Anti-fouling composition

Anti-fouling composition.

The present invention relates to an anti-fouling composition. In particular, the present invention relates to an antifouling composition comprising an enzyme capable of producing a compound that has an antifouling effect.

As discussed in US Patent A-5071479, biocides are needed in very different environments, such as antifungal agents in house paints, algicides in fresh water, and antifouling agents in marine structures exposed to the flora and fauna of seawater. As is known, molds or fungi can grow on house paints or the like, and use the medium paint as a nutrient, or in some cases, the lower substrate, such as wood, as a nutrient. For obvious reasons, this may cause the painted surface to deteriorate the appearance of the painted surface. A biocide can be incorporated into the paint and when the mycelium or fruiting bodies of the fungus contact or penetrate the paint film and thus, through intimate contact with the biocide in the film, the fungi are destroyed. In cooling towers that use fresh water, verdin, molds and algae can be developed if effective compounds are not present to combat their growth.

As discussed in US Patent A-5071479 the growth of marine organisms on the submerged parts of a ship's hull is a particular problem. Such growth increases the frictional resistance of the hull to the passage of water, leading to an increase in fuel consumption and / or a reduction in ship speed. Marine growth accumulates so quickly that the cleaning and repainting solution that is made in dry dock is generally considered too expensive. An alternative that has been practiced with increased efficiency over the years is to limit the amount of fouling by applying a coating paint on the hull that incorporates anti-fouling agents. Anti-fouling agents are biocides that are released from the surface of the paint over a period of time to a lethal concentration for marine organisms on the surface of the hull. The antifouling paint fails only when the biocide concentration available in the surface paint falls below the lethal concentration and with modern paints a useful life of up to two years can be expected.

A biocide used very widely, particularly in marine antifouling agents, is tributyl staph (TBT). However, there is a growing concern about the environmental effects caused by organic scam biocides at their current commercial levels as an active anti-fouling ingredient in coating compositions for aquatic (marine) applications. It has been shown that, due to the wide use of compounds of the particular tributyl type, at concentrations as high as 20% by weight in bottoms of ships, the contamination of the surrounding water due to leaching has reached such a level that it causes degradation of mussels and shell organisms. These effects have been detected along the coast of French Bretafa and a similar effect has been confirmed in US and Far East waters. According to the most recent restriction regulations, with limited exceptions, pleasure boats up to 25 meters in length are no longer allowed to use antifouling paint that contains high levels of tributyl staph compounds.

Research has shown that as long as the leaching rate of the scam can be maintained at or below 4 Ig / cm2 per day, it seems that aquatic life is not affected in the long term. However, it has also been found that in order to be effective in the control of seaweed, as well as with more developed marine organisms, from the painted surface of the bottom of the ships, a certain minimum leaching rate of staph of approximately 9 to 16 Ig / cm2 / day. Normally, this higher leaching rate is achieved with a concentration of the tributyl compound of approximately 15% to 20% by weight of paint.

In view of the effectiveness of TBT, the regulatory authorities have reluctantly agreed that as long as there is not an adequate substitute for the organic anti-fouling active scam ingredients, large ships, that is to say those that have a length greater than 25 meters, still It allows them to use such compounds to minimize fouling. Therefore there is a desire to provide alternative biocides to compounds with TBT base.

US Patent A-4297137 describes that the effects of an anti-fouling composition can be prolonged by moderating the release of the anti-fouling constituents. This document describes antifouling paints that comprise at least one toxic substance for marine organisms that is uniformly incorporated into a discontinuous solid matrix that is insoluble in seawater and is dispersed in the paint. The matrix is ​​formed at least partially from at least one substance that becomes soluble in seawater under the action of enzymes released by marine organisms that must be inhibited and / or by the bacterial film in contact with the paint. Thus when the marine organism is associated with the painted surface, the toxic substance is released and the organisms are inhibited. Like the prior art descriptions, the toxic substances provided in US Pat. No. A-4297137 only include the well-known copper-based and scam compounds, such as TBT.

Abarzua et al., Mar. Ecol. Prog. Ser., Vol. 123: 301-312, 1995 "Biotechnological investigation for the prevention of biofouling. L Biological and biochemical principles for the prevention of biofoulding" propose the extraction of biogenic agents that have antibacterial, anti-algae, antiprotozoal and anti-macroincrustation properties from algae and invertebrates sailors It is proposed that the structure of the extracted agents can be determined, subsequently synthesized and the synthesized agent be used to prevent bioincrustation. No extraction or synthesis method is provided.

EP patent A-0866103 describes a method for controlling the release of compounds that have antimicrobial activity and coating compositions using this system. The method comprises incorporating an enzyme and a substrate into a matrix. The enzyme acts on the substrate to provide a compound. In an envisaged embodiment on the compound another enzyme may subsequently act. The substrate and enzyme (s) produce a compound that has antimicrobial activity.

US Patent A-5747078 is related to food products. The document shows that microbial contamination of food and food, which can cause serious health problems, can be inhibited by a composition comprising a lactoperoxidase system that maintains a continuous release of hydrogen peroxide. Then the hydrogen peroxide reacts with thiocyanate, catalyzed by lactoperoxidase, to produce hypothiocyanate. The hypothiocyanate can then act as an antimicrobial agent. This document provides a background showing the immobilized enzyme system. The document does not talk about anti-fouling agents or any microorganism that has encrusting properties.

The present invention mitigates the problem of prior art.

Aspects of the present invention are defined in the appendix claims. These and other preferred aspects are discussed below.

