of a substituted phenol cleavage product (Shim, H. etal., 1998). In a further example, paraoxon is

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420 nm (Dumas, u _add״iona| example, NPPMK _{(n a}

M. et al., 1997a;G0pal, S. et al., ) _(diSi0udi, _B. et al., ) measured by the release of p-nitrophenol as a dea 9^ _{Mva bean} created to prod־־״ P־ further ־x״־pl־. ^and ζ“₈ adapt the method used in «'־ nltraphenol as a d־־v־g״ ־^{roducl a}_oleavag־ preference of an e״W™ deavage In determining «» 3^a^era' ^b'^nd'ⁿ⁹ ״»״ ״hiral ־1־״־&lt;*״־ (Chen-Goodspeed. phosphoryl group ־״־,־*. ^R- _2M1b- «״. P.etal.־000־; steub״־., . , , 2001a Chen-Goodspeed, M.eta. ״_־MU0S have bean created wherein ׳^־ ״־ ״י .;ה^amp1e ״״j x ™p^|a“^d ״׳ ״י^,r°^ph־nA דה me fluoride comprising ״piety of th־ ־^b_ad־pt־d _method for paraoxon ־3־״־־&lt;־ detection of the CWA analogs cleavage rales us 9 measurement (Li. W.-S. etal., 2001).

Other techniques are X־״w״ t־ th־ ־־־' inhX-y P°‘^a™ °^f

Ashani Y. etal., 1998).

^A ״ ״f various coating . General proc״־« -or empiricafly X In the art. ״־d ™ components andlor coat.ng comp־־'״־־ ^ara procedures include ־ ״־י״־׳״־־־,״ denJty.v־l״^mes0^dsaad^^0rSPeC'’'^C^^raVt’'_ngnt^aiXntily, ־id m coating

Cr purpי.״־ ־־־־ « -^dfca7“ '° °״ ee cXdard techhid׳״־י ״» ־־״«» coaling batch to batch consistency, etc. Examp _{(־ g a ρον׳ό־Γ} coating) various solvent״ ,־quid״־-־) ־ ^a V*־ » ״> ־״ «״ _i״d״d־ tbos־ deseed >־ ״ASTM Bock ofS^er ._{ASTM Boo of}

Xrocaib־״־. Ο־־־־־־־. ^D1555M־“־sXcr Cbemicai. Physical. ־&lt; Standards. Volume 06.01. P״־t - _Book ״, standards. Volume 06.03.

Appearance. D147M8 e״d D21W1. 2^. AST _P ;״־is. Drying O־״. P״־״׳״-־ V״,־me 06.02. Paint - '״־ 3-64״d D1^ ־^AS™ ”״* T _a 05966-02. 2002; and ־P־'‘ ^a^{d C}°^al'^nS

....

289-304, 1995.

48xxxxx xxx

Standard surface specification and/or procedures for preparing a surface (e.g., glass, wood, steel) for empirically measuring a physical and/or visual property of a coating (e.g., a paint, a varnish, a lacquer) and/or film are known to those of ordinary skill in the art (see, for example, ASTM Book of Standards, Volume 06.01, Paint -- Tests for Chemical, Physical, and Optical Properties; Appearance,” D3891-96, D609-00, and D2201-99, 2002; and “ASTM Book of Standards, Volume 06.02, Paint -־ Products and Applications; Protective Coatings; Pipeline Coatings, D358-98, D4227-99, and D4228-99, 2002). Specific procedures for preparing a metal surface and an evaluating a coating (e.g., a primer, a paint) applied to a metal surface are known to those of ordinary skill in the art (see, for example, ASTM Book of Standards, Volume 06.02, Paint-- Products and Applications; Protective Coatings; Pipeline Coatings,” D3276-00, D5161-96, D4417-93, D3322-82, D2092-95, D5065-01, D5723-95, D6386-99, and D6492-99, 2002). Specific procedures for evaluating a coating applied to a plastic surface are known to those of ordinary skill in the art (see, for example, “ASTM Book of Standards, Volume 06.02, Paint Products and Applications; Protective Coatings; Pipeline Coatings, D3002-02, 2002).

Standard procedures for determining the stability of a coating (e.g., a water-borne coating, a UV irradiation cured coating) in a container prior and/or after opening the container are known to those of ordinary skill in the art (see, for example, ASTM Book of Standards, Volume 06.02, Paint — Products and Applications; Protective Coatings; Pipeline Coatings,” D2243-95 and D4144-94, 2002).

Standard procedures for evaluating an applicator (e.g., a brush, a roller, a fabric, a spray applicator, an electrocoat bath) and/or a coating being applied by an applicator are known to those of ordinary skill in the art (see, for example, ASTM Book of Standards, Volume 06.02, Paint - Products and Applications; Protective Coatings; Pipeline Coatings, D6737-01, D5913-96, D5959-96, D5301-92, D5068-02, D5069-92, D4707-97, D5286-01, D6337-98, D4285-83, and D5327-97, 2002; and “ASTM Book of Standards, Volume 06.01, Paint -- Tests for Chemical, Physical, and Optical Properties; Appearance, D1978-91, D5794-95, D4370-01, D4399-90, and D4584-86, 2002.

Standard procedures for preparing a coating (e.g., a paint, a varnish, a lacquer) and/or film layer upon a surface for empirically measuring a physical and/or visual property are known to those of ordinary skill in the art (see, for example, ASTM Book of Standards, Volume 06.01, Paint -- Tests for Chemical, Physical, and Optical Properties; Appearance,” D3924-80, D823-95, and D4708-99, 2002; ASTM Book of Standards, Volume 06.02, Paint -- Products and Applications; Protective Coatings; Pipeline Coatings,” D6206-97, D1734-93, and D4400-99, 2002;

48yyyyy yyy and Paint and Coating Testing Manual, Fourteenth Edition of the Gardner-Sward Handbook,” (Koleske, J. V. Ed.), pp. 415-423, 1995.

Standard procedures for empirically determining the degree and duration of film formation of various coating compositions are known to those of ordinary skill in the art, and may be applied in the practice of the present invention. Example of a standard technique for determining the degree/duration of film formation by loss of a volatile coating component and/or a cross-linking reaction for a coating (e.g., an oil-coating, a UV cured coating, an thermosetting powder coating) include those described in “ASTM Book of Standards, Volume 06.01, Paint - Tests for Chemical, Physical, and Optical Properties; Appearance, D3539-87, D1640-95 and D5895-01e1, 2002; ASTM Book of Standards, Volume 06.02, Paint - Products and Applications; Protective Coatings; Pipeline Coatings, D4217-02, D3732-82, D2091-96, D711-89, D4752-98, and D590996a, 2002; “ASTM Book of Standards, Volume 06.03, Paint - Pigments, Drying Oils, Polymers, Resins, Naval Stores, Cellulosic Esters, and ink Vehicles, D2575-70 and D2354-98, 2002; and Paint and Coating Testing Manual, Fourteenth Edition of the Gardner-Sward Handbook,” (Koleske, J. V. Ed.), pp. 407-414, 1995. Additionally, the temperature generated by a film formation reaction by a coating (e.g., a wood coating) may also be determined by one of ordinary skill in the art (see, for example, ASTM Book of Standards, Volume 06.02, Paint -- Products and Applications; Protective Coatings; Pipeline Coatings,” D3259-95, 2002). Further, standard techniques for evaluating baking conditions on an organic coating and/or film are known to those of ordinary skill in the art, (see, for example, “ASTM Book of Standards, Volume 06.01, Paint Tests for Chemical, Physical, and Optical Properties; Appearance,“ D2454-95, 2002).

In embodiments wherein film formation at room temperature is preferred in a coating, a standard procedure that would be known to one of ordinary skill in that art may be used for measuring film formation rate and/or stages (see for example, “ASTM Book of Standards, Volume 06.01, Paint - Tests for Chemical, Physical, and Optical Properties; Appearance,” D1640-95, 2002. In certain aspects wherein the ability of an oil to undergo film formation is to be determined, a standard procedure described in “ASTM Book of Standards, Volume 06.03, Paint -Pigments, Drying Oils, Polymers, Resins, Naval Stores, Cellulosic Esters, and Ink Vehicles, D1955-85, 2002, may be used. In embodiments wherein the hardness of a film produced by a coating composition is measured (e.g., an organic coating), a standard procedure such as, for example, “ASTM Book of Standards, Volume 06.01, Paint - Tests for Chemical, Physical, and Optical Properties; Appearance, D3363-00, D4366-95, and D1474-98, 2002.

Examples of a standard technique for determining the coating and/or film thickness after application to various surface types are described in ASTM Book of Standards, Volume 06.01,

48zzzzz zzz

Paint - Tests for Chemical, Physical, and Optical Properties; Appearance, D1212-91, D441495, D1005-95, D1400-00, □1186-01, and D6132-97, 2002; “ASTM Book of Standards, Volume

06.02, Paint - Products and Applications; Protective Coatings; Pipeline Coatings, D5235-97,

D4138-94, D2200-95, and D5796-99, 2002; and “Paint and Coating Testing Manual, Fourteenth

Edition of the Gardner-Sward Handbook, (Koleske, J. V. Ed.), pp. 424-438, 1995.

Examples of a standard technique for determining the adhesion of a coating and/or film to various surface types are described in “ASTM Book of Standards, Volume 06.01, Paint -- Tests for Chemical, Physical, and Optical Properties; Appearance, D3359-02, D5179-98, and D219798, 2002; “ASTM Book of Standards, Volume 06.02, Paint — Products and Applications; Protective Coatings; Pipeline Coatings,” D4541-02 D3730-98, D4145-83, D4146-96, and D667701, 2002; and Paint and Coating Testing Manual, Fourteenth Edition of the Gardner-Sward Handbook, (Koleske, J. V. Ed.), pp. 513-524, 1995. Additionally, standard procedures for determining the ability of one or more layers of a multicoat system to function (e.g., adhere, weather) together are described in, for example, ASTM Book of Standards, Volume 06.02, Paint - Products and Applications; Protective Coatings; Pipeline Coatings, D5064-01, 2002.

Various standard techniques for determining the physical properties (e.g., flexibility, tensile strength, toughness, impact resistance, hardness, mar resistance, blocking resistance) relevant to the durability of a film and/or the degree of film formation are known to those of ordinary skill in the art. Such procedures may be used to empirically characterize a film, and determine whether a coating composition produces a film suitable for a given application. Flexibility is the film’s ability to undergo stress from bending and/or flexing without discernable damage (e.g., cracking). Examples of a standard technique for determining the flexibility of a film under mechanical or temperature stress are described in “ASTM Book of Standards, Volume 06.01, Paint -- Tests for Chemical, Physical, and Optical Properties; Appearance, D522-93a and D4145-83, 2002; “ASTM Book of Standards, Volume 06.02, Paint — Products and Applications; Protective Coatings; Pipeline Coatings, D4145-83, D4146-96, and D1211-97, 2002; and Paint and Coating Testing Manual, Fourteenth Edition of the Gardner-Sward Handbook,” (Koleske, J. V. Ed.), pp. 547-554, 1995. Related to flexibility is the tensile strength □f a film, which is the ability of a film to undergo tensile deformation without developing discernable damage (e.g., cracking, tearing). Examples of a standard technique for determining the tensile strength of a film are described in ASTM Book of Standards, Volume 06.01, Paint - Tests for Chemical, Physical, and Optical Properties; Appearance, D2370-98 and D522-93a, 2002; and Paint and Coating Testing Manual, Fourteenth Edition of the Gardner־Sward Handbook, (Koleske, J. V. Ed.), pp. 534-545, 1995. Toughness is the film’s ability to undergo strain imposed in a short period of time (e.g., one second or less) without discernable damage (e.g., breaking, tearing). Examples of a

48aaaaa aaaa standard technique for determining the toughness of a film (e.g., a film for a pipeline) are described in “ASTM Book of Standards, Volume 06.01, Paint - Tests for Chemical, Physical, and Optical Properties; Appearance, D2794-93, 2002; “ASTM Book of Standards, Volume 06.02, Paint - Products and Applications; Protective Coatings; Pipeline Coatings,“ G14-88, 2002; and “Paint and Coating Testing Manual, Fourteenth Edition of the Gardner-Sward Handbook, (Koleske, J. V. Ed.), pp. 547-554, 1995. Impact resistance is the ability of a film to undergo impact with an indenter without developing discernable damage at the dimple site (e.g., cracking). Examples of a standard technique for determining the impact resistance of a film (e.g., a film for a pipeline) are described in “ASTM Book of Standards, Volume 06.01, Paint - Tests for Chemical, Physical, and Optical Properties; Appearance, D2794-93, 2002; ASTM Book of Standards, Volume 06.02, Paint - Products and Applications; Protective Coatings; Pipeline Coatings, G1389 and G14-88, 2002; and “Paint and Coating Testing Manual, Fourteenth Edition of the GardnerSward Handbook, (Koleske, J. V. Ed.), pp. 553-554, 1995. Hardness is the film's ability to undergo an applied static force without developing discernable damage (e.g., a scratch, an indentation). Examples of a standard technique for determining the hardness of a film are described in ASTM Book of Standards, Volume 06.01, Paint - Tests for Chemical, Physical, and Optical Properties: Appearancei D1640-95, D1474-98, D2134-93, D4366-95, and D3363-00. 2002; and Paint and Coating Testing Manual, Fourteenth Edition of the Gardner-Sward Handbook, (Koleske, J. V. Ed.), pp. 555-584, 1995. Mar resistance (mar abrasion resistance) is the film’s ability to undergo an applied dynamic force without developing a change in the film surface appearance (e.g., gloss) due to a permanent deformation (e.g., an Indentation). Examples of a standard technique for determining the mar resistance of a film are described in “ASTM Book of Standards, Volume 06.01, Paint - Tests for Chemical, Physical, and Optical Properties; Appearance. D5178-98 and D6037-96, 2002; and “Paint and Coating Testing Manual, Fourteenth Edition of the Gardner-Sward Handbook, (Koleske, J. V. Ed.), pp. 525-533 and 579-584, 1995. Abrasion resistance (wear abrasion resistance) is the films ability to undergo an applied dynamic force (e.g., washing) without removal of film material. Examples of a standard technique for determining the abrasion resistance (e.g., burnish resistance) of a film are described in ASTM Book of Standards, Volume 06.01, Paint - Tests for Chemical, Physical, and Optical Properties; Appearance, D968-93 and D4060-01, 2002; ASTM Book of Standards.

Volume 06.02, Paint -- Products and Applications; Protective Coatings; Pipeline Coatings, D3170-01, D4213-96, D5181-91, D4828-94, D2486-00, D3450-00, D6736-01, and D6279-99e1, 2002; and “Paint and Coating Testing Manual, Fourteenth Edition of the Gardner-Sward Handbook, (Koleske, J. V. Ed.), pp. 525-533, 1995. Blocking resistance (block resistance) is the ability of a film to resist adhering to a second film, particularly when the two films are pressed together (e.g., a coated door and coated doorframe). Examples of a standard technique for determining the blocking resistance of a film are described in ASTM Book of Standards, Volume

48bbbbb bbbb

06.02, Paint - Products and Applications; Protective Coatings; Pipeline Coatings,” D2793-99 and D3003-01, 2002. Abrasion resistance (wear abrasion resistance) is the film's ability to undergo an applied dynamic force (e.g., washing) without removal of film material. Slip resistance is a coating's (e.g., a floor coating) slipperiness, and can be evaluated as described in Paint and Coating Testing Manual, Fourteenth Edition of the Gardner-Sward Handbook, (Koleske, J. V. Ed.), pp. 600-606, 1995.

Weathering resistance is film's ability to endure and/or protect a surface from an external environmental condition. Examples of environmental conditions that may damage a film and/or surface include contact with varying conditions of temperature, moisture, sunlight (e.g., UV resistance), pollution, biological organisms, or a combination thereof. Examples of a standard technique for determining the weathering resistance of a film (e.g., an automotive film, an external architectural film, a varnish, a wood coating, a steel coating) by evaluating the degree of damage (e.g., fungal growth, color alteration, dirt accumulation, gloss loss, chalking, cracking, blistering, flaking, erosion, surface rust), are described in ASTM Book of Standards, Volume 06.01, Paint Tests for Chemical, Physical, and Optical Properties; Appearance, D4141-01, D1729-96, D66093, D661-93, D662-93, D772-86, D4214-98, D3274-95, D714-02, D1654-92, D2244-02, D523-89, D1006-01, D1014-95, and D1186-01, 2002; “ASTM Book of Standards, Volume 06.02, Paint -Products and Applications; Protective Coatings; Pipeline Coatings,” D3719-00, D610-01, D164197, D2830-96, and D6763-02, 2002; and Paint and Coating Testing Manual, Fourteenth Edition of the Gardner-Sward Handbook,” (Koleske, J. V. Ed.), pp. 619-642, 1995. Additionally, standard techniques are known to those of ordinary skill in the art for determining the resistance of a film to artificial weathering conditions. These procedures are used to contact a film with a simulated weathering condition (e.g., heat, moisture, light, UV irradiation) at an accelerated timetable are described in ASTM Book of Standards, Volume 06.01, Paint - Tests for Chemical, Physical, and Optical Properties; Appearance, DB22-01, D4587-01, D5031-01, D6631-01, D6695-01, D589496, and D4141-01, 2002; “ASTM Book of Standards, Volume 06.02, Paint -- Products and Applications; Protective Coatings; Pipeline Coatings, D5722-95, D3361-01 and D3424-01, 2002, and Paint and Coating Testing Manual, Fourteenth Edition of the Gardner-Sward Handbook” (Koleske, J. V. Ed.), pp. 643-653, 1995.

