Enzymatic peracid generation formulation

0001Cross-reference of related applications This application is incorporated herein by reference in its entirety, respectively, of US Provisional Patent Application Nos. 61 / 102,505 and 61 / 102,512, filed October 3, 2008, respectively. , 61 / 102,514, 61 / 102,520, 61 / 102,531, and 61 / 102,539.

0002The present invention relates to the fields of enzymatic peracid synthesis and in-situ enzyme catalysis. At least one peroxycarboxylic acid is effective for surface disinfection or sterilization, medical device sterilization, food processing equipment sterilization, and in textile and laundry care applications such as bleaching, antifouling, deodorization, disinfection or sterilization. Produce in sufficient concentration to be suitable for use.

0003Peracid compositions have been reported to be effective antibacterial agents. Methods for cleaning, disinfecting, and / or sterilizing hard surfaces, meat products, living plant tissues, and medical devices that resist unwanted microbial growth have been described (eg, Patent Documents 1-5). ). Peracids have also been reported to be useful in preparing bleaching compositions for laundry detergent applications (Patent Documents 6-8).

0004Peracids can be produced by a chemical reaction between a carboxylic acid and hydrogen peroxide (see Non-Patent Document 1). This reaction is usually catalyzed by a strong inorganic acid, such as concentrated sulfuric acid. The reaction of hydrogen peroxide with carboxylic acid is an equilibrium reaction, and the formation of peracid favorably favors the use of excess concentrations of peroxide and / or carboxylic acid, or the removal of water.

0005Some peracid-based disinfectants or bleaches consist of an equilibrium mixture of peracid, hydrogen peroxide, and a corresponding carboxylic acid. One drawback of these commercially available peroxy acid cleaning systems is that they are often unstable in solution over time. One way to overcome stability problems is to generate peracids prior to use by combining multireactive components that are individually stable for long periods of time. Preferably, the individual reaction components are easy to store, relatively safe to handle, and can be mixed to rapidly produce an effective concentration of peracid.

0006It was recently reported that the CE-7 group of carbohydrate esterases has perhydrolase activity. These "perhydrolase" enzymes have been demonstrated to be particularly effective in producing peracids from various carboxylic acid ester substrates when combined with a peroxygen source (each of which is described herein by reference in its entirety). See Patent Documents 9 and 10 and 11 granted to DiCosimo et al., Incorporated in the book). Some members of the CE-7 family of carbohydrate esterases, once the reaction components are mixed, peracetic acid from acetyl esters of alcohols, diols, and glycerols up to 4000-5000 ppm in 1 minute and 9000 ppm in 5-30 minutes. It has been demonstrated that it has sufficient perhydrolysis activity to produce (Patent Document 12).

0007The enzymatic peracid generation system described in Patent Document 13 conferred on DiCosimo et al. Is typically based on the use of multi-reactive components that remain separate until a peracid solution is needed. The use of this approach overcomes the peracid instability problems associated with the storage of many peracid-based disinfectants and bleaches. However, specific formulations that provide long-term stability of perhydrolase activity when using multi-component formulations containing CE-7 carbohydrate esterase remain unaddressed. Organic liquid or log P less than 2 (ie P is [solute]_Octanol/ [Solute]_waterThe long-term storage stability of the CE-7 enzyme, which has perhydrolysis activity when stored in a solvent having (the logarithm of the partition coefficient of the substance between octanol and water) equal to, is of particular concern. Some of the organic ester substrates previously described by DiCosimo et al. Have a log P value of less than 2.

0008The organic liquid or solvent can be detrimental to the activity of the enzyme, either when the enzyme is suspended directly in the organic liquid or solvent, or when a miscible organic / aqueous single-phase liquid or solvent is used. .. Two bibliographic publications reviewing the effects of organic solvents on enzyme activity and structure are Non-Patent Documents 2 and 3. Cowan and Plant, listed above, found that the technique (on page 87) is of little or no value in using organic solvents with log P 2 to stabilize intracellular enzymes in generally organic phase systems. He points out that he is aware of it. Organic solvents with 2-4 log P can be used on a case-by-case basis depending on enzyme stability, and those with log P> 4 are generally useful in organic phase systems.

0009Cowan and Plant (see above) have the effects of direct exposure of enzymes dissolved in single-phase organic-aqueous solvents (on page 91) to solvent concentration, solvent / enzyme surface group interactions, and solvent / enzyme hydration. He further points out that it depends on shell interactions. Single-phase organic-aqueous solvents containing low log P organic solvents must have a low log P value for the solvent so that the solvent mixes perfectly with the aqueous phase to form a single phase. It usually has a negative effect on enzyme stability except in low organic solvent concentration applications. Triacetin has been reported to have a log P of 0.25, similar to that of ethanol (log P -0.26) and isopropanol (log P 0.15) (Cowan and Plant) (Non-Patent Document 4); therefore in triacetin. Storage of enzyme powder is log P <2 (eg, cyclohexanone, log P = 0.94 (Cowan and Plant); 1,2-propanediol, log P = -1.41 (Gunning et al.); 1,3-propanediol, log. P = -1.3 (Non-Patent Document 5; Diethylene glycol butyl ether, log P = 0.56 (Non-Patent Document 6; Triethylene Glycol, log P = -1.75 (Non-Patent Document 7) is expected to result in unacceptable loss of enzyme activity, as is the use of additional co-solvents. Will be.