It has been found that the provision of an integrated system for the generation of an antifouling compound that uses an enzyme from a marine organism provides a stable system that

It has long-term effectiveness in dirty environments such as marine environments

it requires less substrate than prior art systems to provide a given antimicrobial effect. Enzymes from marine organisms, such as algae hexose oxidase (HOX), have low Km values ​​for glucose, specifically 2.7 mM. This low Km means that the enzyme has a very high affinity for glucose. Unlike non-marine enzymes such as non-marine glucose oxidase (GOX), it can have a Km value at least 10 times higher for glucose. In other words, previous enzyme systems have a much lower affinity for glucose than that of the present invention. In antifouling applications, this will make a significant difference, since an enzyme with high glucose affinity will be able to transform all the glucose present. On the other hand an enzyme with lower affinity for glucose is expected to allow leaching of glucose to the surrounding environment. Glucose leaching will counteract the desired antifouling activity because glucose will be a substrate for encrusting organisms. The leaching of glucose therefore results in an increase in scale.

<dl><dt /><dd>it requires fewer enzymes than prior art systems to provide a given antimicrobial effect. Enzymes of marine organisms, such as algae HOX, also have a lower Km value for oxygen again than the Km value for oxygen of prior art systems such as GOX. Again, this higher affinity for the substrate gives HOX algae an advantage. This is because in anti-fouling applications the worst inlay will be in "closed" environments such as ports with low water exchange and high growth of algae and other encrustating organisms. Exactly in those places the inlay was worse, the oxygen content of the water will also be the lowest compared to the open sea. Therefore, enzymes of marine organisms with high affinity for oxygen will be advantageous.</dd></dl>

<dl><dt /><dd>provides improved activity at probable working temperatures. In surface coating compositions such as antifouling paint the antimicrobial component (antifouling) normally has to be active between 15 and 30 ° C. For anti-fouling compositions, this is the temperature of seawater where fouling is a problem. Unlike in prior art systems, enzymes of marine organisms, such as algae HOX, have optimal activity temperature at exactly the optimum temperature for embedding and these enzymes are therefore perfectly suitable as an antifouling agent. The optimum temperature is shown in example 9.</dd></dl>

Use secure and immediately available substrates.

It has improved salt tolerance which leads to more enhanced activity in marine environments.

It is resistant to degradation by embedding organisms. Enzymes of marine organisms, such as algae HOX, are markedly resistant protease enzymes. They will survive treatment with pronase (preparation of broad spectrum proteases) without any loss of activity. This protease resistance is considered especially important in the antifouling application since therefore the enzyme will be resistant to degradation by proteases of the antifouling organisms that are trying to adhere to the coated surface.

In the present specification "encrustants" referred to by the terms "antifouling (s)", "antifouling" and "antifouling" includes organisms that can inhabit and / or grow on the surface to be treated with the present composition. Organisms include microorganisms such as bacteria, fungi and protozoa, and algae and organisms such as algae, plants and animals. The organism can be a marine organism.

The composition of the present invention comprises a precursor enzyme and a precursor substrate, in which the precursor enzyme and the precursor substrate generate a substrate for the enzyme of the present invention by the action of the precursor enzyme on the precursor substrate. This combination of precursor enzyme and precursor substrate will be referred to herein as "generating substrate".

The enzyme of the present system can be obtained or obtainable from a marine microorganism.

Preferably the enzyme of the present system is obtained or obtainable from a seaweed. Preferably the enzyme of the present system is obtained or obtainable from Chondrus crispus.

Preferably, the antifouling compound is hydrogen peroxide.

Preferably, the enzyme is an oxidase. Preferably, the enzyme is selected from glucose oxidase, L amino acid oxidase, D amino oxidase, galactose oxidase, hexose oxidase, pyranose oxidase, malate oxidase, cholesterol oxidase, arylalcohol oxidase, alcohol oxidase, latosterol oxidase, aspartate oxidase, amino oxidase, D glutamate oxidase, ethanolamine oxidase, NADH oxidase, urate oxidase (uricase) and mixtures thereof. Preferably, the enzyme is hexose oxidase.

ENZYME HEXOSA OXIDASA (HOX)

Hexose oxidase (D-hexose: O2-oxidoreductase, EC 1.1.3.5) (also called HOX) is an enzyme that in the presence of oxygen is capable of oxidizing D-glucose and several other reducing sugars including maltose, lactose and cellobiose to its corresponding lactones with subsequent hydrolysis of the respective aldobionic acids. Consequently, HOX differs from another oxidoreductase, glucose oxidase, which can only transform D-glucose, in that the enzyme can use a wide range of sugar substrates. Oxidation catalyzed by HOX can be illustrated as follows:

D-glucose + O2 - and D-gluconolactone + H2O2,

or

D-galactose + O2 - and D-galactonolactone + H2O2

HOX is naturally produced by various species of seaweed. Such species are found inter alia in the Gigartinaceae family. As used herein, the term "HOX" denotes an enzyme that is capable of oxidizing the substrates selected from the group consisting of D-glucose, D-galactose, D-mannose, maltose, lactose and cellobiose.

Preferably, hexose oxidase is obtainable or obtained from Chondrus crispus seaweed.

In one aspect the enzyme hexose oxidase is an enzyme treated in the description EP-A-0832245.

HEXOSA OXIDASA (HOX) PRODUCTION

The gene encoding the HOX enzyme has been cloned from Chondrus crispus seaweed (Stougaard and Hansen 1996, Hansen and Stougaard, 1997). Methylotrophic yeast Hansenula polymorpha (developed in Rhein Biotech, Dusseldorf / Germany as an expression system for heterologous proteins) has also been used to produce the HOX enzyme (the native protein was purified from seaweed (Poulsen and H0strup, 1998) ). WO 96/40935 and WO 98/13478 also describes the cloning and expression in recombinant host organisms of a gene encoding a protein with HOX activity.