Standard techniques for determining a film’s resistance to damage by various chemicals are known to those of ordinary skill in the art. Examples of chemicals that can be used in such procedures include an acid (e.g., 3% acetic acid), a base, an alcohol (e.g., 50% ethyl alcohol, hydrochloric acid, sulfuric acid), a detergent (e.g., a sodium phosphate solution), gasoline, a glycol based antifreeze, an oil (e.g., a vegetable oil, a lubricating petroleum oil, a grease), a solvent, water (e.g., a salt solution, a salt vapor), a polish abrasive, another coating (e.g., graffiti),

48ccccc cccc or a combination thereof. Standard techniques for determining the chemical resistance of a film {e.g., an architectural film, an automotive film, a paint, a lacquer, a varnish, a traffic-coating, a metal surface-film) by evaluating possible damage (e.g., adhesion loss, alteration of gloss, blistering, discoloration, loss of hardness, staining, swelling, wrinkling) are described in, for example, ASTM Book of Standards, Volume 06.02, Paint - Products and Applications; Protective Coatings; Pipeline Coatings, D1308-02, D2571-95, D2792-69, D4752-98, D3260-01, D6137-97, D6686-01, D6688-01, and D6578-00, 2002; ASTM Book of Standards, Volume 06.01, Paint - Tests for Chemical, Physical, and Optical Properties; Appearance, D2370-98, D224801a, and D870-02, 2002; “ASTM Book of Standards, Volume 06.03, Paint - Pigments, Drying Oils, Polymers, Resins, Naval Stores, Cellulosic Esters, and ink Vehicles, D1647-89, 2002; and Paint and Coating Testing Manual, Fourteenth Edition of the Gardner-Sward Handbook,” (Koleske, J. V. Ed.), pp. 662-666, 1995. Additionally, examples □f a standard technique for determining the solvent resistance of a film are described in ״ASTM Book of Standards, Volume 06.02, Paint — Products and Applications; Protective Coatings; Pipeline Coatings, D4752-98 and D5402-93, 2002.

Standard techniques for determining a film's and/or surface's (e.g., metal, wood) resistance to water permeability and/or damage (e.g., corrosion, blistering, adhesion reduction, hardness alteration, color alteration, gloss alteration) by contact with water and/or moisture are described in, for example, “ASTM Book of Standards, Volume 06.01, Paint — Tests for Chemical, Physical, and Optical Properties; Appearance, D870-02, D1653-93, D1735-02, D2247-02, and D4585-99, 2002; and ASTM Book of Standards, Volume 06.02, Paint - Products and Applications; Protective Coatings; Pipeline Coatings, D2065-96, D2921-98, D3459-98, and D6665-01, 2002.

Standard techniques for determining a film’s resistance to damage by a temperature greater than ambient condition are known to those of ordinary skill in the art. Thermal resistance is the film’s ability to undergo stress from a temperature at or below 200°C without discernable damage, while heat resistance is the film's ability to undergo stress from a temperature above 200°C (e.g., fire resistance, fire retardancy, flame resistance) without discernable damage. Standard techniques for determining the thermal and/or heat resistance of a film (e.g., a metalfilm, a wood-lacquer) by evaluating possible damage (e.g., adhesion loss, alteration of gloss, blistering, chalking, discoloration) are described in, for example, ASTM Book of Standards, Volume 06.01, Paint - Tests for Chemical, Physical, and Optical Properties; Appearance, D2370-98, D2485-91, D1360-98, D4206-96, and D3806-98, 2002; and “ASTM Book of Standards, Volume 06.02, Paint - Products and Applications; Protective Coatings; Pipeline Coatings, D1211-97 and D6491-99, 2002.

48ddddd dddd

In some embodiments, it may be desirable to measure the component composition of a coating and/or film such as to verify the presence, absence and/or amount of one or more coating components in a particular formulation. Standard procedures for sampling a coating and/or film, and analyzing the material composition (e.g., a pigment, a binder, liquid component, toxic material), have been described in, for example, ASTM Book of Standards, Volume 06.01, Paint - Tests for Chemical, Physical, and Optical Properties; Appearance, D2371-85, D5380-93, D2372-85, 0269890־, D3723-84, D4451-02, D4563-02, D5145-90, D3925-02, 02348-02, D224590, D3624-85a, D3717-85a, D2349-90, D2350-90, D2351-90, D2352-85, D3271-87, D3272-76, D4017-02, D3792-99, D4457-02, D6133-00, D6191-97, D4764-01, D3718-85a, D3335-85a, 06580-00, E848-94, D4834-88, D4358-84, D2621-87, D3618-85a, D6438-99, D4359-90, D316885, and D4948-89, 2002; ASTM Book of Standards, Volume 06.02, Paint - Products and Applications; Protective Coatings; Pipeline Coatings, D5702-02, 2002; and ASTM Book of Standards, Volume 06.03, Paint -- Pigments, Drying Oils, Polymers, Resins, Naval Stores, Cellulosic Esters, and Ink Vehicles, D1469-00, 2002.

The nonvolatile content of a coating component and/or coating (“total solids content) can provide an estimate, for example, of the volume of film that will be produced by a coating or coating component (e.g., a paint, a clear coating, an electrocoat bath applied coating, a binder solution, an emulsion, a varnish, an oil, a drier, a solvent) and/or the surface area a coating can cover relative to a film’s thickness. The nonvolatile content of coating and/or coating component can be determined by any technique known in the art (see, for example, “ASTM Book of Standards, Volume 06.01, Paint - Tests for Chemical, Physical, and Optical Properties; Appearance, D6093-97, D2697-86, D1259-85, D1644-01, D2832-92, and D4209-82 D5145-90, 2002; ASTM Book of Standards, Volume 06.02, Paint — Products and Applications; Protective Coatings; Pipeline Coatings, D4713-92, D5095-91, 2002; and “ASTM Book of Standards, Volume 06.03, Paint -- Pigments, Drying Oils, Polymers, Resins, Naval Stores, Cellulosic Esters, and Ink Vehicles, D4139-82, 2002. Additionally, the volatile component of a coating can provide an estimate, for example, of VOC release and/or thermoplastic film formation time. The nonvolatile content of coating and/or coating component (e.g., a paint, a clear coating, an automotive coating, an emulsion, a binder solution, a varnish, an oil, a drier, a solvent) can be determined by any technique known in the art (see, for example, “ASTM Book of Standards, Volume 06.01, Paint -- Tests for Chemical, Physical, and Optical Properties; Appearance, D2369-01e1, D2832-92, D3960-02, D4140-82, D4209-82, D5087-02 and D6266-00a, 2002;and “ASTM Book of Standards, Volume 06.02, Paint -- Products and Applications; Protective Coatings; Pipeline Coatings, D5403-93, 2002.

48eeeee eeee

Standard procedures for determining the visual appearance of a coating component, coating and or film (e.g., reflectance, retroreflectance, fluorescence, photoluminescent light transmission, color, tinting strength, whiteness, measurement instruments, computerized data analysis) have been described, for example, in ASTM Book of Standards, Volume 06.01, Paint ־Tests for Chemical, Physical, and Optical Properties; Appearance, E284-02b, E312-02, E80501a, E179-96, E991-98, E1247-92, E308-01, E313-00, E808-01, E1336-96, E1341-96, E1347-97, E1360-90, 033287־, D38700־, E1455-97, E1477-98a, E1478-97 E1164-02, E1331-96, E1345-98, E1348-02, E1349-90, D5531-94, D3964-80, E1651-94, E1682-96, E1708-95, E1767-95, E180896, E1809-01, E2022-01, E2072-00, E2073-02, E2152-01, E2153-01, D1544-98, E259-98, D3022-84, D1535-01, E2175-01, E2214-02, and E2222-02, 2002; “ASTM Book of Standards, Volume 06.02, Paint -- Products and Applications; Protective Coatings; Pipeline Coatings, D4838-88 and D5326-94a, 2002; and “ASTM Book of Standards, Volume 06.03, Paint Pigments, Drying Oils, Polymers, Resins, Naval Stores, Cellulosic Esters, and Ink Vehicles,״ D2090-98, D2090-98 and D6166-97, 2002. Specific techniques for matching two or more colored coatings and/coating components to mlnimalize differences (e.g., metamerism) have been described, for example, in ASTM Book of Standards, Volume 06.01, Paint - Tests for Chemical, Physical, and Optical Properties; Appearance, D4086-92a, E1541-98 D2244-02 2002. Specific techniques for determining differences in the color of a coatings and/coating components, particularly to insure color consistency of a coating composition, “ASTM Book of Standards, Volume 06.01, Paint - Tests for Chemical, Physical, and Optical Properties; Appearance,” D1729-96, D2616-96, E1499-97, and D3134-97, 2002.

Gloss is the film's angular selectivity of reflectance, involving surface-reflected light, responsible for the degree to which reflected highlights or images of objects may be seen as superimposed on a surface (ASTM Book of Standards, Volume 06.01, Paint - Tests for Chemical, Physical, and Optical Properties; Appearance, E284-02b, 2002). An example of a high gloss coating would be a paint film with a glass-like surface apparance, as opposed to a lowgloss (flat) paint. Standard techniques for determining the gloss (e.g., specular gloss, sheen, haze, image clarity, waviness, directionality) of a coating and/or film are described, for example, in “ASTM Book of Standards, Volume 06.01, Paint - Tests for Chemical, Physical, and Optical Properties; Appearance, E284-02b, D523-89, D4449-90, E167-96, E430-97, D4039-93, D576795, and D2244-02, 2002; ASTM Book of Standards, Volume 06.02, Paint - Products and Applications: Protective Coatings; Pipeline Coatings, D392800־a, 2002; and Paint and Coating Testing Manual, Fourteenth Edition of the Gardner-Sward Handbook, (Koleske, J. V. Ed.), pp. 470-480, 1995.

48fffifff

73&lt; סי i

7. Preferred Use of the Invention tn certain embodiments, the compositions and methods of the present invention have use in three primary markets that will benefit from a susceptible surface covered with a selfdecontaminating coating: domestic military, friendly foreign military/civilian, and domestic civilian. It Is contemplated that for military use, a self-decontaminating coating has utility on a surface of a vehicle, a trailer, a barrack, a decontamination shelter, a piece of equipment (e.g., a piece of electronic equipment) or a combination thereof.

It is further contemplated that a biomolecular composition of the present invention may have dual military and/or civilian use in a method for facilitating the disposal of a chemical waste, including but not limited to, a CWA, a pesticide or a combination thereof. A particular dual use embodiment of the present invention includes coating a surface that may be in a facility where there would be an unexceptable delay to the use of a piece of equipment, a space (e.g., a room, a command center, a computer center), a vehicle (e.g., a public transportation vehicle, an emergency vehicle) or a combination thereof if the facility was subjected to or suspected of exposure to, a dangerous chemical (e.g., a nerve agent). In some aspects, the piece of equipment, the space, and/or the vehicle may be used by a military personnel, an emergency personel or a combination thereof. In specific facets, the piece of equipment, the space, and/or the vehicle is a critical piece of equipment, space and/or vehicle. It is contemplated that such a facility may be contacted with a chemical from a chemical weapon attack (e.g., a CWA gas attack), an accidental release of a chemical, or a combination thereof. Examples of such facilities include a control room at a military base, an airport, a nuclear power plant, a hospital, or a combination thereof. It is an aspect of the disclosure that a facility (i.e., a space, a vehicle, a piece of equipment) that may be subject to exposure to a chemical (e.g., a nerve agent) may be coated with the disclosed compositions and would then be detoxified and safe after contact with the chemical.

Civilian applications contemplated include a coating of a surface in contact with air, such as for example, a ventilation intake or an air filter, as well as a surface (e.g., an interior surface, an exterior surface) comprised in a hospital clean room, a community safe room, a control room for a nuclear plant, a control room for a chemical plant, a control room for a power plant, a control room for a water plant, a government building, an industrial building, a facility for public transportation (e.g., a train, a subway, a plane, an airport), and a surface of an equipment by a first responder, or any combination of the forgoing.

It is contemplated that for each formulation of a coating and a biomolecular composition of the present invention, enzymatic decontamination parameters based on chemical (e.g., CWA

48ggggg gggg simulant) degradation assessment will be established in a range of exterior weathering conditions. If a specific formulation of enzyme composition in a coating remains active after exposure to exterior weathering conditions, there is a significant utility for using the bioactive painted surfaces in exterior and field application. For example, it is contemplated that a biomolecular composition of the present invention incorporated in standard formulations of waterbased or latex-based paint will result in minimal to no changes in the durability of the paint based on standard exterior weathering conditions. In a general aspect of the present invention, a weathering study may indicate a need to reformulate a composition to improve a particular property (e.g., enhance biomolecular composition stability). In this aspect, it is contemplated that standard methods, known to those of skill in the art (e.g., encapsulation), may be used to increase stability and re-test the resulting formulation. Application of such methods can be used to modify various formulations to produce a composition with one or more properties optimized to a particular application, as described herein and as would be know to one of ordinary skill in the art in light of the present disclosures.

8. Combinations of Decontamination Compositions and Methods

In certain embodiments, a composition or method of the present invention may be combined with another composition method for decontamination (e.g., detoxification, degradation) of a chemical. In preferred aspects, the additional composition or method comprises one for decontamination of a pesticide or chemical warfare agent. Such additional compositions and methods are known in the art (Yang, Y.C. etal., 1992), and may be applied prior, during and/or after application of a composition and/or method of the present invention. In particularly preferred embodiments, such a combination of a composition and/or method disclosed herein with a traditional composition and/or method produces greater decontamination than that achieved without such a combination.

Additional compositions that are contemplated include, but are not limited to, a caustic agent; a decontaminating foam (e.g., Sandia, Decon Green); an application of intensive heat and carbon dioxide for a sustained period; an incorporation of a material into a coating that, when exposed to sustained high levels of UV light, degrades a chemical; a chemical agent resistant coating; or a combination thereof. Examples of a caustic agent include, a bleaching agent, DS2, or a combination thereof.

As used herein, a caustic agent is a composition capable of destroying usually via a chemical reaction, a material, unfortunately including animal tissue such as skin. Thus, application of a caustic agent is often accompanied by the wearing of protective gear for those not contaminated or suspected of being contaminated, as would be understood by those of

48hhhhh hhhh ordinary skill in the art. Certain caustic agents, such as for example, a bleaching agent or decontamination solution 2 (“DS2), have specifically been formulated and/or used to decontaminate chemical warfare agents. Both G agents and VX can be decontaminated with these caustic agents. As used herein, a “bleaching agent” refers to a reactive chemical compound capable breaking a double bond in another chemical compound, which is often a useful property for degrading a toxic or otherwise undesirable chemical. Examples of a bleaching agent include a bleach powder, a bleach solution, or a combination thereof. A bleach powder may comprise, but is not limited to, Ca(OCI)CI and Ca(OCI)₂ (“high test hypochlorite, ΉΤΗ״); Ca(0CI)2 and CaO (“super tropical bleach, STB”); Ca(OCI)₂ and MgO (Dutch powder); or a combination thereof. A bleach solution may comprise, but is not limited to, NaOCI (bleach), usually 2% to 6% wt in water; a HTH slurry, usually 7% HTH wt in water; a STB slurry, usually 7% to 70% wt in water; activated solution of hypochlorite (“ASH), usually 0.5% Ca(OCI)₂ and 0.5% sodium dihydrogen phosphate buffer and 0.05% detergent in water; self-limited activated solution of hypochlorite (“SLASH”), usually 0.5% Ca(OCI)₂ and 1.0% sodium citrate and 0.2% citrate acid and 0.05% detergent in water; or a combination thereof. Bleach, Dutch powder, ASH and SLASH are generally applied to skin and equipment for decontamination, while HTH and STB are generally applied to equipment and terrain for decontamination. VX is preferably decontaminated at an acid pH, wherein it is more soluble (Yang, Y.C. et al., 1992).