In a preferred embodiment, the hexose oxidase enzyme comprises the amino acid sequence presented in SEQ ID NO: 1 or one of its variants, homologue, derivative or fragment. In a preferred embodiment, the hexose oxidase enzyme comprises the amino acid sequence presented in SEQ ID NO: 1.

In a preferred embodiment, the hexose oxidase enzyme is encoded by a nucleotide sequence presented in SEQ ID NO: 1 or one of its variants, homologue, derivative or fragment. In a preferred embodiment, the hexose oxidase enzyme is encoded by a nucleotide sequence presented in SEQ ID NO. 1.

In a preferred embodiment, the hexose oxidase enzyme is encoded by a nucleotide sequence capable of hybridizing the nucleotide sequence presented in SEQ ID No. 1 or one of its variants, homologue, derivative or fragment or a sequence complementary to the hybridization sequence . In a preferred embodiment, the hexose oxidase enzyme is encoded by a nucleotide sequence capable of hybridizing to the nucleotide sequence presented in SEQ ID NO: 1 or a sequence complementary to the hybridization sequence.

The enzyme, preferably the hexose oxidase enzyme can be prepared in a manner described in British Patent Application No. 9927801.2.

VARIANTS / HOMOLOGIES / DERIVATIVES (AMINO ACIDS SEQUENCE)

The amino acid sequences of the present invention are presented in SEQ ID No. 1 or are sequences obtainable from the HOX enzyme of the present invention but also include homologous sequences obtained from any source, for example, homologous, viral, related bacterial proteins cellular and synthetic peptides, as well as their variants or derivatives.

Thus, the present invention addresses variants, homologues or derivatives of the amino acid sequences presented herein, as well as variants, homologues or derivatives that encode the nucleotide sequences for those amino acid sequences.

In the context of the present invention, a homologous sequence is taken to include an amino acid sequence with at least 75, 85 or 90% identical, preferably at least 95 or 98% identical at the amino acid level above at least, for example, the Amino acid sequence as presented in SEQ ID N ° 1 of the sequence listed herein. In particular, homology should normally be considered with respect to those regions of the sequence that are known to be essential for enzymatic activity rather than for non-essential neighboring sequences. These regions include but are not limited to putative FAD binding domains in HOX such as SGGH79C, LGGH146I and LGGH320A. Although homology can also be considered in terms of similarity (for example, amino acid residues having similar chemical properties / functions), in the context of the present invention it is preferred to express homology in terms of sequence identity.

Homology comparisons can be carried out by eye, or more normally, with the help of readily available sequence comparison programs. These commercially available computer programs can calculate% homology between two or more sequences.

The% homology can be calculated on continuous sequences, for example one sequence is aligned with the other sequence and each amino acid of one sequence is directly compared with the corresponding amino acid of the other sequence, one residue at a time. This is called an "no gaps" alignment. Normally, such alignments without gaps are carried out only on a relatively short number of remains.

Although this is a very simple and consistent method, it fails to take into account that, for example, in a pair of identical sequences, an insertion or deletion will cause the next amino acid residue to leave the alignment, this potentially results in a large reduction. % homology when a global alignment is carried out. As a consequence, most sequence comparison methods are designed to produce optimal alignments that take into account possible insertions and deletions without unduly penalizing the total homology result. This is achieved by inserting "gaps" in the alignment sequence to try to maximize local homology.

However, these more complex methods assign "penalty gaps" to each gap that occurs in the alignment so that, for the same number of identical amino acids, a sequence of alignment with the least possible gaps - which reflects a higher ratio Between the two sequences compared you will achieve a higher score than one with many gaps. "Costs of related holes" are normally used and bear a relatively high cost for the existence of a hole and a smaller penalty for each subsequent remainder in the hole. This is the most commonly used hollow punctuation system. The high penalty per hole of course will produce optimal alignments with fewer gaps. Most alignment programs allow the modification of the penalty for gaps. However, it is preferred to use the default values ​​when using such programs for sequence comparison. For example, when using the GCG Wisconsin Bestfit package (see below), the omitted gap penalty for amino acid sequences is -12 for one hole and -4 for each extension.

The calculation of the maximum% of homology therefore firstly requires the production of an optimal alignment, taking into account the penalty for gaps. A suitable computer program for carrying out such alignment is the GCG Wisconsin Bestfit package (University of Wisconsin USA; Devereux et al., 1984, Nucleic Acids Research 12: 387). Examples of other softwares that can perform sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et al., 1999 ibid -Chapter 18), FASTA (Atschul et al., 1990, J. Mol. Biol ., 403-410) and the GENEWORKS comparison tool collection. Both BLAST and FASTA for online or offline research (see Ausubel et al., 1999 ibid, pages 7-58 to 7-60). However, it is preferred to use GCG Bestfit program.

Although the final% homology can be measured in terms of identity, the alignment process itself is usually not based on a comparison of all or no pairs. In contrast, a similarity scoring matrix with scale that assigns points in each pair comparison based on chemical similarity or evolutionary distance is generally used. An example of such a commonly used matrix is ​​the BLOSUM62 matrix, the omitted matrix of the BLAST program collection. GCG Wisconsin programs generally use either public defaults or a custom comparison chart of symbols if provided (see user manual for details). It is preferred to use public defaults for the

5 GCG package, or in the case of other software, to the omitted matrix BLOSUM62.

Once the software has produced an optimal alignment, it is possible to calculate the% homology, preferably the% identity of the sequence. Normally the software does this as part of the comparison sequence and generates a numerical result.

The terms "variant" or "derivative" in relation to the amino acid sequences of the present invention

10 they include any substitution, variation, modification, replacement, deletion or addition of one (or more) amino acids of or to the sequence provided that the resulting amino acid sequence has an enzymatic activity, preferably having at least the same enzymatic activity as the amino acid sequence presented in SEQ ID No. 1.

SEQ ID No. 1 may be modified for use in the present invention. Normally, modifications are made

fifteen so that they maintain the enzymatic activity of the sequence. Amino acid substitutions can be made, for example, from 1, 2 or 3 to 10 or 20 substitutions provided that the modified sequence retains the required enzymatic activity. Amino acid substitutions may include the use of analogies that occur unnaturally.

SEQ ID No. 1 of the present invention may also have deletions, insertions or substitutions of residues of

twenty amino acids that produce an invaluable change and result in a functionally equivalent enzyme. Deliberate amino acid substitutions can be made based on similarity in polarity, charge, solubility, hydrophobia, hydrophilicity, and / or the antipathic nature of the moieties provided that the enzymatic activity of the HOX enzyme is retained. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with

25 Unloaded polar groups that have similar hydrophilic values ​​include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine and tyrosine.

Conservative substitutions can be made, for example according to the following table. The amino acids of the same block in the second column and preferably in the same line in the third column can be substituted between them:

<dl><dt>ALIFATICO </dt><dd>NO POLAR GAP </dd></dl>

<dl><dt>ILV </dt><dd /></dl>

<dl><dt>POLAR WITHOUT LOAD </dt><dd>CSTM </dd></dl>

<dl><dt>NO </dt><dd /></dl>

<dl><dt>POLAR LOADED </dt><dd>FROM </dd></dl>

<dl><dt>Kr </dt><dd /></dl>

<dl><dt>AROMATIC </dt><dd>HFWY </dd></dl>

VARIANTS / HOMOLOGIES / DERIVATIVES (NUCLEOTIDE SEQUENCE)

An expert will understand that numerous different amino acid sequences can encode the same HOX enzyme as a result of the degeneracy of the genetic code. In addition, it is understood that experts can, using routine techniques, make nucleotide substitutions that do not affect the HOX enzyme encoded by the

35 nucleotide sequence of the invention to reflect the codon use of any particular host organism in which the HOX enzyme of the present invention is expressed.

The terms "variant", "homologous" or "derivative" in relation to the nucleotide sequences presented in SEQ ID No. 1 of the present invention include any substitution, variation, modification, replacement, deletion or addition of one (or more) nucleic acids from or to the sequence provided the nucleotide sequence

40 The resulting code encodes an HOX enzyme that has an enzymatic activity, preferably having at least the same enzymatic activity as the nucleotide sequence presented in SEQ ID NO: 1 of the sequence listed.

As indicated above, with respect to sequence homology, there is preferably at least 75%, more preferably at least 85%, more preferably at least 90% homology in the sequences shown in the sequence of the list hereof memory. More preferably there is at least 95%, at least more preferably 98% homology. Nucleotide homology comparisons can be

carry out as described above. A preferred sequence comparison program is the GCG Wisconsin Bestfit program described above. The omitted punctuation matrix has a match value of 10 for each identical nucleotide and 9 for each non-match. The penalty of creation of the omission hole is -50 and the penalty of each extension omission hole is -3 for each nucleotide.

The present invention also encompasses nucleotide sequences that are capable of selectively hybridizing to the sequences presented herein, or any variant, fragment, or derivative, or the complement of any of the foregoing. The nucleotide sequences are preferably 15 nucleotides in length, more preferably at least 20, 30, 40 or 50 nucleotides in length.

SUBSTRATUM

Preferably, the substrate is selected from peptides, L amino acids, and carbohydrates / sugars, including hexoses, preferably glucose, galactose, lactose, 2-deoxyglucose, pyranose, xylan, cellulose, inulin, starch, dextran, pectin and their mixtures

In a very preferred embodiment, the enzyme / substrate combination is selected from glucose / hexose oxidase, glucose / glucose oxidase, L amino acid / L amino acid oxidase, galactose / galactose oxidase, lactose / galactoxidase / hexose oxidase, lactose / - galactoxidase / glucose oxidase, 2-deoxyglucose / glucose oxidase, pyranose / pyranose oxidase, and mixtures thereof.

In one aspect the antifouling composition is generated by the action of the enzyme on the substrate that is present in the composition. Thus the antifouling compound is generated by a "one stage" process. In some cases the substrate can be prepared in situ. In these cases, the composition further comprises a precursor enzyme and a precursor substrate in which the precursor enzyme and the precursor substrate are selected such that the precursor enzyme generates the substrate. In this last aspect the anti-fouling composition is generated by a "two stage" process.

In the one-step process, the enzyme is preferably selected from hexose oxidase, glucose oxidase, L amino acid oxidase, galactose oxidase, pyranose oxidase, and mixtures thereof.

In the one-stage process, the substrate is preferably selected from a hexose, preferably glucose, L-amino acid, galactose, 2-deoxyglucose, pyranose, and mixtures thereof.

In the one-step process preferably the enzyme / substrate combination is selected from glucose / hexose oxidase, glucose / glucose oxidase, L amino acid / L amino acid oxidase, galactose / galactose oxidase, 2-deoxyglucose / glucose oxidase, pyranose / pyranose oxidase , and their mixtures.

In the two-stage process preferably the enzyme is hexose oxidase.