DS2 was developed to function at various temperatures (I.e., -25°C to 52°C), particularly those below the freezing point of most aqueous compositions. It usually comprises 70% diethylenetriamine (H₂NCH₂CH₂NHCH₂CH₂NH₂), 28% ethylene glycol monomethyl ether (CH₃OCH₂CH₂OH), and 2% sodium hydroxide (NaOH). DS2 is noncorrosive to many metals, but is damaging to many paints, leathers, rubber materials, plastics and skin. Contact with a paint is generally limited to 30 minutes or less. An aqueous rinse is generally used to remove DS2, and exposure to air and/or water degrades DS2 (Yang, Y.C. et al., 1992).

Various other decontamination compositions and methods are known to those of skill in the art. Examples of a decontaminating foam include Sandia, Decon Green, or a combination thereof. Examples of an incorporation of a material include incorporation of TiO₂ and porphyrins into acetonitrile coatings that, when exposed to a sustained high level of UV light in an oxygen environment (e.g., air), degrade a chemical agent (e.g., mustard). Addition of water to the acetonitrile coating comprising TiO₂ and porphyrins will aid the degradation of VX to non-toxic compounds (Buchanan, J. H. etal., 1989; Fox, M. A., 1983). Additionally, CARCs have been developed to withstand repeated decontamination efforts.

48iiiiiiiii

Decontamination compositions are often prepared and packaged in equipment for easy of handling. Such an equipment packages include, but are not limited to, kits (e.g., a toweiette package) and delivery apparatus (e.g., a sprayer). Examples of specific decontamination equipment packages that may be used in combination with a composition or method of the present invention include a ABC-M11 portable decontamination apparatus, which comprises DS2, a devise for spraying DS2, and a vehicle mounting bracket; a ABC-M12A1 power-driven, skidmounted decontamination apparatus, which comprises a personnel shower unit, a pump, a tank, a M2 water heater, and delivers water, foam, DS2, STB, and/or deicing liquid; a M258A1 personal decontamination kit, which comprises towelettes soaked with a decontamination solution (i.e., 72% ethanol, 10% phenol, 5% NaOH, 0.2% ammonia, and 12% water), ampules of a decontaminating solution (5% ZnCI₂, 45% ethanol, 50% water) for adding to a towlette soaked with chloramines-B (PhS(O)₂NCINa), packing foil, and a plastic carrying case; a M280 individual equipment decontamination kit, which comprises twenty fold the contents of the M258A1 kit; a M291 skin decontamination kit, which comprises six XE-555 resin (i.e., styrene/divinyl benzene copolymer, a strong acid cation-exchange resin and a strong base anion-exchange resin for absorption and chemical detoxification) filled fiber pads packaged in foil; a M13 portable decontamination apparatus, which comprises DS2, a container and an equipment/vehlcle mount, and is capable of dispensing DS2; a M17 lightweight, transportable decontamination apparatus, which comprises hoses, cleaning jets, personnel showers, a collapsible rubberized fabric tank, and is capable of dispensing water; or a combination thereof. The ABC-M11, M13 and M280 decontamination equipment packages are generally used for equipment (e.g., vehicles), the M258A1 and M17 decontamination equipment packages are generally used for equipment and/or personnel, and the ABC-M12A1 and M291 decontamination equipment packages are generally used for personnel (Yang, Y.C. et al., 1992).

9. Removing a Coating or Film

In certain embodiments, it may be desirable to remove a coating and/or film from a surface such as a non-film forming coating, a temporary film, a self-cleaning film, a coating and/or film that has been damaged, contaminated with an OP compound, or is otherwise no longer desired or no longer is suitable for use. Various coating removers (e.g., a paint remover) are known to those of ordinary skill in the art, and often comprise solvents described herein capable of dissolving a coating component (e.g., a binder) integral to a film's structural integrity. Standard procedures for determining the effectiveness of a coating remover have been described, for example, in ASTM Book of Standards, Volume 06.02, Paint - Products and Applications; Protective Coatings; Pipeline Coatings, D6189-97, 2002.

48jjjjjjjjj

Example 24

Assay for Active Phosphoric Trlester Hydrolase Expression In Cells

Routine analysis of parathion hydrolysis in whole cells is accomplished by suspending cultures in 10 milli-Molar (mM) Tris hydrocholoride at pH 8.0 comprising 1.0 mM sodium EDTA (TE buffer'¹). Cell-free extracts are assayed using sonicated extracts in 0.5 milliLiters (“ml) of TE buffer. The suspended cells or cell extracts are incubated with 10 microLiters (μΓ) of substrate, specifically 100 pg of parathion in 10% methanol, and p-nitrophenol production is monitored at a wavelength of 400 nm. To induce the opd gene under lac control, 1.0 pmol of isopropyl-p-Dthiogalactopyranoside (Sigma) per ml is added to the culture media.

Example 25

Preparation of Enzyme Powder

In a typical preparation, a single colony of bacteria that expresses the opd gene is selected and cultured in a rich media. After growth to saturation, the cells are concentrated by centrifugation at 7000 rotations per minute (rpm) for 10 minutes for example. The cell pellet is then resuspended In a volatile organic solvent such as acetone one or two times in order to dessicate the cells and to remove a substantial portion of the water contained in the cell pellet. The pellet may then be ground or milled to a powder form. The powder may be frozen or stored at ambient conditions for future use, or may be added immediately to a surface coating formulation. Additionally, the powder may be freeze dried, combined with a cryoprotectant (e.g., cryopreservative), or a combination thereof.

Example 26

Two-Pack OPH Paint Coating: OPH Powder and Latex Paint

In an example of use of the powder prepared as described in Example 25, 3 mg of the milled powder was added to 3 ml of 50% glycerol. The suspension was then added to 100 ml of Olympic® premium interior flat latex paint (Olympic®, One PPG Place, Pittsburg, PA 15272 USA). This paint with biomolecular composition was then used to demonstrate the activity of the paint biomolecular composition in hydrolysis of a pesticide or a nerve agent analog.

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Example 27 Application of OPH Paint to a Surface

In a first set of assays, a paint product as prepared in Example 26 was applied to a hard, metal surface. The surface used in the present example was a non-galvanized steel surface that was cleaned through being degreased, and pretested with a primer coat. A control surface was painted with the identical paint with no biomolecular composition. Paraoxon, an organophosphorus nerve gas analog was used as an indicator of enzyme activity. Paraoxon, which is colorless, is degraded to form p-nitrophenol, which is yellow in color, plus diethyl phosphate, thus giving a visual indication of enzyme activity. In multiple assays, the surface with control paint remained white, indicating no production of p-nitrophenol, and the surface painted with the paint and biomolecular composition turned yellow within minutes, indicating an active OPH enzyme in the paint. This demonstration has shown that the surface remains active for more than 65 days, which was the maximum duration of the protocol.

In a further demonstration, the surfaces were treated as described above and each surface was then treated with paraoxon, an OP insecticide. Approximately 100 flies were then placed on each surface under a plastic cover. In each procedure, within three hours, virtually all the flies on the control surface with no paint biomolecular composition were killed by the paraoxon. In contrast, only approximately 5% of the flies on the enzyme comprising surface had died.

In a demonstration of enzyme stability in the paint, a series of wood dowels were dipped into the paint with OPH enzyme composition. The dowels were then placed in tubes containing paraoxon to indicate enzyme activity as described above. In each case, a positive yellow color was seen except in those dowels painted with no biomolecular composition as controls. The control solution remained clear in every case.

In order to demonstrate the shelf life of both the dry biomolecular composition and the paint with biomolecular composition, the biomolecular composition was aged from 0 to 20 days prior to mixing in the paint. The mixed paint and biomolecular composition was then also aged from 0 to 20 prior to painting individual dowels. The enzyme composition retained strong activity after 20 days aging prior to being mixed in the paint, and for 20 days after mixing the maximum time used in the assay.

48111U1111

Example 28

Buffered Enzyme Paint

As the hydrolysis reaction that degrades nerve agents proceeds, the local pH decreases. Without limiting the invention to any particular mechanism, it is contemplated that due to the law of mass action, or to the optimum pH of the enzyme, the reaction is slower as the pH decreases. Because this effect could prevent or inhibit some surfaces from becoming completely decontaminated, active paint formulations have been prepared that include one or more buffering agents.

In initial procedures, the following compositions were used: 10 mg enzyme powder as described in Example 25, 100 μΙ 0.1 M buffer, 800 μΙ H_ZO, and 100 μΙ paraoxon for a 1000 μΙ reaction volume.

Reactions were run for 1.5 to 2 hours and both pH and product concentration were measured. The concentration of product (p-nitrophenol) is measured by absorbance at 400 nm.

Ammonium bicarbonate, both monobasic and dibasic phosphate buffers, Trizma base and five zwitterionic buffers have been used in the active paint compositions. All the buffers were effective at allowing the reaction to proceed further to completion, thus demonstrating the advantage of addition of a buffering agent to the active paint compositions.

Example 29

NATO Demonstration of Soman Detoxification Using OPH-Painted Surfaces

At the September 22, 2002, meeting of the NATO Army Armaments Group in Cazaux, France, painted metal surfaces were assayed with soman using standard NATO procedures and protocols. For the assays, 10 cm x 10 cm metal plates primed with standard NATO specification paints were coated with paint containing OPH. Control plates plus two different versions of the OPH enzyme composition differing in soman detoxification specificity were used. These surfaces were allowed to dry for several hours at room temperature and then assayed according to standard NATO assay protocol (described below), modified to account for the unique character of the surfaces treated with a paint comprising OPH.

The form of OPH in the biomolecular composition contains both the changes of the previously described H254R mutant and the H257L mutant, and is corresponding designated the “H254R, H257L mutant.” The H254R, H257L mutant demonstrates a several-fold enhanced rates

48mm tn nunmm mm of R-VX catalysis relative to either the H254R mutant or the H257L mutant, and a 20-fold enhancement of activity relative to wild-type OPH. This version of the OPH biomolecular composition has been assayed in paints treated with soman or R-VX, and are described below.

Following standard protocols, OPD painted surfaces were uniformly contaminated with an isopropanol solution containing the chemical warfare agent soman. The concentration of soman on each contaminated surface was 1.0 mg/cm². The contaminated plates were maintained at or slightly above room temperature (>2013) without any forced air-flow for various perio ds of time. A zero-time, 15 minutes, 30 minutes, and 45 minutes sample was taken for each control and biomolecular composition-containing plate series. In order to terminate the reaction and isolate residual soman on the plate surface, each plate was submerged in a container of isopropanol at the end-point and placed on a shaker to thoroughly extract any residual nerve agent. The solubilized portions were then quantified for soman. These assays showed that both the forms of OPH biomolecular composition were highly effective in detoxifying soman on metal surfaces. The two different OPH biomolecular compositions assayed detoxified the soman at levels over 65% and 77% after 45 minutes (Nato Army Armaments Group Project Group 31 on Non-Corrosive, Biotechnology-Based Decontaminants for CBW Agents, 2002). Additional assays with a CWA simulant indicated that had the NATO assay run for one to two hours, substantially all of the soman would have been detoxified.

Example 30 Aberdeen Proving Ground

A demonstration of an OPH biomolecular composition has been conducted at SBCCOM in Aberdeen, MD. In these assays, a primed wooden stick was coated with paint containing OPH biomolecular composition. The painted sticks used were 2 milimeter (mm”) in diameter x 15 mm in length. By estimating that the paint layer was 0.25 mm thick, the resulting surface area was approximately 125 mm². After coating the stick with paint containing OPH biomolecular composition and allowing the paint to dry, the coated stick was inserted into a microfuge tube containing 100 μΙ of 3.24 mM Russian-VX agent in saline and 900 μΙ phosphate buffer at pH 8.3. The tubes containing R-VX and the painted sticks were allowed to sit overnight in a hood at room temperature. Appropriate controls were run simultaneously.

The following morning, the contents of the microfuge tubes were assayed for free thiols by the Eilman method. 10 mM DTNB [molecular weight (“MW) 396.3] was prepared in 10 mM phosphate buffer at pH 8.0 for use as the indicator of enzyme activity. OPH paint's cleavage of R-VX releases a free thiol that reacts with DNTP to produce a colored product detectable

48nnnnn nnnn spectrophotometrically at 405 nm. Ten μΙ of the microfuge tube contents, 100 μΙ DTNB solution and 890 μΙ phosphate buffer at pH 8.3 were read for thiol release at 405 nm using a Varian Carey 300 Spectrophotometer. The spectrophotometer was blanked with an unpainted stick control reaction. The molar equivalent of the R-VX hydrolyzed was determined using an extinction coefficient of 14,150 and the Beer-Lambert equation to calculate the product concentration. Results indicated that overnight exposure to OPH paint coated sticks resulted in decontamination of Russian VX from 32.4 μΜ in the original tube to less than 1 μΜ.

Example 31

NATO Protocols for Organophosphorus CWA Decontamination

This example describes a method for determining the decontamination properties of a coating, specifically paint, comprising an phosphoric triester hydrolase biomolecular composition of the present invention. NATO assay requirements will be followed as closely as possible. Although actual assaying protocols among NATO countries vary somewhat, standard to ail is the level of contamination. For exterior surfaces it is 10grams per meter squared (“g/m²). For interiors it is 1g/m². Basic elements of NATO assaying procedures are as follows:

A. Coated Surface

A 10x10 cm metal plate coated with a coating that may comprise a biomolecular composition of the present invention.

B. Contamination

Usually achieved with a multi-channel micropipette that can dispense 1 pl drops, with 100 drops per 10 x 10 cm metal plate.

C. Incubation

The plates will be placed into a sealed incubator, at 25C or 30Ό, for a period ranging from 30 minutes to 3 hours.

D. Decontamination

The decontamination protocol varies according to the system being assayed. For example, spraying of decontamination solutions will last between 5 seconds to 20 seconds, depending on the pressure of the system.

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Ε. Sampling

For standard solution-based decontamination, the assays will be normally prepared in a way that run-off decontaminant will be collected after it comes in contact with the plates and the CWA agent or CWA simulant. A set of plates will be removed for analysis at intervals, with the most common being 15 minutes and 30 minutes. Any residual liquid on the plates will be added to the run-off. For enzyme btomolecular composition assays, the plates will be not rinsed after decontamination, although the rinse is standard with other decontaminants. This rinsate would also be collected for analysis. A set of plates without decontamination will be used as 0 minute, 15 minute, and 30 minute controls.

F. Analysis

The run-off liquid and rinsate will be immediately extracted with a solvent, such as, for example, chloroform, hexane, etc., known to dissolve the CWA agent or CWA simulant. The plates themselves can be subjected to two types of analysis: contact hazard and off-gas hazard. For contact hazard, the plates will be covered with an absorbent material. For example, the French government uses silica gel TLC plates, and the government of the USA uses a dental dam as the absorbent material. In either case, the absorbent material is held in place with a weight and incubated for 15 minutes to 30 minutes at 25־C or 30C. The absorbent will be removed and extracted with solvent. The plates will be then extracted with solvent to determine residual agent absorbed into the paint, and thus the contact hazard. If surface decontamination efficiency, specifically the amount of residual agent detectable, is the only variable being assessed, the plates will be immediately extracted with solvent, eliminating the contact hazard step. All of the solvent samples will be analyzed by Gas Chromatography (“GC) with a flame photometric detector (“FPD) and a phosphorus filter for nerve agents. Some countries use Gas Chromatography-Mass Spectrometry (GC-MS) for the analysis.

Example 32 Large-Scale Batch Fermentation to Produce OPH

Batch Culture-Rich Medium comprised 24 g/L yeast extract; 12 g/L casein hydrolysate; 4 ml/L glycerol; 2.31 g/L KH₂PO&lt;; 12.54 g/L K₂HPO₄; 0.24 g/L CoCI₂6H₂O; 2 g/L glucose; 0.2 ml/L PPG2000; and 100 pg/ml ampicillin.

Batch Culture-5 L scale was grown at the following conditions: 30°C; 400-450 rpm agitation; CO controlled at 20%; uncontrolled initial pH between 6.8-6.9: 5 Lpm (1 wm) aeration; and atmospheric pressure. Over a time period of 0 to 50 hours, the Escherichia coli strain's growth was measured by optical density at 600 nm, the specific paraoxonase activity was determined

48ppppp

PPPP (pmol m!'¹ min'¹), the volumetric paraoxonase activity was determined (pmol ml'¹ min'¹), the pH measured over a range of pH 6 to pH 9, the agitation measured over a range of 0 rpm to 500 rpm, and the dissolved oxygen measured over a range of 0% to 100%.

Batch Culture-400 L scale was grown at the following conditions: 30°C; 150200־ rpm agitation; DO at 0-100%; uncontrolled initial pH 6.58; 200-300 Lpm {0.5-0.75 vvm) aeration; and tank pressure at 0-10 psi. Over a time period of 0 to 30 hours, the Escherichia coli strain’s growth was measured by optical density at 600 nm, the specific paraoxonase activity was determined (pmol ml‘¹ min'¹), the volumetric paraoxonase activity was determined (pmol ml'¹ min'¹), the pH measured over a range of pH 6 to pH 6, the agitation measured over a range of 0 rpm to 200 rpm, the dissolved oxygen measured over a range of 0% to 100%, the aeration rate measured over a range of 0 to 300 Lpm, and the tamk pressure measured over a range of 0 psi to 12 psi.

Example 33 Large-Scale Fed-Batch Fermentation to Produce OPH

Fed Batch Culture-Defined Medium comprised 13.3 g/L KH_ZPO₄: 4 g/L (NH₄)₂SO₄; 1.7 g/L citric acid; 10 g/L glycerol; 1.2 g/L MgSO₄'7H₂O; 0.024 g/L MnCI₂4H₂O; 2.26 mg/L CuCI₂H₂O; 5 mg/L H₃BO₃; 4.5 mg/LThiamineHCI; 4 mg/L Na₂M00₄'7H_zO; 0.06 g/L Fe(lll) citrate; 8.4 mg/L EDTA; 4 mg/L CoCI₂'6H₂O; 8 mg/L Zn(acetate)_zH_zO; and 100 pg/ml ampicillin.

Feed: 500 g/L carbon source and 10 g/L MgSO₄7H₂O.

Batch Culture-5 L scale was grown at the following conditions: 30°C; 200-1000 rpm agitation; DO controlled at 20%; pH controlled at 6.5; 5 Lpm (1 wm) aeration: and atmospheric pressure. Feed was initiated as the 16^th hour, with the feed rate profile a constant rate with stepwise increments. Over a time period of 0 to 70 hours, the Escherichia coli strain's growth was measured by optical density at 600 nm, the specific paraoxonase activity was determined (pmol ml'¹ min'¹), the volumetric paraoxonase activity was determined (pmol ml¹ min ¹), the pH measured over a range of pH 6 to pH 9, and the addition of the feed measured from 0 ml to 1000 ml.

Example 34 Coating Formulation

It is contemplated that any described coating composition may be altered (e.g., by direct addition and/or coating component substitution) to incorporate the biomolecular composition of the present invention. The previous embodiments of the invention primarily described

48qqqqq qqqq compositions and techniques for preparing, testing, and using a coating prepared de novo. However, it is contemplated that the biomolecular composition of the present invention may be incorporated into a standard coating by direct addition, as described in Example 26. In specific aspects, it is contemplated that such added biomolecular composition may comprise 0.001% to 40% or more, including all intermediate ranges and combinations thereof, by weight or volume, of the final composition produced by a combination of a coating and the biomolecular composition of the present invention.

Alternatively, it is contemplated that a previously described coating composition may be altered by substitution (“replacement”) of one or more coating components, particularly a binder and/or a particulate material coating component (e.g., a pigment, a rheological control agent, a dispersant) by the biomolecular composition of the present invention. It is contemplated that 0.001% to 100%, including alt intermediate ranges and combinations thereof, of the binder and/or particulate material coating component may be substituted by biomolecular composition of the present invention. Additionally, the concentration of a biomolecular composition of the present invention may exceed 100%, by weight or volume, of the substituted coating component. In specific aspects, a coating component may be substituted with a biomolecular composition of the present invention equivalent to 0.001% to 500%, including all intermediate ranges and combinations thereof, of the coating component. For example, a 20% (e.g., 2 kg) of a dispersant may be replaced by 10% (e.g., 1 kg) of the biomolecular composition of the present invention to produce a coating with similar dispersion properties as a non-substituted formulation. In an addition example, 70% of a specific pigment (e.g., 7 kg) may be replaced by the equivalent of 127% (e.g., 12.7 kg) of the biomolecular composition of the present invention to produce a coating with similar hiding power as a non-substituted formulation. The various assays described herein, or as would be known to one of ordinary skill in the art in light of the present disclosures, may be used to determine the properties of a coating and/or film produced by direct addition and/or coating component substitution by the biomolecular composition of the present invention.

The following is an example of a exterior gloss alkyd house paint that comprises various particulate materials (e.g., silica, a shading pigment, bentonite clay) that may incorporate a biomolecule composition of the present invention. This example of an exterior gloss alkyd house paint comprises a grind and a letdown. The grind comprises by weight or volume: a first alkyd 232.02 lb or 29.9 gallons; a second alkyd 154.2 lb or 20 gallons; an aliphatic solvent (e.g., duodecane) 69.55 lb or 1.7 gallons; lecithin 7.8 lb or 0.91 gallons; TiO2 185.25 lb or 5.43 gallons; 10 micron silica 59.59 lb or 2.7 gallons; bentonite clay 18.00 lb or 1.44 gallons; a second alkyd 97.22 lb or 12.61 gallons; a first alkyd 69.84 lb or 9.00 gallons; and mildewcide 7.8 lb or 0.82 gallons. The letdown comprises by weight or volume: aliphatic solvent (e.g., dudecane) 19.50 lb

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ΓΤ or 3.00 gallons; a first drier (e.g., 12% solution cobalt) 2.00 lb or 0.23 gallons; a second drier (e.g., 18% solution Zr) 2.92 lb or 0.32 gallons; a third drier 3 (e.g., 10% solution Ca) 8.00 lb or 0.98 gallons; methyl ethyl ketoxime (Anti skinning agent) 3.22 lb or 0.42 gallons; an aliphatic solvent 9.75 lb or 1.50 gallons; and a shading pigment 0.3 lb or 0.04 gallons. In some embodiments, the particulate material of the coating formulation may be partly or fully substituted by the biomolecule composition of the present invention. In other embodiments, the above formulation may be enhanced by direct addition of a biomofecuie composition of the present invention.

In another example, the following exterior flat latex house paint may be modified to incorporate a biomolecule composition of the present invention. This example of an exterior flat latex house paint formulation, in typical order of addition, by weight or volume: water, 244.5 lb or 29.47 gallons; hydroxyethylcellulose, 3 lb or 0.34 gallons; glycols, 60 lb or 6.72 gallons; polyacrylate dispersant, 6.8 lb or 0.69 gallons; biocides, 10 lb or 1 gallons; non-ionic surfactant, 1 lb or 0.11 gallons; titanium dioxide, 225 lb or 6.75 gallons; silicate mineral, 160 lb or 7.38 gallons; calcined clay, 50 lb or 2.28 gallons; acrylic latex, @ 60%, 302.9 lb or 34.42 gallons; coalescent,

9.3 lb or 1.17 gallons; defoamers, 2 lb or 0.26 gallons; ammonium hydroxide, 2.2 lb or 0.29 gallons; 2.5% HEC solution, 76 lb or 9.12 gallons. In some embodiments, the particulate material (e.g., silicate mineral, calcined clay, titanium dioxide) of this coating formulation may be partly or fully substituted by the biomolecule composition of the present invention. In other embodiments, the above formulation may be enhanced by direct addition of a biomolecule composition of the present invention.

It is contemplated that any such previously described coating formulation may be modified to incorporate a blomolecular composition of the present invention. Examples of described coating compositions include over 200 industrial water-borne coating formulations (e.g., air dry coatings, air dry or force air dry coatings, anti-skid of non-slip coatings, bake dry coatings, clear coatings, coil coatings, concrete coatings, dipping enamels, lacquers, primers, protective coatings, spray enamels, traffic and airfield coatings) described in “Industrial waterbased paint formulations, 1988, over 550 architectural water-borne coating formulations (e.g., exterior paints, exterior enamels, exterior coatings, interior paints, interior enamels, interior coatings, exterior/interior paints, exterior/interior enamels, exterior/interior primers, exterior/interior stains), described in Water-based trade paint formulations, 1988, the over 400 solvent borne coating formulations (e.g., exterior paints, exterior enamels, exterior coatings, exterior sealers, exterior fillers, exterior primers, interior paints, interior enamels, interior coatings, interior primers, exterior/interior paints, exterior/interior enamels, exterior/interior coatings, exterior/interior varnishes) described in “Solvent-based paint formulations, 1977; and the over

48ssssss sss

1500 prepaint specialties and/or surface tolerant coatings (e.g., fillers, sealers, rust preventives, galvanizers, caulks, grouts, glazes, phosphatizers, corrosion inhibitors, neutralizers, graffiti removers, floor surfacers) described in Prepaint Specialties and Surface Tolerant Coatings, by

Ernest W. Flick, Noyes Publications, 1991.

Example 35 Ranges

To provide a description of the present invention that is both concise and clear, various examples of ranges have been identified herein with the phrase “including all intermediate ranges and combinations thereof. Examples of specific values (e.g., %, kDa, °C, pm, kg/L, Ku) that can be within a cited range by the reference to including all Intermediate ranges and combinations thereof include 0.000001, 0.00001, 0.0001, 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, 1.0, 1.1,1.2, 1.30, 1.31,

1.32, 1.33, 1.34, 1.35, 1.36, 1.37, 1.38, 1.39, 1.40, 1.41, 1.42, 1.43, 1.44, 1.45, 1.46, 1.47, 1.48,

1.49, 1.50, 1.51, 1.52, 1.53, 1.54, 1.55, 1.56, 1.57, 1.58, 1.59, 1.60, 1.61, 1.62, 1.63, 1.64, 1.65,

1.66, 1.67, 1.68, 1.69, 1.70, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2,

3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1,4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67. 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105,106, 107,108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127,

128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145,146,

147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164,165,

166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183,184,

185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202,203,

204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221,222,

223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240,241,

242, 243, 244, 245, 246, 247, 248, 249, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475,500,

525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950,975,

1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, or more.

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However, general ranges for each type of unit (e.g., %, kDa, °C, pm, kg/L, Ku) are contemplated. Examples of values that can be within a cited percentage range, as applicable, include 0.001% to 100%, including all intermediate ranges and combinations thereof. Examples of values that can be within a cited molecular mass range, as applicable, in kilo Daltons (“kDa”), include 0.50 kDa to 110 kDa, including all intermediate ranges and combinations thereof. Examples of values that can be within a cited temperature range, as applicable, in degrees Celsius (°C), that can be within a cited range include of 0°C to 500°C, including all intermediate ranges and combinations thereof. Examples of values that can be within a thickness range (e.g., coating and/or film thickness upon a surface), as applicable, in micrometers (“pm), that can be within a cited range include of 1 pm to 2000 pm, including all intermediate ranges and combinations thereof. Examples of values that can be within a cited density range, as applicable, in kilograms per liter (kg/L”), include 0.50 kg/L to 20 kDa, including ail intermediate ranges and combinations thereof. Examples of values that can be within a cited shear rate range, as applicable, in Ku, include 20 Ku to 300 Ku, including all intermediate ranges and combinations thereof.

Example 36 Elastomers

It is contemplated that a biomolecular composition may also be incorporated into an elastomer. Elastomers (rubbers) are polymers that can undergo large, but reversible, deformations upon a relatively low physical stress. It is contemplated that an elastomer composition may incorporate a biomolecular composition of the present invention, such as by preparation with the biomolecular composition and/or direct addition such as by a multi-pack composition. Elastomers (e.g., tire rubbers, polyurethane elastomers, polymers ending in an anionic diene, segmented polyerethane-urea copolymers, diene triblock polymers with styrenealpha-methylstyrene copolymer end blocks, poly(p-methylstyrene-b-E-methylstyrene), polydimethylsiloxane-vinyl monomer block polymers, chemically modified natural rubber, polymers from hydrogenated polydienes, polyacrylic elastomers, polybutadienes, transpolyisoprene, poly isobutene, cis-1,4-polybutadiene, polyolefin thermoplastic elastomers, block polymers, polyester thermoplastic elastomer, thermoplastic polyurethane elastomers) and techniques of elastomer synthesis and elastomer property analysis have been described, for example, in Walker, Β. M., ed., Handbook of Thermoplastic Elastomers, Van Nostrand Reinhold Co., New York, 1979; Holden, G., ed., et. al., Thermoplastic Elastomers, 2^nd Ed., Hanser Publishers, Verlag, 1996.

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Example 37 Fillers and Filled Polymers

A filler is a bulk material in a composition. Extender pigments are used as a filler for coatings. In certain embodiments, a biomolecular composition may be used as a filler for various compositions. Examples of compositions that use fillers that are contemplated herein for incorporation of a biomolecular composition of the present invention, include a composition comprising a polymer, thermoplastic material, a thermostat material, an elastomer, or a combination thereof. Such filler comprising materials have been described in Gerard, J.F., ed., Fillers and Filled Poiymenrs-Macromolecular Symposia 169, Wiley-VCH, Verlag, 2001; Slusarski, L., ed., Fillers for the New Millenium-Macromolecular Symposia 194, Wiley-VCH, Verlag, 2003; and Landrock, A. H., Adhesives Technology Handbook, Noyes Publications, New Jersey, 1985.

Example 38 Adhesives and Sealants

An adhesive is a composition that is capable of holding at least two surfaces together in a strong and permanent manner. A sealant is a composition capable of attaching to at least two surfaces, filling the space between them to provide a barrier or protective coating. In certain embodiments, a biomolecular composition may be used as a component of an adhesive or a sealant, such as, for example, by direct addition, substitution of an adhesive or sealant component (e.g., a particulate material), or a combination thereof.

Examples of adhesives and sealants (e.g. caulks, acrylics, elastomers, phenolic resin, epoxy, polyurethane, anarobic and structural acrylic, high-temperature polymers, water-based industrial type adhesives, water-based paper and packaging adhesives, water-based coatings, hot melt adhesives, hot melt coatings for paper and plastic, epoxy adhesives, plastisol compounds, construction adhesives, flocking adhesives, industrial adhesives, general purpose adhesives, pressure sensitive adhesives, sealants, mastics, urethanes, ) for various surfaces (e.g., metal, plastic, textile, paper), adhesive and sealant components (e.g.,antifoams, antioxidants, extenders, fillers, pigments, flame/fire retardants, oils, polymer emulsions, preservatives, bactericides, fungicides, resins, rheological/viscosity control agents, starches, waxes, acids, aluminum silicates, antiskinning agents, calcium carbonates, catalysts, crosslinking agents, curing agents, clays, corn starch, starch derivatives, defoamers, antifoams, dispersing agents, emulsifying agents, epoxy resin diluents, lattices, polybutenes, polyvinyl acetates, preservatives, acrylic resins, epoxy resins, ester gums, ethylene/viny! acetate resins, maleic resins, natural resins, phenolic resins, polyamide resins, polyethylene resins,

48wwv ww polypropylene resins, polyterpene resins, powder coating resins, radiation coating resins, urethane resins, vinyl chloride resins, emulsion resins, dispersion resins, resin esters, rosins, silicas, silicon dioxide, stabilizers, surfactants/surface active agents, talcs, thickeners, thixotropic agents, waxes) techniques of preparation and assays for properties, have been described in Skeist, I., ed., Handbook of Adhesives, 3^rd Ed., Van Nostrand Reinhold, New York, 1990; Satriana, M.J. Hot Malt Adhesives: Manufacture and Applications, Noyes Data Corporation, New Jersey, 1974; Petrie, E. M., Handbook of Adhesives and Sealants, McGraw-Hill, New York, 2000; Hartshorn, S. R., ed., Structural Adhesives-Chemistry and Technology. Plenum Press, New York, 1986; Flick, E. W., Adhesive and Sealant Compound Formulations, 2^nd Ed., Noyes Publications, New Jersey, 1984; Flick, E., Handbook of Raw Adhesives 2^nd Ed., Noyes Publications, New Jersey, 1989; Flick, E., Handbook of Raw Adhesives, Noyes Publications, New Jersey, 1982; Dunning, H. R., Pressure Sensitive Adhesives- Formulations and Technology, 2^nd Ed., Noyes Data Corporation, New Jersey, 197Z; and Flick, E. W״ Construction and Structural Adhesives and Sealants, Noyes Publications, New Jersey, 1988.

Example 39 Textiles

It is contemplated that a biomolecular composition may also be incorporated into a material applied to a textile, such as, for example, a textile finish. Materials for application to a textile, textile finishes (e.g., soil-resistant finishes, stain-resistant finishes) and finish components (e.g., antioxidants, defoamers, antimicrobials, wetting agents, flame retardants, softeners, soil repellents, hand modifiers, antistatic agents, biocides, fixatives, scouring agents, dispersants, defoamers, anticracking agents, binders, stiffeners, cohesive agents, fiber lubricants, emulsifiers, antistats, yarn to hard surface lubricants) as well as assays for determining their properties are described, for example, in Johnson, K., Antistatic Compositions for Textiles and Plastics, Noyes Data Corporation, New Jersey, 1976; Rouette, H.K., Encyclopedia of Textile Finishing, Springer, Veriag, 2001; Textile Finishing Chemicals: An Industrial Guide, by Ernest W. Flick, Noyes Publications, 1990; and Handbook of Fiber Finish Technology”, by Philip E. Slade, Marcel Dekker, 1998. A specific example of a textile finish is the trademark formulations of water repellent and/or oil repellent finish known as Scotchguard™ (3M Corporate Headquarters, Maplewood, Minnesota, U.S.A.).