In the two-stage process preferably the substrate is glucose.

In the two-stage process, preferably the precursor enzyme is amyloglucosidase.

In the two-stage process preferably the precursor substrate is starch.

Thus, in the two-stage process, preferably the precursor substrate / precursor enzyme / enzyme combination is starch / amyloglucosidase / hexose oxidase.

Preferably, the precursor substrate in the two step process is selected from oligomers and substrate polymers for oxidative enzymes, starch, lactose, cellulose, dextrose, peptide, inulin, and mixtures thereof.

The provision of precursor substrates is particularly preferred because they provide prolonged and / or continuous release of substrate by the action of the precursor enzyme on the precursor substrate.

Native starch is particularly preferred as a precursor substrate. Native starch provides densely clustered crystals that can be applied directly to a surface coating. In addition, native starch is insoluble in water.

Cellulose is also particularly preferred as a precursor substrate. Cellulose is a common component in paints and the use of cellulose as a precursor substrate reduces the number of additional components that must be added to a paint composition.

Preferably, the precursor enzyme in the two-stage process is selected from exoactive enzymes capable of degrading oligomeric or polymeric substrates to monomeric units, for example galactosidase, peptidase; amyloglucosidase, and mixtures thereof.

Optionally, the composition further comprises a binder to immobilize at least one of the constituents, optionally to immobilize the enzymes.

The compositions of the present invention can be formulated as coatings, lacquers, dyes, enamels, and the like, hereafter referred to generically as "coating (s)".

Thus, in one aspect the present invention provides a coating consisting of a composition as defined above.

Preferably, the coating is formulated for the treatment of a surface selected from exterior wood, external surface of a central heating system, and a hull of a marine ship.

The coating includes a liquid vehicle (solvent) to dissolve or suspend the composition.

The liquid vehicle can be selected from any liquid that does not interfere with the activities of any essential component of the composition. In particular, the liquid vehicle must not interfere with the activity of the essential enzymes and / or anti-fouling component. Suitable liquid vehicles are described in US Patent A-5071479 and include water and organic solvents including aliphatic hydrocarbons, aromatic hydrocarbons, such as xylene, toluene, mixtures of aliphatic and aromatic hydrocarbons having melting points between 100 and 320 ° C , preferably between 150 and 230 ° C; highly aromatic petroleum distillates, for example naphtha solvent, distilled tar oil and mixtures thereof; alcohols such as butanol, octanol and glycols; vegetable and mineral oils ; Ketones such as acetone; petroleum fractions such as mineral liquor and kerosene, chlorinated hydrocarbons, glycol esters, glycol ester ethers, their derivatives and mixtures.

The liquid vehicle may contain at least one polar solvent, such as water, mixed with an organic solvent of low oil or oil-like volatility, such that the mixture of aromatic and aliphatic solvents found in white alcohol, also commonly called mineral liquor.

The vehicle can typically contain at least one of a diluent, an emulsifier, a humectant, a dispersant or other surface active agent. Examples of suitable emulsifiers are described in US Patent A-5071479 and include ethylene nonylphenol oxide esters, sorbitol polyoxyethylene esters or sorbitan polyoxyethylene fatty acid esters, their derivatives and mixtures.

Any surface coating material may be incorporated into the composition and / or coating of the present invention. Examples of recognized coating materials are polyvinyl chloride resins in a solvent based system, chlorinated gums in a solvent based system, acrylic resins and methacrylate resins in solvent or aqueous based systems, vinyl chloride systems. vinyl acetate copolymer as aqueous dispersions or solvent-based systems, butadiene copolymers such as butadiene-styrene gums, butadiene-acrylonitrile gums, and butadiene-styrene-acrylonitrile gums, drying oils such as flax oil, acid resins, asphalt, epoxy resins, urethane resins, polyester resins, phenolic resins, their derivatives and mixtures.

The composition and / or coating of the present invention may contain pigments selected from inorganic pigments such as titanium dioxide, ferric oxide, silicon, talc or clay from China, organic pigments such as carbon black or sea water insoluble dyes. , its derivatives and mixtures.

The composition and / or coating of the present invention may contain materials such as rosin to provide controlled release of the antifouling compound, since rosin is very slightly soluble in seawater.

The composition and / or coating of the present invention may contain plasticides, rheological characteristics modifiers, other conventional ingredients and mixtures thereof.

The composition and / or coating of the present invention, particularly the coating, also comprises an adjuvant that is conventionally used in compositions used to protect materials exposed to an aquatic environment. These adjuvants can be selected from additional fungicides, auxiliary solvents, processing additives such as defoamers, fixatives, plasticizers, UV stabilizers or stability improvers, water soluble or water insoluble dyes, color pigments, dryers, scale inhibitors, thickening or antiprecipitate agents such as carboxymethyl cellulose, polyacrylic acid or polymethyl acrylic acid, antioxidant agents, their derivatives and mixtures.

The additional fungicide (s) used in the composition and / or coating of the present invention is preferably soluble in the liquid vehicle.

In one aspect the present invention provides a marine antifouling agent consisting of a composition as defined above.

Preferably, the antifouling is self-polishing.

In one aspect of the present invention, the generating substrate or substrate and / or the enzyme is encapsulated. Preferably the substrate / substrate generator and / or enzyme is encapsulated by a semipermeable membrane.

The generator / enzyme substrate / substrate may be individually encapsulated independently of each other or may be encapsulated together. In the previous embodiment, the generator substrate or substrate can be activated by the antifouling agent. For example, the encapsulated material can be selected so that in contact with an antifouling agent, the generator substrate or enzyme can be released upon contact with the other substrate / generator substrate or enzyme. Thus, a composition can be provided so that it only provides an antifouling compound or increases the provision of antifouling compound when it comes into contact with an antifouling agent.