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Example 40 Waxes

It is contemplated that a biomolecular composition may also be incorporated into a material applied to a surface after manufacture, such as, for example, a wax. Waxes (e.g., natural waxes, fossil waxes, earth waxes, peat waxes, montana waxes, lignite paraffins, petroleum waxes, synthetic waxes, commercial modified, blended, and compounded waxes, emulsifiable waxes, waxy alcohols, waxy acids, metallic soaps, compounded waxes, paraffin wax compounds, ethyl cellulose and wax mixtures, compositions with resins and rubber) and methods of wax preparation and assays for wax properties have been described, for example, in Warth, A. H., The Chemistry and Technology of Waxes, Reinhold Publishing Corporation, New York, 1956; Bennet, H., Industrial Waxes Volume II Compounded Waxes and Technology, Chemical Publishing Co., New York, 1975.

Example 41 Additional OPAAs

It additional embodiment is it contemplated that the following organisms produce an OPAA that may be used in a biomolecular composition of the present invention: Acinetobacter calcoaceticus ATCC 19606, Aeromonas hydrophila ATCC 7966, Aeromonas proteolytica, Arm. A isolate 1, Arm. A isolate 2, Bacillus subtilis (fr. Zuberer), Bacillus subtilis, ATCC 18685, Bacillus subtilis BRB41, Bacillus subtilis Q, Bacillus thuringensis (fr. Zuberer), Burkholderia cepacia LB400, Burkholderia cepacia T, Citrobacter diversus, Citrobacter freundii ATCC 8090, Edwardsiella tarda ATCC 15947, Enterobacter aerogenes ATCC 13048, Enterobacter cloacae 963־, Enterobacter liquefaciens 363, Enterobacter liquefaciens 670, Erwinia carotovora EC18967, Erwinia herbicola, Erwinia herbicola (agglomerans), Escherichia coli E63, Hafnia alvei ATCC 13337, Klebsiella pneumoniae ATCC 13883, Lactobacillus easel 686, Lactococcus lactis subsp. lactis plL253, Proteus morganaii, Proteus vulgaris ATCC 13315, Pseudomonas aeriginosa ATCC 10145, Pseudomonas aeriginosa ATCC 27853, Pseudomonas flourescens, Pseudomonas putida ATCC 18633, Pseudomonas putida PpY101, Pseudomonas sp. P, Salmonella typhimurium ATCC 14028, Serratia marcescens ATCC 8100, Serratia marcescens HY, Serratia marcescens Nima, Shigella flexneri ATCC 12022, Shigella sonnei ATCC 25931, Staphylococcus aureus ATCC 25923, Staphylococcus sp. S, Streptococcus faecalis ATCC 19433, Vibrio parahaemolyticus TAMU 109, Yersinia enterocolitica ATCC 9610, Yersinia enterocolitica TAMU 84, Yersinia frederiksenii TAMU 91, Yersinia intermedia ATCC 29909, Yersinia intermedii TAMU 86, Yersinia kristensenia ATCC 33640, Yersinia kristensenia TAMU 95, Yersinia sp. ATCC 29912, Vibrio proteolyticus ATCC 15338, Thermus sp. ATCC 31674, Streptomyces

48xxxxx xxxx

I73׳h 8/1 cinnamonensis subsp. Proteolyticus ATCC 19893, Deinococcus proteolyticus ATCC 35074, Clostridium proteolyticum ATCC 49002, Aeromonas jandaei ATCC 49568, Aeromonas veronii biogroup sobria ATCC 9071, Pseudoaltermonas haloplanktis ATCC 23821, Xanthomonas campestris ATCC 33913, Pseudoalteromonas espejiana ATCC 27025, Shewanella putrefasciens ATCC 8071, Stenotrophomonas maltophilus ATCC 13637, Ochrobactrum anthropi ATCC 19286, Desulfovibrio vulgaris, or a combination thereof.

Example 42 Dowel Assay - Paraoxonase

This example describes assay procedure for quantitative assessment of surface activity of a composition comprising a biomolecular composition using medicine sticks/dowels. The equipment used is a U.V. Spectrophotometer, a U.V. 1 cm pathlength cuvettes, 3 ml and 100 pl volume, and 1.5 ml eppendorf tubes. The reagents used include paraoxon (MW 275.21, ChemService cat#PS-610), 99% CHES (“2-[cyclohexylamino]ethanesulfonic acid), (MW 207.3, Sigma cat # C-2880), and C0CL_z 6H_ZQ (MW 237.9, Sigma cat # C-3169). 1 M C0Cl_z, sterile, can be prepared as 23.79 g C0Cl_z per 100 ml ddH_z0 that is filter sterilized or autoclaved. 200 mM CHES, pH 9.0, sterile can be prepared as 4.15 g + 80 ml ddH_z0, pH to 9.0 with NaOH, where the total volume with ddH_z0 is 100 ml, and can be filter sterilized or autoclaved. The assay buffer is 20 mM CHES, pH 9.0, 50 μΜ C0Cl_z.

In a 1.5 mL Eppendorf tube add: paraoxon to 1 mM ( ex: 126 μΙ of 12 mM paraoxon) and assay buffer to 1.5 ml (ex: 1374 μΙ CHES buffer). Add a 5 mm length of treated stick to start the reaction, mix by inverting. Take 10 μΙ samples at 1 minute intervals, diluting with 90 pL CHES buffer into a 100 μΙ cuvette. Record the absorbance at 400 nm (A₄₀0_nn1), blanking against CHES buffer + paraoxon. A small amount of hydrolysis of paraoxon without biomolecular composition may occur. Mix by inversion before each time point.

Alternatively, in a 3 ml cuvette, add:paraoxon to 1 mM (ex: 168 μΙ of 12 mM paraoxon), andassay buffer to 2.0 ml (ex: 1832 μΙ CHES buffer). Add a 5 (or 15 mm) length of treated stick to start the reaction. Record the (A,00nm) a* th® following time points: 0, 15, 30, 45, 60, 120, 180, 240, 300, 360, 420 and 480 minutes. Mix by inversion at regular intervals. If absorbances above 2.5 are observed, dilute 10 pL samples with 90 pL CHES buffer in a 100 pL cuvette, as for fast reactions, above.

48yyyyy yyyy

The following results (Table 12) demonstrate 90% degradation of the paraoxon over the time frame of measurement by a paroxonase bimolecular additive of the present invention as determined by the dowel assay.

<td colspan="6"> Table 12. Results</td>

<td> Time</td><td colspan="3"> Replicates</td><td></td><td></td>

<td> (seconds)</td><td> A</td><td> B</td><td> C</td><td> umoles p-NP</td><td> Std Dev</td>

<td> 0</td><td> 0.0218</td><td> 0.0218</td><td> 0.0224</td><td> 0.0220</td><td> 0.0003</td>

<td> 120</td><td> 0.1794</td><td> 0.1518</td><td> 0.1253</td><td> 0.1522</td><td> 0.0271</td>

<td> 240</td><td> 0.4359</td><td> 0.3953</td><td> 0.3418</td><td> 0.3910</td><td> 0.0472</td>

<td> 360</td><td> 0.7529</td><td> 0.6541</td><td> 0.6218</td><td> 0.6763</td><td> 0.0683</td>

<td> 480</td><td> 0.9494</td><td> 0.8971</td><td> 0.8894</td><td> 0.9120</td><td> 0.0327</td>

<td> 600</td><td> 0.9724</td><td> 0.9688</td><td> 0.9659</td><td> 0.9690</td><td> 0.0032</td>

<td> 720</td><td> 0.9706</td><td> 0.9706</td><td> 0.9729</td><td> 0.9714</td><td> 0.0014</td>

<td> 840</td><td> 0.9700</td><td> 0.9694</td><td> 0.9782</td><td> 0.9725</td><td> 0.0049</td>

<td> 960</td><td> 0.9535</td><td> 0.9535</td><td> 0.9435</td><td> 0.9502</td><td> 0.0058</td>

<td> 1080</td><td> 0.9600</td><td> 0.9935</td><td> 0.9912</td><td> 0.9816</td><td> 0.0187</td>

<td> 1200</td><td> 0.9500</td><td> 0.9665</td><td> 0.9682</td><td> 0.9616</td><td> 0.0101</td>

<td colspan="3"> p-NP = reaction product</td><td></td><td></td><td></td>

[0131] An antifungal peptide refers specifically to a contiguous amino acid sequence from 3 to 100 amino acid residues In length, including ail intermediate ranges, and which is capable of exerting antifungal activity, as defined above. For simplicity, where the context permits, the term antifungal peptide aiso refers to antifungal polypeptides (i.e., a contiguous amino acid sequence from 101 to 10,000 amino acid residues in length, including all intermediate ranges, and antifungal proteins which are proteinaceous molecules having a contiguous amino acid sequence of more than 10,000 amino acid residues length. Preferably such peptides, polypeptides and proteins are not encoded by the genome of an organism.

[0132] Antifungal peptldic agent refers to a peptide, polypeptide or protein having the ability to inhibit the growth of one or more genera and/or species of fungi. It is also intended to encompass mixtures of such peptides, polypeptides and proteins, together with any associated stabilizers, carriers, and inactive peptides /polypeptides/proteins. Where the context allows, the term antifungal peptldic agent may also refer to a peptide library aliquot containing a mixture of peptides in which at least two of the N-terminal amino acid residues are known, if the peptidic agent is a mixture of peptides, at least one will have antifungal activity.

[0133] Antifungal activity refers to inhibition of fungal cell attachment and/or growth, and is may also refer to fungal cell killing, as the context permits. Accordingly, some antifungal peptidic agents can also be denoted as fungistatic agents or fungicides. [0134] Inhibition of fungal growth refers to cessation or reduction of fungal cell proliferation, and can also include inhibition of expression of cellularly produced proteins in static fungal cell colonies. Such inhibition can provide or facilitate disinfection, decontamination or sanitization of inanimate objects, which refer to the process of reducing the number of fungus microorganisms to levels that no longer pose a threat (e.g., to property or human health). Use of a bioactive antifungal agent can be accompanied by manual removal of mold-contaminated building materials, in some instances.

[0135] The term biocide as used herein refers to a substance that kills microorganisms and their spores. Depending on the type of microorganism killed, a biocidal substance may be further defined as a bactericide, fungicide, or algaecide. The term biostatic refers to a substance that prevents the growth of the microorganism and its spores, and encompasses bacterlstatic, fungistatic and algaestatic compounds.

[0136] A fungicide is a biocidal substance used to kill or inactivate a specific microbial group, the fungi. The term fungistatic, Is used to denote substances that prevent fungal microorganisms from growing or reproducing, but do not result in substantial inactivation or killing.

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[013η An effective amount refers to a concentration of antifungal peptide that is capable of exerting the desired antifungal effect, as defined above.

[0138] An inanimate object refers to structures and objects other than living organisms. Examples of inanimate objects are architectural structures having painted or unpainted surfaces such as the exterior and Interior walls of buildings, Industrial equipment, outdoor sculptures and furniture, construction materials for indoor or outdoor use, such as wood, stone, brick, wall board (sheetrock), ceiling tiles, concrete, unglazed tile, stucco, grout, roofing tiles, shingles, painted or treated wood, synthetic composite materials, leather and textiles.

[0139] A base or substrate refers to any surface that can potentially support the infestation and/or growth of a fungus or spore under favorable conditions for such infestation or growth. It is intended to include exterior surfaces of objects as well as interior surfaces of porous and semiporous objects (e.g., high surface area porous stone structures), constitutes a surface on which a coating can be directly applied and/or impregnated.

[0140] The term coating has its usual meaning and specifically includes the process of applying (e.g., brushing, dipping, spreading, spraying) or otherwise producing a coated surface, which may also be referred to as a coating, coat, covering, film or layer on a surface.

[0141] Where the context so indicates, the term coating may instead refer to the coating composition or mixture that is applied. For example, a coating composition may be capable of undergoing a change from a fluent to a nonfluent condition by removal of solvents, vehicles or carriers, by setting, by chemical reaction or conversion, or by solidification from a molten state. The coating or film that is formed may be hard or soft, elastic or inelastic, permanent or transitory. Where the context allows, the act of coating also includes impregnating a surface or object by causing a coating material to extend or penetrate into the object, or into the interstices of a porous, cellular or foraminous material. The general composition and properties of conventional coating materials are described in U.S. Patent Application No. 10/655,345 filed September 4, 2003, which is hereby Incorporated herein by reference. Additionally, the use of the term coating (“coat,” “surface coat, surface coating”) is also intended to be consistent with its use in PAINT and Coating Testing Manual, Fourteenth Edition of the Gardner-Sward Handbook (Koleske, J. V. Ed.), p. 696,1995; and in ASTM Book of Standards, Volume 06.01, Paint - Tests for Chemical, Physical, and Optical Properties; Appearance,” D16-00, 2002, i.e., a liquid, liquefiable or mastic composition that is converted to a solid protective, decorative, or functional adherent film after application as a thin layer. Examples of a coating include a clear coating and a paint.

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[0142] A paint generally refers to a “pigmented liquid, liquefiable or mastic composition designed for application to a substrate in a thin layer which is converted to an opaque solid film after application. Used for protection, decoration or identification, or to serve some functional purpose such as the filling or concealing of surface irregularities, the modification of light and heat radiation characteristics, etc, [Paint and Coating Testing Manual, Fourteenth Edition of the Gardner-Sward Handbook (Koleske, J. V. Ed.), p. 696, 1995]. Surface treatments, particularly coatings and paints, have been described in U.S. 2004-0109853 Al. ¹

Elastomers or rubbers are polymers that can undergo large, but reversible, deformations upon a relatively low physical stress. Elastomers (e.g., tire rubbers, polyurethane elastomers, polymers ending in an anionic diene, segmented polyerethane-urea copolymers, diene triblock polymers with styrene-alpha-methylstyrene copolymer end blocks, poly (p-methy!styrene-b-p־methylstyrene), polydimethylsiloxane-vinyi monomer block polymers, chemically modified natural rubber, polymers from hydrogenated polydienes, polyacrylic elastomers, polybutadienes, trans-polyisoprene, polyisobutene, cis-1, 4-polybutadiene, polyolefin thermoplastic elastomers, block polymers, polyester thermoplastic elastomer, thermoplastic polyurethane elastomers) and techniques of elastomer synthesis and elastomer property analysis have been described, for example, in Walker, B. M., ed., Handbook of Thermoplastic Elastomers, Van Nostrand Reinhold Co., New York, 1979; Holden, G., ed., et. al., Thermoplastic Elastomers, 2nd Ed., Hanser Publishers, Verlag, 1996.

[0143] An adhesive is a composition that is capable of uniting, bonding or holding at least two surfaces together, preferably in a strong and permanent manner (e.g״ glue, cement, paste).

[0144] A sealant Is a composition capable of attaching to at least two surfaces, filling the space between them to provide a barrier or protective coating (e.g., by filling gaps or making a surface nonporous).

[0145] A fungal-prone material is a substance that is capable of serving as a food source for a fungus, or is a material that contains one or more such substance. For example, in the context of a paint or coating composition, a fungal-prone material may be a binder containing a carbon-based polymer that serves as a nutrient for a fungus.

[0146] All patents, published patent applications and other publications cited herein are hereby incorporated herein by reference to the extent that they describe materials and methods supplementary to that set forth herein. One skilled in the art will readily appreciate that the present invention is well adapted to carry out any objects and obtain the ends and advantages mentioned as well as those inherent therein. The preferred 52 antifungal compositions and methods described herein are exemplary and intended to be representative of other embodiments which will be apparent to those skilled in the art in light of the present disclosure. For Instance, In light of the present disclosure and representative examples, changes in the disclosed compositions and methods and other uses will occur to those skilled in the respective arts of preparing and using paints and coatings, textile finishes, waxes, elastomers, adhesives and sealants which are encompassed within the spirit of the invention and defined by the scope of the appended claims. The present examples, therefore, are not to be considered as limiting the scope of the present invention.

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Tyr Phe Lys Leu Arg Phe 1 5 <210> 39 <211> 7 <212> PRT <213> peptide <400> 39

Phe His Phe Lys Leu.Arg Phe

5 <210> 40 <211> 7 <212> PRT <213> peptide <400> 40 lie Hie Phe Lys Leu Arg Phe ^{1 5} <210> 41 <211> 6 <212> PRT <213־> peptide <400> 41

Phe Arg Leu Lys Phe His 62

Patent provided by Sughrue Mlon, PLLC - http://www.sughrue.com

<td> 1</td><td> 5</td>

<td> &lt;210&gt;. &lt;211&gt; &lt;212&gt; &lt;213&gt;</td><td> 42 S PRT peptide</td>

<td> &lt;400&gt;</td><td> 42</td>

Arg Phe Lys Leu Arg Phe

<td> 1</td><td> 5</td>

<td> &lt;210&gt; &lt;211&gt; &lt;212&gt; &lt;213&gt;</td><td> 43 6 PRT peptide</td>

<td> &lt;400&gt;</td><td> 43</td>

Ser Phe Lys Leu Arg Phe

<td> 1</td><td> 5</td>

<td> &lt;210&gt; &lt;211&gt; &lt;212&gt; &lt;213&gt;</td><td> 44 6 PRT peptide</td>

<td> &lt;400&gt;</td><td> 44</td>

Met Phe Lys Leu Arg Phe

<td> 1</td><td> 5</td>

<td> &lt;210&gt; &lt;211&gt; c212&gt; &lt;213&gt;</td><td> 45 6 PRT peptide</td>

<td> &lt;400&gt;</td><td> 45</td>

Thr Phe Lys Leu Arg Phe

<td> 1</td><td> 5</td>

<td> &lt;210&gt; &lt;211&gt; &lt;212&gt; &lt;213&gt;</td><td> 46 6 PRT peptide</td>

<td> &lt;400&gt;</td><td> 46</td>

Gin Phe Lys Leu Arg Phe

<td> 1</td><td> 5</td>

<td> &lt;210&gt; &lt;211&gt; &lt;212&gt; &lt;213&gt;</td><td> 47 6 PRT peptide</td>

<td> &lt;400&gt;</td><td> 47 63 Patent provided by Sughrue Mion, PLLC - http://www.sughrue.com</td>

Trp Phe Lys Leu Arg Phe

5 <210> 48 <211> 44 <212> PRT , ~ . ,.

<213> Horseshoe Crab Tachystatm A Peptide <

220>

<221> Misc_feature <222> (10) - - (10) <223> n = anything <400> 48

Tyr Ser Arg Cys Gin Leu Gin Gly Phe Asn Cys Val Val Arg Ser Tyr 1 ⁵

Gly Leu Pro Thr He Pro Cys Cys Arg Gly Leu Thr Cys Arg Ser Tyr . ר .j kJ

Phe Pro Gly Ser Thr Tyr Gly Arg Cys Gin Arg Tyr <210>49 <211> 25 <212> PRT <213> Androctonus australis <220>

<221> Misc_feature <222> (22).(22) <223> n ־־ anything <400> 49

Arg Ser Val Cy. «9 01־ XI Π. “5 ^Β1γ “^y³ t- 1o. 1a

Tyr Tyr Lys Cys Thr Asn Arg Pro Tyr <210> 50 <211> 13 <212> PRT, <213> Synthetic Tritrpticin <400>50

Val Arg Arg Phe 1

Pro Trp Trp Trp Pro 5

Phe Leu Arg Arg 10 <210>

<211>

<212>

<213>

PRT

Human HNP-3 Defensin

Patent provided by Sughrue Mion, PLLC - http://www.sughrue.com <400> 51

<td> Asp Cys Tyr 1</td><td> Cys</td><td> Arg He Pro Ala Cys 5</td><td> He Ala 10</td><td> Gly</td><td> Glu</td><td> Arg Arg Tyr 15</td>

<td> Gly Thr Cys</td><td> He 20</td><td> Tyr Gin Gly Arg Leu 25</td><td> Trp Ala</td><td> Phe</td><td> Cys</td><td> Cys 30</td>

<td> &lt;210&gt; 52</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> &lt;211&gt; 38</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> &lt;212&gt; PRT</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td colspan="3"> &lt;213&gt; Phytolacca americana</td><td></td><td></td><td></td><td></td>

<td> &lt;4Q0&gt; 52</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> Ala Gly Cys 1</td><td> He</td><td> Lys Asn Gly Gly Arg 5</td><td> Cys Asn 10</td><td> Ala</td><td> Ser</td><td> Ala Gly Pro 15</td>

<td> Pro Tyr Cys</td><td> Cys</td><td> Ser Ser Tyr Cys Phe</td><td> Gin He</td><td> Ala</td><td> Gly</td><td> Gin Ser Tyr</td>

<td></td><td> 20</td><td> 25</td><td></td><td></td><td></td><td> 30</td>

<td> Gly Val Cys</td><td> Lys</td><td> Asn Arg</td><td></td><td></td><td></td><td></td>

<td> 35</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> &lt;210&gt; 53</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> &lt;211&gt; 23</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> &lt;212&gt; PRT</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td colspan="2"> &lt;213&gt; Syntheti</td><td> c construct Magainin</td><td> 2</td><td></td><td></td><td></td>

<td> &lt;400&gt; 53</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> Gly He Gly 1</td><td> Lys</td><td> Tyr Leu His Ser Ala 5</td><td> Lys Lys 10</td><td> Phe</td><td> Gly</td><td> Lys Ala Trp 15</td>

Val Gly Glu lie Met Asn Ser 20

<td> &lt;210&gt; &lt;211&gt; &lt;212&gt; &lt;213&gt;</td><td colspan="2"> 54 13 PRT Bob taurus</td>

<td> &lt;400&gt;</td><td> 54</td><td></td>

<td colspan="2"> lie Leu Pro Trp Lys 1 5</td><td> Trp Pro Trp Trp Pro Trp Arg Arg 10</td>

<td> &lt;210&gt; &lt;211&gt; &lt;212&gt; &lt;213&gt;</td><td> 55 44 PRT Heliothis virescens</td>

<td> &lt;400&gt;</td><td> 55</td>

Asp Lys Leu lie Gly Ser Cys Val Trp Gly Ala Val Asn Tyr Thr Ser

Patent provided by Sughrue Mion, PLLC . http://www.sughrue.com

PCT/US2004/0217U

Asp Cys Asn

Gly Glu Cys Lys Arg Arg Gly Tyr Lys Gly Gly His Cys 20 ²⁵³⁰

Gly Ser Phe Ala Asn Val Asn Cys Trp CyB. Glu Thr ^Y 35 <210>56 <211> 44 <212> PRT <213> Heliothis virescens <400>56 _Mp Lys be־ 17־ ־11 ״« w־ V.1 Trp Gly Al. V.1 A־n Tyr ־« 1 ⁵

Asp cy־ A.״ Gly Olu cy־ «S ^GlY °^1Y “^{Y Hi־}

Gly Ser Phe Ala Asn Val Asn Cys Trp Cys Glu Thr <210> 57 <211> 46 <212> PRT, <213> Seed of pea Sativum defensm 1 (psdl) <400> 57

Lys Thr Cys Glu His Leu Ala Asp Thr Tyr Arg Gly Val Cys Phe Thr 1 ⁵

Μ» Al« Ser Cy־ A־p A־p Hi־ Cy־ by. A־n by־ Ala Hl־ beu 11־ Ser 20

Gly Tbr cy־ Hl־ A־־ Trp by־ Cy־ Phe Cys Thr Gin As־ cy.

₃540 <210>58 <211> 18 <212> PRT <213> Synthetic Gomesin <400>58 mn Cys Arg Arg Leu Cys Tyr Lys 1 5

Gin Arg Cys Val Thr Tyr Cys Arg

15

Gly Arg

Patent provided by Sughrue Mion, PLLC - http7iwww.sughrue.com <210>59 <211> 25 <212> PRT <213> Bovine Lactoferricin B <400>59

Phe Lys Cys Arg Arg Trp

Gin Trp Arg Met Lys Lys Leu Gly Ala Pro

1015

Ser He Thr Cys Val Arg Arg Ala Phe

2025 <210>60 <211>12 <212> PRT <213> Synthetic PW2 <400> 60

His Pro Leu Lys Gin Tyr Trp Trp Arg Pro Ser He !510 <210>61 <211> 20 <212> PRT <213> Human Hepcidin 20 <400>61

He Cys He Phe CyB Cys Gly Cys Cys His Arg Ser Lys Cys Gly Met

¹⁵ 10 5ר

Cys Cys Lys Thr 20 <210> 62 <211> 25 <212> PRT <213> Human Hepcidin 25 <400> 62

Asp Th-r His Phe Pro He Cys He Phe Cys Cys Gly Cys Cys His Arg !5 10 15

Ser Lys Cys Gly Met Cys Cys Lys Thr

25 <210>

<211>

<212>

<213>

PRT

Amaranthus caudatus <400>

Val Gly Glu Cys Val Arg Gly

Arg

Pro Ser

Gly Met

Cys

Cys

Ser

Cys 1 67

Patent provided by Sughrue Mion, PLLC - http://www.sughrue.com

5

Gin Phe Gly Tyr Cys Gly Lys Gly Tyr Cys Gly Arg ^{25 3} <210> 64 <211> 7B <212> PRT <213 > Amaranthus caudatus <400> 64

Gly Tyr Phe Cys 1

Glu Ser Cys Arg Lys 5 lie lie Gin Lys Leu Glu Asp

15

Met Val Gly Pro Gin Pro Asn Glu Asp Thr Val Thr Gin Ala Ala Ser 20

Gin Val Cys Asp Lys Leu Lys He Leu Arg Gly Leu Cys Lys Lys He 35 ⁴⁰⁴⁵

Met Arg Ser Phe Leu Arg Arg lie Ser Trp Asp He Leu Thr Gly Lys

5^5S

Lys Pro Gin Ala lie Cys Val Asp He Lys lie Cys Lys Glu <210> 65 <211> 23 <212> PRT .

<213> African clawed frog Magainin 2 <400> 65

Gly He Gly Lys Phe Leu His Ser Ala Lye ₅ !Ο

Lys Phe Gly Lys Ala Phe

Val Gly Glu

He Met Asn Ser .20 <210> 66 <211> 26 <212> PRT <213> Honey bee venom Melittm B <400> 66

Gly He Gly Ala Val Leu Lys Val Leu Thr Thr Gly beu Pro Ala Leu

He Ser Trp He Lys Arg Lys Arg Gin Gin ²⁵

Patent provided by Sughrue Mion, PLLC ־ http://www.sughrue.com

<td colspan="3"> WO 2005/007758</td><td rowspan="2"> PCT/US2004/021711</td>

<td> &lt;210&gt; &lt;211&gt; &lt;212&gt; &lt;213&gt;</td><td colspan="2"> 67 21 PRT Podisus maculiventris</td>

<td> &lt;400&gt;</td><td> 67</td><td></td><td></td>

<td colspan="2"> Gly Ser Lys Lys Pro Val Pro lie 1 5</td><td> lie Tyr 10</td><td> Cys Asn Arg Arg Thr Gly 15</td>

Lys Cys Gin Arg Met 20 <210>68 <211> 38 <212> PRT <213 > Common ice plant Antimicrobial peptide 1 <400>68

Ala Lys Cys lie Lys Asn Gly Lys Gly Cys Arg Glu Asp Gin Gly Pro !5 10

Pro Phe Cys Cys Ser Gly Phe Cys Tyr Arg Gin Val Gly Trp Ala Arg

2530

Gly Tyr Cys Lys Asn Arg <210> 69 <211> 13 <212> PRT <213> Bovine Melanotropin alpha (Alpha-MSH) <400> 69

Ser Tyr Ser Met Glu His Phe Arg Trp Gly Lys Pro Val

5 10 <210> 70 <211> 33

PRT <213> Rabbit Corticostatin III (MCP-1) <400> 70

Val Val Cys Ala Cys Arg Arg Ala Leu Cys Leu Pro Arg Glu Arg Arg

10 15

Ala Gly Phe Cys Arg He Arg Gly Arg He His Pro Leu Cys Cys Arg

25 30

Arg

Patent provided by Sughrue Mion, PLLC - http://www.sughrue.com

PCT7US2004/021711 <210> 71 <211>33

9. T1?RT <213> Rabbit Corticostatin IV (MCP-2) <400> 71 val Val Cy־״ ־ Cy־ Arg Arg Ala Dau Cy־ Leu Pro Lav Glu «9 «9 t»1U ⁵ »1. Gly Ph־ cy־11 9» ־ AT9 Gly «9 h. ⁹“ ״־ “־ ־1־ ׳>n2□

Arg <210>72 <211>35 <212> PRT״ . ״ <213> Chinese oak'silk moth Cecropm B <400> 72

Lys Trp Lys He Phe Lys Lys He Glu Lys Val Gly Arg Asn lie Arg 1 ⁵_Mn Gly 11־11 ־ W Al. Gly Pr־ Ala Val Ala Val Leu Gly Glu Ala ²⁵

Lys Ala Leu 35 <210> 73 <211> 48 <212> PRT <213 > Bovine Semmalplasmin <400> 73 _Ser Asp Glu Lys Ala Ser Pro Asp Lys His His Arg Phe Ser Leu Ser

Arg Tyr Ala Lya L־u Ala A־־ Arg L־u Ala A־n Pro Lya L־u Leu Glu

Thr Ph־ Leu Ser Lya Trp 11־ Gly Asp Arg Gly Aan Arg 8־r Val Ly־ ⁴⁰“ <210> <2H> <212> <213>

PRT

Rabbit HP-3A defensin <400> 74

Patent provided by Sughrue Mion, PLLC - http://www.sughrue.com

Gly lie Cys Ala Cys Arg Arg Arg 1 5

Ser Gly Tyr Cys Arg Val Asn Gly

Phe Cys Pro Asn Ser Glu Arg Phe

15

Ala Arg Tyr Val Arg Cys Cys Ser 25 30

Arg Arg <210> 75 <211> 30 <212> PRT <213> Human HNP-1 Defenain <400> 75

Ala Cys Tyr Cys Arg lie Pro Ala

5

Cys He Ala Gly Glu Arg Arg Tyr

15

Gly Thr Cys lie Tyr Gin Gly Arg 20

Leu Trp Ala Phe Cys Cys

30 <210> 76 <211> 29 <212> PRT <213 > Human HNP-2 Defenain <400> 76

Cys Tyr Cys Arg lie Pro Ala Cys

5

He Ala Gly Glu Arg Arg Tyr Gly

15

Thr Cys He Tyr Gin Gly Arg Leu 20

Trp Ala Phe Cys Cys 25 <210>77 <211> 33 <212 > PRT <213 > Human HNP-4 Defensin <400>77

Val Cys Ser Cys Arg Leu Val Phe

Cys Arg Arg Thr Glu Leu Arg Val

15

Gly Asn Cys Leu He Gly Gly Val 20

Ser Phe Thr Tyr Cys Cys Thr Arg 25 30

Val <210> 78 <211> 24 <212> PRT

Patent provided by Sughroe Mion, PLLC - http^/www.sughrue.com

WO 2005/007758 PCTAJS2004/021711 <213 > Human HiBtatin 5 <400> 73

ASP ser His Ala Lys Arg His His Gly Tyr Lys Arg Lys Phe His Glu 15 10 ¹⁵

Lys His His Ser His Arg Gly Tyr <210> 79 <211> 32 <212> PRT <213> Human Histatin 3 <400>79

Asp Ser His Ala Lys Arg His Hie

Gly Tyr Lys Arg Lys Phe His Glu

LVS His His Ser His Arg Gly Tyr Arg Ser Asn Tyr Leu Tyr Asp Asn

25 30 <210> 80 <211> 12 <212> PRT <213> Histatin 8 <400> 80 !!vs Phe His Glu Lys His His Ser His Arg Gly Tyr

510 <210>81 <211>38 <212> PRT <213> Bovine Tracheal antimicrobial peptide <400> Bl

Asn Pro Val Ser Cys Val Arg Asn Lys Gly lie Cys Val Pro He Arg !5 1015

CyB Pro Gly Ser Met Lys Gin He Gly Thr Cys Val Gly Arg Ala Val

2530

Lys Cys Cys Arg Lys Lys <210> 82 <211> 37 <212> PRT <213> Garden four-o'clock AMP1 (MJ-AMP1) <400> 82

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Gin. Cys He Gly Asn Gly □ly Arg 1 5

Tyr CyB Cys Ser Gly Phe Cys Leu

PCI7US2004/021711

Asn Glu Asn Val Gly Pro Pro 10 15

Gin Pro Gly Gin Gly Tyr Gly 30

Tyr Cys Lys Asn Arg

<td> &lt;210&gt; &lt;211&gt; &lt;212&gt; &lt;213&gt;</td><td> B3 36 PRT Garden four-o'clock AMP2</td>

<td> &lt;400? Cys He</td><td> B3 &gt; Gly Asn Gly Gly Arg Cys</td>

5

AMP2)

Glu Asn Val Gly Pro Pro Tyr 10 15

Cys CyB Ser Gly Phe Cys Leu Arg

Pro Asn Gin Gly Tyr Gly Val

Cys Arg Asn Arg

<td></td><td> 35</td>

<td> &lt;210&gt;</td><td> 84</td>

<td> &lt;211&gt;</td><td> 33</td>

<td> &lt;212 &gt;</td><td> PRT</td>

<td> &lt;213&gt;</td><td> Maize MBP-1</td>

<td> &lt;400?</td><td> 84</td>

Arg Ser Gly Arg Gly Glu Cys Arg 1 5

Gin Cys Leu Arg Arg His Glu

15

Gly Gin Pro Trp Glu Thr Gin Glu 20

Met Arg Arg CyB Arg Arg Arg 30

Gly <210> B5 <211> 23 <212> PRT <213> Rape AFP2 <400? B5

Gin Lys Leu Cys Glu Arg Pro Ser

5

Thr Trp Ser Gly Val Cys Gly 10 15

Asn Asn Asn Ala Cys Lys Asn 20

Patent provided by Sughrue Mion, PLLC - http://www.sughrue.com <210> 96 <211> 27 <212> PRT <213> Turnip AFP1 <400> B6 nl קר Lys Leu Cys Glu Arg Pro Ser Gly Thr Trp 1 510