The composition of the present invention can be provided as a ready-to-use product or as a concentrate. The ready-to-use product may be in the form of an aqueous solution, aqueous dispersion, oil solution, oil dispersion, emulsion or an aerosol preparation. The concentrate can be used, for example, as a coating additive, or it can be diluted before use with additional solvents or suspending agents.

An aerosol preparation according to the invention can be obtained in a usual manner by incorporating the composition of the present invention comprised, or dissolved or suspended in a suitable solvent, in a volatile liquid suitable for use as a propellant, for example the mixture of chlorides and fluorides derived from methane and ethane commercially available under the "Freon" brand, or compressed air.

As discussed in US Patent A-5071479 the composition and / or coating of the present invention may include additional ingredients known to be useful in preservatives and / or coatings. Such ingredients include fixatives such as carboxy methyl cellulose, polyvinyl alcohol, paraffin, co-solvents, such as ethyl glycol acetate and methoxypropyl acetate, plasticides such as benzoic acid esters and phthalates, for example, dibutyl phthalate, dioctyl phthalate and didodecyl phthalate, its derivatives and mixtures. Optionally it can also include dyes, color pigments, scale inhibitors, chemical stabilizers or dryers (dryers) such as cobalt octoate and cobalt naphthenate, depending on specific applications.

The composition and / or coating of the present invention can be applied by any of the techniques known in the art including by brush, spray, roller coating, dipping and combinations thereof.

The combinations of the present invention can be prepared simply by mixing the various ingredients at a temperature at which they are not adversely affected. The conditions of preparation are not critical. The equipment and methods conventionally used in the manufacture of coatings and similar compositions can be advantageously employed.

The invention will now be described, by way of example only, with the following examples.

EXAMPLES

The antifouling effect of an antifouling composition of the present invention is tested according to the following examples. These examples show the effectiveness of the present composition in preventing fouling. The examples also provide optimization of the antifouling properties of the present composition.

The hexose oxidase (HOX) used in each of the present examples is available from DaniscoCultor. HOX is a fermented product from Hansenula polymorfa yeast that expresses the gene encoding the HOX enzyme cloned from Chondrus crispus seaweed.

Example 1 - Preparation of an antifouling composition ("one stage")

Soluble or immobilized hexose oxidase or other hydrogen peroxide that generates enzymes such as glucose oxidase is tested as an anti-fouling compound that generates enzymes in an anti-fouling composition. Hexose oxidase can be immobilized for example by binding to an anion exchanger, O Sepharose FFTM (available from Pharmacia) using 20 mM triethanolamine buffer, pH 7.3. Alternatively, the hexose oxidase or alternative hydrogen peroxide that generates enzymes binds covalently to a suitable vehicle such as epoxy activated Shepharosa ™ (Pharmacia, Sweden), carbodiimide activated agarose (Bio-Rad, USA). Other traditional procedures known in the art for immobilization can also be used.

The range of concentrations used in 0.0001 to 1000 U of hexose oxidase / hydrogen peroxide activity that generates enzymes per ml of anti-fouling composition. One unit of enzyme activity is defined as the amount of enzyme that produces 1 Imol of H2O2 per minute at 25 ° C.

To establish the suitability for use in the present invention the activity of the enzyme can be analyzed as follows. The activity of hexose oxidase (HOX) is measured according to the following procedure.

HOX analysis is based on the measurement of hydrogen peroxide generated in glucose oxidation.

Hydrogen peroxide oxidizes -dianisidine in the presence of peroxide to form a dye. HOX

-D-glucose + H2O2 + O2 - D-glucono-delta-lactone + H2O2 POD

5 H2O2 + o-dianisidine -2 H2O + o-dianisinaox Reagents

<dl><dt>1. </dt><dd>100 mM phosphate buffer, pH 6.3 </dd></dl>

<dl><dt>2. </dt><dd>100 mM D-glucose (SIGMA, G-8270) in 100 mM phosphate buffer, pH 6.3 </dd></dl>

<dl><dt>3. </dt><dd>o-Dianisidine (SIGMA, D-3252), 3.0 mg / ml in distilled water </dd></dl>

10 4. Peroxidase (SIGMA, P-8125), 0.10 mg / ml in 100 mM phosphate buffer, pH 6.3

Analysis

120 Reagent 1

150 Reagent 2

10 Reagent 3

fifteen 10 Il reagent 4 and

10 Enzyme Dissolution

The analysis is carried out on a microtiter plate. The reaction is initiated by the addition of enzymatic solution. The mixture is incubated at 25 ° C for 15 minutes with stirring. The blank test contains all components with water instead of enzymatic solution. The formation of the dye is measured on a plate of

twenty microtiter read at 405 nm. The linearity of the reaction can be checked using a kinetic program on the microplate reader.

A standard curve of hydrogen peroxide can be constructed using various concentrations of H2O2 (MERCK).

Example 2 - Preparation of an anti-fouling composition ("two stages")

25 Glucose and galactose are analyzed in concentrations of 0.01 to 100 Ig per ml of anti-fouling composition as substrates to generate a substrate for hexose oxidase in the systems described in example 1. To provide a system that generates a continuous substrate, starch is used, preferably granules of intact wheat, corn or potato starch, in a concentration of 0.01 ng to 100 Im per ml of anti-fouling composition, together with amyloglucosidase (GRINDAMYLTM AG 1500 Ba ery Enzyme de DaniscoCultor or other

30 commercial product of amyloglucosidase). The components are present in concentrations that provide 0.000001 to 10 AGU per ml of antifouling composition.