Ser Gly Val

Cys Gly IS

Asn Asn Asn Ala Cys Lys Asn Gin Cys Ile Asn

2025 <210>97 <211> 27 <212> PRT <213> Turnip AFP2 <400>87

Gin Lys Leu Cys 1

Glu Arg Pro Ser Gly 5

Thr Trp Ser Gly Val Cys

15

Gly

Asn Asn Asn Ala Cys Lys Asn Gin Cys lie Arg

25 <210> BB <211> 33 <212> PRT <213> Two coloured leaf frog Adenoregulin <400>83

Gly Leu Trp Ser Lys Ile Lys Glu Val Gly Lys Glu Ala Ala Lys Ala !5 1015

Ala Ala Lys Ala Ala Gly Lys Ala Ala Leu Gly. Ala Val Ser Glu Ala

2530

Val <210> B9 <211> 16 <212> PRT <213> Pig Protegrin 2 <400> B9

Arg Gly Gly Arg 1

Leu Cys Tyr Cys 5

Arg Arg Arg Phe Cys 10 lie

Cys Val 15 <210> 90 <211> IB <212> PRT

Patent provided by Sughrue Mion, PLLC - http://www.sughrua.com <213> Pig Protegrin 3 <400> 90

Arg Gly Gly Gly Leu Cys Tyr Cys Arg Arg Arg Phe Cys Val Cys Val

10 ¹⁵

Gly Arg

<td> &lt;210&gt;</td><td> 91</td>

<td> &lt;211&gt;</td><td> 38</td>

<td> &lt;212&gt;</td><td> PRT</td>

<td> &lt;213&gt;</td><td> Crab eating macaque Histatin 1</td>

<td> &lt;400&gt;</td><td> 91</td>

<td rowspan="2"> Asp Ser His 1</td><td colspan="3"> Glu Glu Arg His His Gly Arg His Gly His His Lys Tyr</td>

<td> 5</td><td> 10</td><td> 15</td>

<td colspan="3"> Gly Arg Lys Phe His Glu Lys His His Ser 20 25 Asn Tyr Leu Tyr Asp Asn 35 &lt;210&gt; 92 &lt;211&gt; 24 &lt;212&gt; PRT &lt;213&gt; African clawed frog Peptide PGQ</td><td> His Arg Gly Tyr Arg Ser 30</td>

<td> &lt;400&gt; 92</td><td colspan="3"></td>

<td> Gly Val Leu 1 Ala Leu Asn &lt;210&gt; 93 &lt;211&gt; 20 &lt;212&gt; PRT &lt;213&gt; Bull</td><td> Ser Asn Val He Gly Tyr 5 Ala Val Leu Lys Gin 20 frog Ranalexin</td><td> Leu 10</td><td> Lys Lys Leu Gly Thr Gly 15</td>

<td> &lt;400&gt; 93</td><td colspan="3"></td>

<td> Phe Leu Gly 1</td><td> Gly Leu lie Lys He Val 5</td><td> Pro 10</td><td> Ala Met He Cys Ala Val 15</td>

<td> Thr Lys</td><td> Lys</td><td> Cys 20</td>

<td> &lt;210&gt;</td><td> 94</td><td></td>

<td> &lt;211&gt;</td><td> 30</td><td></td>

<td> &lt;212&gt;</td><td> PRT</td><td></td>

Patent provided by Sughrue Mion, PLLC - http://www.sughrue.com <213 > Guinea pig GNCP-2 <400> 94

Arg Cys He Cys Thr Thr Arg Thr

5

Gly Thr Cys Leu Phe Gin Asn Arg

Cys Arg Phe Pro Tyr Arg Arg Leu

15

Val Tyr Thr Phe Cys Cys

30 <210> 95 <211> 18 <2125 PRT <213> Pig Protegrin 4 <400> 95

Arg Gly Gly Arg Leu Cys Tyr Cys

5

Arg Gly Trp He Cys Phe Cys Val

15

Gly Arg <210> 96 <211> 18 <212> PRT <213> Pig Protegrin 5 <400> 96

Arg Gly Gly Arg Leu Cys Tyr Cys

5

Arg Pro Arg Phe Cys Val Cys Val

15

Gly Arg <210> 97 <211> 27 <212> PRT <213> Bovine BMAP-27 <400> 97

Gly Arg Phe Lys Arg Phe Arg Lys Lys Phe Lys Lys Leu Phe Lys Lys

15 10 5 ר

Leu Ser Pro Val He Pro Leu Leu His Leu Gly

25 <210> 98 <211> 28 <212> PRT <213> Bovine BMAP-28 <400> 98

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Gly Gly Leu Arg Ser Leu Gly Arg Lys ⁵

He Leu Arg Ala Trp Lys Lys

15

Tyr Gly Pro He He Val Pro lie He Arg He Gly <210>99 <211>39 <212> PRT <213> Asian toad Buforin 1 <400> 99

Ala Gly Arg Gly Lys Gin Gly

5

Gly Lys Val Arg Ala Lys Ala Lys Thr

15

Arg Ser Ser Arg Ala Gly Leu Gin Phe ²⁵

Pro Val Gly Arg Val His Arg 30

Leu Leu Arg Lys Gly Asn Tyr 35 <210> 100 <211> 21 <212> PRT <213> Asian Toad Buforin II <400> 100

Thr Arg Ser Ser Arg Ala Gly Leu Gin Phe Pro Val Gly Arg Val His

¹⁵ 10 5 ו

Arg Leu Leu Arg Lys 20 <210> 101 <211> 34 <212> PRT <213> Bovine BMAP-34 <400> 101

Gly Leu Phe Arg Arg Leu Arg Asp 1 ⁵

Ser He Arg Arg Gly Gin Gin Lys

15 lie Leu Glu Lys Ala Arg Arg He Gly Glu Arg He Lys Asp He Phe

25 30

Arg Gly <210> 102 <211> 19 <212> PRT

Patent provided by Sughrue Mion, PLLC - http://www.sughrue.com <213> Trichoderma longibrachiatum Tricholongin <400> 102

Ala Gly Phe Ala Ala Gin Ala Ala Ala Ser Leu Ala Pro Val Ala Ala 15 10 15

Gin Gin Leu <210> 103 <211> 34 <212> PRT <213> Sauvage's leaf frogn Dermaseptin 1 <400> 103

Ala Leu Trp Lys Thr Met Leu Lye Lys Leu Gly Thr Met Ala Leu His

15 10 5ד

Ala Gly Lys Ala Ala Leu Gly Ala Ala Ala Asp Thr He Ser Gin Gly

2530

Thr Gin <210>104 <211> 45 <212> PRT <213> Para rubber tree Pseudo-hevein (Minor hevein) <400> 104

Glu Gin Cys Gly Arg Gin Ala Gly Gly Lys'Leu Cys Pro Asn Asn Leu

10

Cys Cys Ser Gin Tyr Gly Trp Cys Gly Ser Ser Asp Asp Tyr Cys Ser

2530

Pro Ser Lys Asn Cys Gin Ser Asn Cys Lys Gly Gly Gly

4045 <210> 105 <211> 33 <212> PRT <213> Wrinkled frog Gaegurin-1 <400> 105

Ser Leu Phe Ser Leu He Lys Ala Gly Ala Lys Phe Leu Gly Lys Asn

10 15

Leu Leu Lys Gin Gly Ala Cys Tyr Ala Ala Cys Lys Ala Ser Lys Gin 20 25 30

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Cys <210> 10S <211> 36 <213> Two-colored leaf frog Skin peptide tyrosine-tyrosine <400> 106

Tyr pro Pro Lys Pro Glu Ser Pro Gly Glu Asp Ma Ser Pro Glu Glu

Met M Lys JF Leu Tbx Ma Leu K3 Hie Tyr 11־ Asa Leu Val ito 95

Arg Gin Arg Tyr <210> 107 <211> 50 <212> PRT <213> Penoeid shrimp Penaeidin-1 <400>107

Tyr Arg Gly Gly Tyr Thr Gly Pro lie Pro Arg Pro Pro Pro lie Gly

Arg pro Pro Leu Arg Leu Val Val Cys Ala Cys Tyr Arg Leu Ser Val ³ 7530

S־r Asp Me Arg A־» Cys Cys 11־ Lys Phe Gly S־r Cy. Cys His Leu iO4b

Val Lys 50 <210> 108 <211> 33 <213> Golden hamster Neutrophil defensin 1 (HANP-1) <400>108

Val Thr Cys Phe Cys Arg Arg Arg Gly Cys Ala Ser Arg Glu Arg His _τ5 lie Gly Tyr Cys Arg Phe Gly Asn Thr He Tyr Arg Leu Cys Cys Arg 2025

Arg

Patent provided by Sughrue Mion, PLLC - http://www.sughrue.com <210> 109 <211> 33 <212> PRT <213> Golden, hamster Neutrophil defensin 3 (HANP-3) <400>109

Val Thr Cys Phe Cys Arg Arg Arg Gly Cys Ala Ser Arg Glu Arg Leu !5 lie Gly Tyr Cys Arg Phe Gly Asn Thr lie Tyr Gly Leu Cys Cys Arg 20 ²⁵³⁰

Arg <210>110 <211> 21 <212> PRT <213> Oriental weatherfish Misgurm <400>110

Arg Gin Arg Val Glu Glu Leu Ser Lys Phe Ser Lys Lys Gly Ala Ala

Ala Arg Arg Arg Lys 20 <210> 111 <211> 41 <212> PRT <213> Japanese morning glory PN-AMP 111

Gin Gin Cys Gly Arg Gin Ala Ser Gly Arg Leu Cys Gly Asn Arg Leu

5 10 <400>

Cys Cys Ser Gin Trp Gly Tyr Cys Gly Ser Thr Ala Ser Tyr Cys Gly

2530

Ala Gly Cys Gin Ser Gin Cys Arg Ser

3540 <210> 112 <211> 19 <212> PRT . ,. .

<213> Rainbow trout Histone H2B.-1 (HLP-1) (Fragment) <400> 112

Pro Asp Pro Ala Lys Thr Ala Pro Lys Lys Gly Ser Lys Lys Ala Val

¹⁵ 10 5ד

Thr Lys Ala

Patent provided by Sughwe Mion, PLLC - http://www.sughrue.com <210> 113 <211>17 <2Ϊ3> Rainbow trout Histone H2B-3(HLP-3)(Fragment) <400>113

Pro Asp Pro Ala Lys Thr Ala Pro Lys Lys Lys Ser Lys Lys Ala Val * ד ΛID

Thr <210> 114 <211> 30

2S» SLu־ »W» neutrophil ά2 «1־־־£־ (F.HftD-2) <400> 114

Ala Cys Tyr Cys Arg He Pro Ala Cys Leu Ala Gly Glu Arg Arg Tyr

¹⁵ ס! 5.ץ

Gly Thr Cys Phe Tyr Met Gly Arg Val Trp Ala Phe Cys Cys 20 <210> 115 <211> 35 <212 > PRT, , <213> pseudacanthotermes spiniger Termicin <400>115

Ala Cys Asn Phe Gin Ser Cys Trp Ala Thr Cys Gin Ala Gin His Ser 15 1015

He Tyr Phe Arg Arg Ala Phe Cys Asp Arg Ser Gin Cys Lys Cys Val

25

Phe Val Arg Gly 35 <210> 116 <211> 25 <212> PRT . . . , <213> pseudacanthotermas spiniger Spmgerin <400> ' 116

His Val Asp Lys Lys Val Ala Asp Lys Val Leu Leu Leu Lys Gin Leu

Arg He Met

Arg Leu Leu Thr Arg Leu ²⁵

Patent provided by Sughrue Mion, PLLC - http://www.sughrue.com <210> <211> <212 > <213>

PRT

Southern bell frog Aurein 1.1 <400>

Gly Leu Phe Asp lie lie Lys Lys He Ala Glu Ser lie <210>118 <211> 30 <212 > PRT <213> Ponerine ant Ponericin G1 <400>118

Gly Trp Lys Asp Trp Ala Lys Lys Ala Gly Gly Trp Leu Lys Lys Lys rj 1015

Glv Pro Gly Met Ala Lys Ala Ala Leu Lys Ala Ala Met Gin

2530 <210> 119 <211> 24 <212> PRT, <213> Rio Grande leopard frog Brevinin-IBB <400> 119

Phe Leu Pro Ala lie Ala Gly Met Ala Ala Lys Phe Leu Pro Lys He

Phe Cys Ala He Ser Lys Lys Cys <210>

<211>

<212>

<213>

0

PRT

Green frog Rana1exin-1CB <400>

Phe Leu Gly Gly Leu Met Lys Ala Phe Pro Ala He He Cys Ala Val

Thr Lys Lys Cys 20 <210>

<211>

<212>

<213>

PRT

Green frog Ranatuerin-2CA <400>

Patent provided by Sughrue Mion, PLLC - http://www.sughrue.com

Gly Leu Phe Leu Asp Thr Leu Lye Gly 1 5

Ala Ala Lys Asp Val Ala Gly

15

Lys Leu Leu Glu Gly Leu Lys Cys Lys ²⁵

He Ala Gly Cys Lys Pro <210> 122 <211> 27 <212> PRT <213> Green frog Ranatuerin-2CB , <400> 122

Gly Leu Phe Leu Asp Thr Leu Lys Gly Leu Ala Gly Lys Leu Leu Gin

5 10 ¹⁵

Gly Leu Lys Cys He Lys Ala Gly Cys Lys Pro ²⁵ <210> 123 <211> 40 <212> PRT <213> Ginkgo Ginkbilobin <400> 123

Ala Asn Thr Ala Phe Val 1 5

Ser Ser Ala His Asn Thr Gin Lys 10

He Pro 15

Ala Gly Ala Pro Phe Asn Arg Asn Leu Arg Ala Met Leu Ala Asp Leu

25 ³⁰

Arg Gin Asn Ala Ala Phe Ala Gly

40 <210>124 <211> 20 <212> PRT <213> Malabar spinach Alpha-basrubrin (Fragment) <400>124

Gly Ala Asp Phe Gin Glu Cys Met Lys Glu His Ser Gin Lys Gin His

1015

Gin His Gin Gly 20

<td> &lt;210&gt; &lt;211&gt; &lt;212&gt; &lt;213&gt;</td><td> 125 24 PRT Paradoxical frog Pseudin 1</td>

<td> &lt;400&gt;</td><td> 125</td>

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PCT/US2004/0217U

Gly Leu Asn Thr Leu Lys Lye Val 1 5

Phe Gin Gly Leu His Glu Ala He ¹⁵

Lys Leu He Asn Asn His Val Gin 20

Lys Ala Trp Lys Ser Lys Leu Ala ¹⁵ <210> 126 <211> 45 <212> PRT <213> Scorpion Parabutoporin <400> 126

Phe Lys Leu Gly Ser Phe Leu Lye 15

Lys Lys Leu Arg Ala Lys Gly Lys Glu Met Leu Lys Asp Tyr Ala Lys 20 ²⁵³

Gly Leu Leu Glu Gly Gly Ser Glu Glu Val Pro Gly Gin <210>127 <211> 44 <212> PRT , . ._ <213> African yellow leg scorpion Opistoporm 1 <400>127

Gly Lys Val Trp Asp Trp lie Lys Ser Thr Ala Lye Lys Leu Trp Asn

Ser Glu Pro Val Lys Glu Leu Lys Asn Thr Ala Leu Asn Ala Ala Lys 20 25

Asn Leu Val Ala Glu Lys He Gly Ala Thr Pro Ser ,=40 <21Q> 12B <211> 44 <212> PRT , ._ <213 > African yellow leg scorpion □pistoponn 2 <400>128

Gly Lys Val Trp Asp Trp He Lys Ser Thr Ala Lys Lys Leu Trp Asn 1 ^{5 10}

Ser Glu Pro Val Lys Glu Leu Lys Asn Thr Ala Leu Asn Ala Ala Lys 20 2530

Asn Phe Val Ala Glu Lys He Gly Ala Thr Pro Ser

Patent provided by Sughrue Mion, PLLC - http://www.sughrue.com <210>129 <211> 12 <212> PRT <213 > Rainbow trout Histone H2A (Fragment) <400>129

Ala Glu Arg Val Gly Ala Gly Ala Pro Val Tyr Leu !5 <210>130 <211> 33 <212> PRT <213> Sea hare Dolabellanin B2 <400> 130

Ser His Gin Asp Cys Tyr Glu Ala Leu His Lys Cys Met Ala Ser His

Ser Lys Pro Phe Ser Cys Ser Met Lys Phe His Met Cys Leu Gin Gin <210> 131 <211> 35 <212> PRT <213> Noctuid moth Cecropin A <400> 131

Arg Trp Lys Val Phe Lys Lys He Glu Lys Val Gly Arg Asn He Arg ^a 10 15

Asp Gly Val lie Lys Ala Ala Pro Ala He Glu Val Leu Gly Gin Ala

25 30

Lys Ala Leu <210>

<211>

<212>

<213>

PRT

Human HNP-5 Defensin <400>

Gin Ala Arg Ala Thr Cys Tyr Cys Arg Thr Gly Arg Cys Ala Thr Arg

1° ¹⁵

Glu Ser Leu Ser Gly Val Cys Glu He Ser Gly Arg Leu Tyr Arg Leu

Patent provided by Sughrue Mion, PLLC - htlp://www.sughrue.com

Cys Cys Arg 35 <210>133 <211> 35 c212> PRT <213> Human HNP-6 Defensin <400>133

Ser Thr Arg Ala Phe Thr Cys His Cys Arg Arg Ser Cys Tyr Ser Thr !5 1015

Glu Tyr Ser Tyr Gly Thr Cys Thr Val Met Gly He Asn His Arg Phe

2530

Cys Cys Leu <210> 134 <211> 84 <212> PRT _<213> Holotrichia diomphalia Holotricin 3 <400> 134

Tyr Gly Pro Gly Asp Gly His Gly Gly Gly His Gly Gly Gly His Gly _χ 5 1015

Gly Gly Hie Gly Asn Gly Gin Gly Gly Gly His Gly His Gly ProGly ²⁵³⁰

Gly Gly Phe Gly Gly Gly His Gly Gly Gly His Gly Gly Gly Gly Arg 35 4045

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Pro Gly His Gly Ala Gly 50 5560

Glv Gly Tyr Pro Gly Gly His Gly Gly Gly His His Gly Gly Tyr Gin 65 70 ⁷⁵³⁰

Thr His Gly Tyr

<td> &lt;210&gt; &lt;211&gt; &lt;212&gt; &lt;213&gt;</td><td colspan="2"> 135 45 PRT Bovine Lingual antimicrobial peptide</td>

<td colspan="2"> &lt;400&gt; 135 Gly Phe Thr Gin Gly Val Arg Asn Ser</td><td> Gin Ser Cys Arg Arg Asn. Lys</td>

Patent provided by Sughrue Mion, PLLC - http://www.sughrue.cQm

5

Gly lie Cys Val 20

Pro lie Arg Cys

Pro Gly Ser Met Arg Gin lie Gly

30

Thr Cys Leu Gly Ala Gin Val Lys Cys

40

Cys Arg Arg Lys <210? 136 <211> 29 <212> PRT <213> Rat RatNP-3 <400>136

Cys Ser Cys Arg Thr Ser Ser Cys Arg Phe Gly Glu Arg Leu Ser Gly

10IS

Ala Cys Arg Leu Asn Gly Arg He Tyr Arg Leu Cys Cys

2025 <210?137 <211> 31 <212> PRT <213> Guinea pig GNCP-1 <400> 137

Arg Arg Cys He Cys Thr Thr Arg Thr CyB Arg Phe Pro Tyr Arg Arg

10

Leu Gly Thr Cys He Phe Gin Asn Arg Val Tyr Thr Phe Cys Cys

2530 <210> 13Θ <211>47 <212> PRT <213? Penoeid shrimp Penaeidin-4a <400> 13Θ

His Ser Ser Gly Tyr Thr Arg Pro Leu Pro Lys Pro Ser Arg Pro He

1015

Phe He Arg Pro lie Gly Cys Asp Val Cys Tyr Gly He Pro Ser Ser

2530

Thr Ala Arg Leu Cys Cys Phe Arg Tyr Gly Asp Cys

3540

Cys His Arg 45 <210>

<211>

<212>

<213>

PRT

Bovine hexapeptide

Patent provided by Sughrue Mion, PLLC - http://www.sughrue.com

PCT7US2004/021711 <400> 139

Arg Arg Trp Gin Trp Arg 1 ⁵ <210> 140 <211> IB <212> PRT <213> P-18 <400> 140

Lys Trp Lys Leu Phe Lys Lys He Pro Lys Phe Leu His Leu Ala Lys

5 ר

Lys Phe <210> 141 <211> 20 <212> PRT <213> Human MUC7 20-Mer <4D0> 141

I,_־u a־ Hl־ Gin ItfB «» H־ “a ^{Ser LYSHi0} c 10Lys Arg Cys Arg <210> 142 <211>21 <212> PRT.

<213> Rana nigromaculata Nigrocm 2 <400>142

Sly Leu L־u Sar Lys Val Leu Gly Val Gly by־ by־ Val lam CyB Gly . 5 10

Val Ser Gly Leu Cys <210> 143 <211> 33 <212> PRT <213> Rana nigromaculata Nigrocin 1 <400> 143

Gly Leu Leu Asp Ser lie Lys Gly Met Ala lie Ser Ala Gly Lys Gly ״ ' r 10 ¹=

Ala Leu Gin Asn Leu Leu Lys Val Ala Ser Cys Lys Leu Asp Lys Thr 88 Patent provided by Sughrue Mion, PLLC - http://www.sughrue.com

Cys

<td></td><td> PCT/US2004/021711</td>

<td> 25</td><td> 30</td>

<td> &lt;210&gt; &lt;211&gt; &lt;212&gt; &lt;213&gt;</td><td> 144 10 PRT lactoferrin</td><td> (Lf) peptide 2</td>

<td> &lt;400&gt;</td><td> 144</td><td></td>

<td colspan="2"> Phe Lys Cys Arg Arg</td><td> Trp Gin Trp Arg Met</td>

<td> 1</td><td> 5</td><td> 10</td>

<td> &lt;210&gt; &lt;211&gt; &lt;212&gt; &lt;213&gt;</td><td> 145 IB PRT Impatiens balsamina Ib-AMP3</td><td></td><td></td>

<td> &lt;400&gt;</td><td> 145</td><td></td><td></td>

<td colspan="2"> Arg His Arg Cys Cys Ala Trp Gly Pro 1 5</td><td> Gly Arg Lys Tyr Cys 10</td><td> Lys Arg 15</td>

Trp Cys

<td> &lt;210&gt; &lt;211&gt; &lt;212&gt; &lt;213&gt;</td><td> 146 18 PRT Impatiens balsamina Ib-AMP4</td><td></td><td></td>

<td> &lt;400&gt;</td><td> 146</td><td></td><td></td>

<td colspan="2"> Gly Arg Arg Cys Cys Gly Trp Gly Pro 1 5</td><td> Gly Arg Arg Tyr 10</td><td> Cys Arg Arg 15</td>

<td> Trp Cys</td><td> J</td><td></td><td></td>

<210> 147 <211> 14 <212> PRT <213 > Synthesis dhvar4

147 .־400>

Lys Arg Leu Phe Lys Lys Leu Leu Phe Ser Leu Arg Lys Tyr

<td> 1</td><td> 5</td><td> 10</td>

<td> &lt;210&gt;</td><td> 14 Θ</td><td></td>

<td> &lt;211&gt;</td><td> 14</td><td></td>

<td> &lt;212&gt;</td><td> PRT</td><td></td>

<td> &lt;213&gt;</td><td> Synthesis dhvar5</td><td></td>

Patent provided by Sughrue Mion, PLLC * http://www.sughrue.com <400> 148

Leu Leu Leu Phe Leu Leu Lys Lys Arg Lys Lys Arg Lys Tyr <210> 149 <211> 6 <212> PRT <213> Synthetic peptide <220?» <221> Misc_feature <222> (1).»(5) <223> X = anything <400> 149

Xaa Xaa Xaa Xaa Xaa Cys

5 <210> 150 <211> 6 <212> PRT <213> Synthetic peptide <220>

<221> Misc_feature <222> (1).(4) <223> X = anything <400> 150

Xaa Xaa Xaa Xaa Phe Cys 1 ⁵ <210> 151 <211> 6 <212> PRT <213> Synthetic peptide <220>

<221> Misc_feature <222> (1).(4) <223> X = anything <400>151

Xaa Xaa Xaa Xaa Asn Cys <210>152 <211>6 <212> PRT <213> Synthetic peptide <220 <221> MiscjEeature <222> (1).(4) <223> X = anything

Patent provided by Sughrue Mion, PLLC - http://www.sughrue.com <400> 152

Xaa Xaa Xaa Xaa Trp Cys

5 <210> 153 <211> 6 <212> PRT <213> Synthetic peptide <220>

<221> Misc_feature <222> (1).(4) <223> X = anything <400>153

Xaa Xaa Xaa Xaa He Cys <210> 154 <211>6 <212> PRT <213> Synthetic peptide <220>

<221> Misc_feature <222> (1).(4) <223> X = anything <400> 154

Xaa Xaa Xaa Xaa Thr Cys ⁵ <210> 155 <211> 6 <212> PRT <213> Synthetic peptide <220>

<221> Misc_feature <222> (1) -. (4) <223> X = anything <400>155

Xaa Xaa Xaa Xaa Tyr Cys <210>156 <211> 6 <212> PRT <213> Synthetic peptide <220:.

<221> Mi8c_feature <222> (1).(4) <223> X = anything

Patent provided by Sughrue Mion, PLLC - http://www.sughaje.c01n

156 <0ס4>

Xaa Xaa Xaa Xaa Val Cys

5 <210> 157 <211> 6 <212> PRT <213> Synthetic peptide <220>

<22Misc_feature <222> (1)-(4) <223> X = anything <400>157

Xaa Xaa Xaa Xaa Met Cys <21D>158 <211>6 <212 > PRT <213> Synthetic peptide <220>

<221> Misc_feature <222> (1).(4) <223> X = anything <4O0>158

Xaa Xaa Xaa Xaa Gly Cys 15 <210>159 <211> 6 <212> PRT <213> Synthetic peptide <220>

<221> Misc_feature <222> (1).(4) <223 > X = anything <400>159

Xaa Xaa Xaa Xaa Glu Cys 1 ⁵ <210>160 <211>6 <212> PRT <213> Synthetic peptide <22O>

<221> Misc_feature <222> (1) . . (4) <223> X = anything

Patent provided by Sughrue !Vlion, PLLC - http://www.sughrue.com

PCT7US2004/021711 <400> 160

Xaa Xaa Xaa Xaa Ser Cys

5 <210> 161 <211> 6 <212> PRT <213> Synthetic peptide <220>

<221> Misc_feature <222> (1) . (4) <223> X = anything <400>161

Xaa Xaa Xaa Xaa Leu Cys 15 <210> 162 <211>6 <212> PRT <213> Synthetic peptide <220>

<221> Misc_feature <222> (1).(4) <223> X = anything <400>162

Xaa Xaa Xaa Xaa Pro Cys 15 <210> 163 <211>6 <212> PRT <213> Synthetic peptide <220>

<221> Misc_feature <222> (1) . (4) <223> X = anything <400> 163

Xaa Xaa Xaa Xaa His Cys 1 5

<td> &lt;210&gt; &lt;211&gt; &lt;212&gt; &lt;213&gt;</td><td> 164 6 PRT Synthetic peptide</td>

<td> &lt;220&gt; &lt;221&gt; &lt;222&gt; &lt;223&gt;</td><td> Miscofeature (1) . . (4) X = anything</td>

Patent provided by Sughrue Mion, PLLC - http://www.sughnje.conn <400> 164

Xaa Xaa Xaa Xaa Cys Cys

5 <210> 165 <211> 6 <212> PRT <213> Synthetic peptide <220>

<221> Misc_feature <222> (1)--(4) <223> X = anything <400> 165

Xaa Xaa Xaa Xaa Ala Cys

5 <210> 166 <211> 6 <212> PRT <213> Synthetic peptide <220>

<221> Misc_feature <222> (1).(4) <223> X = anything <400> 166

Xaa Xaa Xaa Xaa Lys Cys 1 ⁵ <210> 167 <211> 6 <212> PRT <213> Synthetic peptide <220>

<221> Misc_feature <222> (1).(4) <223> X = anything <400>167

Xaa Xaa Xaa Xaa Gin Cys <210>168 <211>6 <212> PRT <213> Synthetic peptide <220>

<221> Misc_feature <222> (1)--(4) <223> X “ anything

Patent provided by Sughrue Mion, PLLC - htlp;//www.sugt1me.com

PCI7US2004/021711 <400> 168

Xaa Xaa Xaa Xaa Arg Cys

5 <210> 169 <211> 6 <212 > PRT <213> Synthetic peptide <220>

<221> Misc_feature <222> ¢1).(4) <223־> X = anything <400> 169

Xaa Xaa Xaa Xaa Asp Cys

5 <210> 170 <211> 6 <212> PRT <213> Synthetic peptide <220>

<221> Misc_feature <222> (1).(4) <223> X = anything <400> 170

Xaa Xaa Xaa Xaa Cys Tyr ⁵ <210> 171 <211> 6 <212> PRT <213> Synthetic peptide <220>

<221> Misc_feature <222> (1).(4) <223> X = anything <400> 171

Xaa Xaa Xaa Xaa Cys Gin 1 5 <210> 172 <211> 6 <212> PRT <213> Synthetic peptide <220>

<221> Misc_feature <222> (1) . - (4) <223> X = anything

Patent provided by Sughrue Mion, PLLC - http://www.sugh1ue.com

PCT7US2004/021711 <400> 172

Xaa Xaa Xaa Xaa Tyr Ser

5 '

<td> &lt;210&gt; &lt;211&gt; &lt;212&gt; &lt;213&gt;</td><td> 173 6 PRT Synthetic peptide</td>

<td> &lt;220&gt; &lt;221&gt; &lt;222&gt; &lt;223&gt;</td><td> Misc_feature (1) . . (4) X = anything</td>

<td> &lt;400&gt;</td><td> 173</td>

Xaa Xaa Xaa Xaa Tyr Asp 1 5 <210> 174 <211> 6 <212> PRT <213 > Synthetic peptide <220>

<221> Misc_feature <222> (1). (4) <223> X = anything <400>174

Xaa Xaa Xaa Xaa Thr Qin 15 <210>175 <211>6 <212> PRT <213> Synthetic peptide <400>175

Trp Thr Phe Arg Tyr Cys 15 <210> 176 <211> 6 <212> PRT <213> Synthetic peptide <400> 176

Cys Tyr Arg Phe Thr Trp 1 5 <210> 177 <211> 11 <212> PRT <213> Glonterella aingulata

Patent provided by Sughrue Mion, PLLC ־ http://www.sughrue.com

<td> &lt;220&gt; &lt;221&gt; &lt;222&gt; &lt;22 3 &gt;</td><td colspan="2"> Misc_feature (9). . (9) N = anything</td>

<td> &lt;400&gt;</td><td> 177</td><td></td>

<td colspan="2"> Gly Tyr Phe Ser Tyr Pro His</td><td> Gly Asn Leu Phe</td>

<td> 1</td><td> 5</td><td> 10</td>

<td> &lt;210&gt;</td><td> 178</td>

<td> &lt;211&gt;</td><td> 13</td>

<td> &lt;212&gt;</td><td> PRT</td>

<td> &lt;213&gt;</td><td> Saccharomyces cerevisiae</td>

<400> 178

Trp His Trp Leu Gin Leu Lys Pro Gly Gin Pro Met Tyr

<td> 1</td><td> 5</td><td> 10</td>

<td colspan="2"> &lt;210&gt; 179 &lt;211&gt; 13 &lt;212&gt; PRT &lt;213&gt; Saccharomyces kluyveri &lt;400&gt; 179 Trp His Trp Leu Ser Phe Ser Lys</td><td> Gly Gin Pro Met Tyr</td>

<td> 1</td><td> 5</td><td> 10</td>

<td> &lt;210&gt; &lt;211&gt; &lt;212&gt; &lt;213&gt;</td><td> 180 19 PRT Synthetic peptide</td>

<td> &lt;220&gt;</td><td></td>

<td> &lt;221&gt;</td><td> mod_res</td>

<td> &lt;222&gt;</td><td> ¢2) . . (4)</td>

<td> &lt;223&gt;</td><td> Nle</td>

<td> &lt;400&gt;</td><td> 180</td>

<td colspan="2"> Tyr Asn Leu Glu Asp His Pro Gin Gly Asp His Pro Lys Leu Gin Leu</td>

<td> 1</td><td> 5 10 15</td>

Trp His Trp <210> 181 <211> 15 <212> PRT <213> Synthetic peptide <220>

<221> MOD_RSS <222> (2) . . (4) <223> Nle

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PCT7US2004/021711 <400> 181

Tyr Asn Leu Glu Pro Gin Gly

5

Pro Lys Leu Gin Leu Trp His 10