1 AGU is defined as the activity of amyloglucosidase that releases 1 Imol of glucose per minute from maltose (0.5% w / v) in 50 mM sodium acetate, pH 5.0 (adjusted with concentrated acetic acid) at 40 ° C. the analysis is stopped by transferring 200 Il of test mixture to 100 Il of 0.1 M hydrochloric acid

35 hydrochloric and the amount of glucose released is measured using glucose dehydrogenase reagent (Merc no 12193) or another glucose detection system.

Example 3 - Generation of hydrogen peroxide by HOX-containing paint

The following experiment was carried out to analyze the ability of hexose oxidase (HOX) to generate hydrogen peroxide.

40 At 11.0 g of paint (wall paint with Sadolin Glans 7 water base and Histor 9010 oil base, respectively) 2.0, 0.5 and 1 g, respectively, of HOX (DaniscoCultor fermented product of Hansenula polymorpha) spray dried on starch (10 U / g). Water-based paint also added 5 g of water per treatment.

Plastic disposable transfer pipettes (Sarstedt) were immersed (the head part) in the paint. 45 Transfer pipettes were allowed to air dry for 3 hours.

Then the activity of hexose oxidase (HOX) was measured by immersion of the head of the pipette covered with paint in a glass tube with 2 ml of HOX test reagent, see below, the only HOX activity comes from HOX in the painting.

The tubes were incubated at room temperature.

5 As white paint was used without adding HOX.

The result of the experiment is shown in Table 1. HOX is homogeneously distributed in the paint, since the entire surface of the paint immediately turned red when I contacted the HOX test reagent. The color developed is immediately observed indicating that the paint has no inhibitory effect on HOX activity. The experiment proves that HOX is capable of generating peroxide from

10 hydrogen from an added exogenous substrate (here glucose) even when immobilized in a paint matrix after drying.

Table 1

Activity

Water-based paint, white 0

0.2 g HOX +

0.5 g HOX ++

1.0 g HOX +++

Oil-based paint, white 0

0.2 g HOX +

0.5 g HOX ++

1.0 g HOX +++

Control, reagent analysis plus HOX free +++

The concentration range used is 0.0001 to 1000 U of hexose oxidase activity or an alternative enzyme that generates hydrogen peroxide per ml of antifouling composition.

fifteen Example 4 - Model system for coating

A dialysis tube containing an antifouling composition is used as a model system for a coating to prevent scale on the surface of a covered material.

An anti-fouling composition in the dialysis tube is used to generate a concentration of hydrogen peroxide on the surface of the effective dialysis tube to create anti-fouling.

twenty The dialysis tube used has a cut-off value of 10,000 Da. The dialysis tube is either a dialysis tube or a dialysis box (such as Slide-A-LyzerTM available from Pierce; IL, USA).

The dialysis tube is immersed in a glass flask with 1 to 5 liters of sea or lake water collected as indicated above. The glass flask is slowly shaken with a magnetic stirrer and incubated at room temperature near a window to allow daylight to penetrate. It is monitored visually

25 Embedding on the dialysis tube for up to 4 weeks based on the appearance of a microbial growth layer on the dialysis tube and is assessed on a scale of 1 to 5 as described above. As a negative control, a dialysis tube containing tap water was used.

Optionally, the catalase immobilized in nitrocellulose membrane pieces, which have subsequently been blocked with 0.1% Tween 20, are added to lake or sea water to prevent the accumulation of peroxide of

30 hydrogen in the water surrounding the dialysis tube. The concentration of catalase used is in the range of 0.000001 to 100 CU, where 1 CU is defined as the activity of catalase that degrades 1 mol of hydrogen peroxide per minute at 30 ° C in 50 mM phosphate buffer. sodium, pH 7.0, as described for catalase in the Sigma catalog: Biochemicals Organic Compounds for Research and Diagnostic Reagents, Sigma Chemical Company 1995, page 221.

35 The compositions of the present invention are effective in preventing fouling.

Example 5 - HOX stability in paint.

The painted heads of the transfer pipettes described in example 1 were kept at room temperature for 2 months and then "analyzed" in the reagent mixture as described in example 2.

Table 2

Activity Water-based paint, white 0 0.2 g HOX + 0.5 g HOX ++ 1.0 g HOX Not determined Oil-based paint, white 0 0.2 g HOX +++ 0.5 g HOX +++ 1.0 g HOX +++ Control, reagent analysis plus free HOX Not determined

From the results of table 2 it is clear that HOX was stable for two months at temperature atmosphere in a dry paint matrix. Example 6 - Coating test.

5 Establishment of a test system for an anti-fouling composition. Samples of 0.5 to 5 ml of lake or sea water were collected in test tubes from Lake Brabrands in Aarhus, Denmark, and from the Baltic Sea in Aarhus. On the day the water samples were taken, the antifouling composition that was tested was added to the test tubes and sealed with ParafilmTM.

The test tubes were incubated at room temperature near a window to allow daylight

10 will penetrate The inlay is visually monitored for up to 4 weeks based on the appearance of a microbial growth layer on the walls of the test tube. For comparison, a test tube with 0.1% sodium azide and a test tube without antifouling composition, respectively, were used as positive and negative controls.

These test tubes were rated from 1 to 5, respectively, on a scale of 1 to 5 activity

fifteen highly effective antifouling to no activity, respectively. Commercial marine antifouling coating material is used without the addition of antifouling biocide. The antifouling compositions according to the present invention are mixed with the coating material and applied to the surface of metal, glass and plastic plates according to the instructions of the manufacturer of the coating material.

twenty The coated plates are immersed in water in a lake or in seawater. Embedding on the plates is monitored visually for up to 2 years based on the appearance of a layer of microbial growth on the plates and assessing on a scale of 1 to 5 as described above. As a negative control, a coating without anti-fouling composition was used.

The compositions of the present invention are effective in preventing fouling. 25 Example 7 - Stability of the anti-fouling composition in aquarium water. 10.0 g of paint (based on Histor 9010 oil) were added 500 mg of starch (Merc 1253), 50 mg of

HOX (DanisCultor fermented product from Hansenula polymorpha) spray dried on starch (10 U / g). A disposable transfer plastic pipette (Sarstedt) was immersed (the head part) in the paint.

30 The transfer pipettes were allowed to air dry for 24 hours. Then it was kept in 250 ml of water from an aquarium in a Kautex bottle for two months. The bottle was left in a daylight window. After two months, the pipette head was washed, air dried and then "tested" in a complete HOX reagent mixture. The HOX still showed full activity.

Example 8 - Proof of the generator substrate concept. To provide a continuous starch of the generating substrate system, preferably intact granules of wheat, corn or potato starch in a concentration of 0.01 ng to 100 mg per ml of antifouling composition, as well as amyloglucosidase (AMG) (GRINDAMYLTM AG 10000 Ba ery Enzyme de

Danisco Cultor or other commercial amyloglucosidase product) in concentrations that provide

0.000001 to 100 AGU per ml of antifouling composition are used together with HOX. To 10.0 g of paint (based on Histor 9010 oil) 500 mg of starch (Merc ), HOX (DanisCultor fermented product from Hansenula polymorpha) spray-dried over starch (10 U / g) and AMG were added

5 (10,000 AGU / g) as indicated in the table. A disposable transfer plastic pipette (Sarstedt) was immersed (the head part) in the paint. The transfer pipettes were allowed to air dry for 3 hours.

Then the activity of hexose oxidase (HOX) was measured by immersing the head of the paint-covered pipette in a glass tube with 2 ml of HOX test reagent without glucose, see example 1 for the test reagent, the HOX's only activity came from HOX in the paint and the only substrate for HOX

generated by AMG in the paint by hydrolysis of starch to glucose in the paint. The tubes were incubated at room temperature for 48 hours. As white, paint without HOX or added AMG was used. Table 3 Activity

fifty mg HOX + 10 mg AMG + 50 mg HOX + 20 mg AMG + 50 mg HOX -White (without enzymes) -

fifteen The results given in table 3 show that the combination of HOX and AMG works as intended. AMG is a generator of glucose from immobilized starch in the paint and HOX is a generator of hydrogen peroxide from the glucose generated.

Example 9 - Activity temperature. Hexose oxidase (purified HOX) was evaluated in relation to activity as a function of temperature and

twenty compared to a commercial glucose oxidase (Amano 081443/00018). Procedure: Sample: The enzyme sample was dissolved in water and dislodged on a PD10 column using 20 mM of

pH 6.3 phosphate buffer and diluted to 0.4 U / ml. It is attached to an Elisa plate:

25 150 μl 100 mM glucose in 100 mM phosphate buffer, pH 6.3 120 μl 100 mM phosphate buffer, pH 6.3 10 l 0-dianisidine (3 mg / ml in water) 10 μl peroxide (0.10 mg / ml in 100 mM phosphate buffer, pH 6.3) 10 he sample

30 It was tested 10 minutes at 30 ° C and measured at 405 nm. Results The results of the activity measurement as a function of temperature are shown in Table 4 and in the

Figure 1.

Table 4

<dl><dt>Temperature </dt><dd>Hexose oxidase Commercial Glucose Oxidase </dd></dl>

<dl><dt>° C </dt><dd>Relative activity,% Relative activity,% </dd></dl>

<dl><dt>10 </dt><dd> 70 67 </dd></dl>

<dl><dt>25 </dt><dd> 98 67 </dd></dl>

<dl><dt>27,5 </dt><dd> 100 72 </dd></dl>

<dl><dt>32,5 </dt><dd> 98 78 </dd></dl>

<dl><dt>37,5 </dt><dd> 94 78 </dd></dl>

<dl><dt>42 </dt><dd> 83 85 </dd></dl>

<dl><dt>45 </dt><dd> 81 100 </dd></dl>

<dl><dt>50 </dt><dd> 59 94 </dd></dl>

The results of activity versus temperature clearly illustrate a difference in the activity profile.

Hexose oxidase has its optimum temperature between 25-35 ° C, which is almost coincident with the maximum inlay temperature. On the contrary it seems that GOX has an optimal temperature at 50 ° C that is well above the temperatures that can be reached at sea.

<dl><dt>SEQ ID No. 1 </dt><dd /></dl>

<dl><dt>Molecule Name: hoxpic </dt><dd>1644 bbs linear DNA </dd></dl>

<dl><dt>Printed sequence: 1-1644 (complete) </dt><dd>Date of printing 04 June 1999 </dd></dl>

<dl><dt>Description: </dt><dd /></dl>

LIST OF SEQUENCES 0135 110 Danisco A / S 120 Composition

130 P006441EPA 140 07001141.6 141 02-06-200 150 GB 9913050.2

5 151 160 2 170 patent version 3.4 210 1 211 1644

10 212 DNA 213 Chondrus crispus 220 221 CDS 222 (1) .. (1964)

15 �400� 1

210 2 211 546 212 PRT 213 Chondrus crispus 400 2