<p num="0010"><patcit num="1"><text>U.S. Pat. No. 6,545,047</text></patcit><patcit num="2"><text>U.S. Pat. No. 6,183,807</text></patcit><patcit num="3"><text>U.S. Pat. No. 6,518,307</text></patcit><patcit num="4"><text>U.S. Pat. No. 5,683,724</text></patcit><patcit num="5"><text>U.S. Patent Application Publication No. 2003/0026846</text></patcit><patcit num="6"><text>U.S. Pat. No. 3,974,082</text></patcit><patcit num="7"><text>U.S. Pat. No. 5,296,161</text></patcit><patcit num="8"><text>U.S. Pat. No. 5,364,554</text></patcit><patcit num="9"><text>International Publication No. 2007/070609 Pamphlet</text></patcit><patcit num="10"><text>U.S. Patent Application Publication No. 2008/0176299</text></patcit><patcit num="11"><text>U.S. Patent Application Publication No. 2008/176783</text></patcit><patcit num="12"><text>DiCosimo et al., US Patent Application Publication No. 2009/0005590</text></patcit><patcit num="13"><text>U.S. Patent Application Publication No. 2009/0005590</text></patcit></p>

<p num="0011"><nplcit num="1"><text>Organic Peroxides, Daniel Swern, ed., Vol.1, pp 313-516; Wiley Interscience, New York, 1971</text></nplcit><nplcit num="2"><text>C. Laane et al., Biotechnol. Bioeng. 30: 81-87 (1987)</text></nplcit><nplcit num="3"><text>Cowan, DAand Plant, A., Biocatalysis in Organic Phase Systems., Ch.7 in Biocatalysis at Extreme Temperatures, Kelly, RWWand Adams, M.eds., Amer.Chem.Soc.Symposium Series, Oxford University Press, New York, NY, pp 86-107 (1992)</text></nplcit><nplcit num="4"><text>YM Gunning et al., J. Agric. Food Chem. 48: 395-399 (2000)</text></nplcit><nplcit num="5"><text>SJ.Kuo et al., J.Am.Oil Chem.Soc.73: 1427 ~ 1433 (1996)</text></nplcit><nplcit num="6"><text>N.Funasaki et al., J.Phys.Chem.88: 5786 ~ 5790 (1984)</text></nplcit><nplcit num="7"><text>L. Braeken, et al., ChemPhysChem 6: 1606-1612 (2005)</text></nplcit></p>

<p num="0012"> Therefore, the problem to be solved is that a mixture of peracid-generating enzymes, which retains significant perhydrolysis activity even when the enzyme is stored in a mixture with a carboxylic acid ester substrate, is used for peracid production. To formulate the product to be used in an ester substrate.</p>

<p num="0013"> The problem described is a method of spray-drying an aqueous formulation containing at least one enzyme structurally classified as a CE-7 enzyme and having perhydrolysis activity, wherein the formulation is a spray-dried formulation (enzyme powder). Was solved by the discovery of a method further comprising an oligosaccharide excipient that stabilizes perhydrolase activity when combined with a carboxylic acid ester substrate used for peracid production.</p><p num="0014"> In one aspect, it is a method for stabilizing the perhydrolysis activity of the enzyme when present in a formulation consisting of the enzyme and a carboxylic acid ester. (a) Provided is an aqueous formulation that is structurally classified as a CE-7 enzyme and comprises at least one enzyme having perhydrolysis activity, at least one oligosaccharide excipient, and optionally at least one surfactant. With the process; (b) The aqueous formulation of (a) is spray dried to give the enzyme powder that substantially retains the perhydrolysis activity of the at least one enzyme when present in the formulation consisting of the carboxylic acid ester and the enzyme powder. With the manufacturing process Methods are provided that include.</p><p num="0015"> Another embodiment is structurally classified as a CE-7 enzyme, with at least one enzyme having perhydrolysis activity and at least one oligosaccharide excipient, and optionally a spray-dried formulation of at least one surfactant. It is about an enzyme powder containing, which substantially retains the perhydrolysis activity of at least one enzyme when present in a formulation consisting of a carboxylic acid ester and the enzyme powder.</p><p num="0016"> A further embodiment is for formulations containing the enzyme powders discussed above mixed with carboxylic acid esters. In another aspect, the formulation comprises an enzyme powder mixed with a carboxylic acid ester selected from the group consisting of monoacetin, diacetin, triacetin, monopropionin, dipropionin, tripropionin, monobutyrin, dibutyrin, tributyrin, and mixtures thereof.</p><p num="0017"> An additional aspect is (a) Provided is an aqueous formulation that is structurally classified as a CE-7 enzyme and comprises at least one enzyme having perhydrolysis activity, at least one oligosaccharide excipient, and optionally at least one surfactant. With the process; (b) The step of spray-drying the aqueous formulation of (a) to produce an enzyme powder; (c) With the step of combining the enzyme powder of (b) with an aqueous solution containing a carboxylic acid ester and a peroxygen source. It is about the manufacturing method of the disinfection formulation containing.</p><p num="0018"> Another aspect is (a) (1) (i) Enzyme powders discussed above; and (ii) Carboxylic acid ester With formulations containing; (2) With a peroxygen source With the process of providing a set of reaction components, including; (b) A step of combining the reaction components under suitable aqueous reaction conditions to produce a peroxycarboxylic acid; It is about the method of producing a peroxycarboxylic acid from a carboxylic acid ester containing.</p><p num="0019"> A further embodiment is a method of disinfecting or sterilizing a hard surface or inanimate object using an enzymatically produced peroxycarboxylic acid composition. (a) (1) (i) Enzyme powders discussed above; and (ii) Carboxylic acid ester Formulations containing; (2) Peroxygen source With the process of providing a set of reaction components, including; (b) A step of combining the reaction components under suitable aqueous reaction conditions, thereby forming a peroxycarboxylic acid product; (c) With the step of optionally diluting the peroxycarboxylic acid product; (d) A step of contacting the hard surface or inanimate object with the peroxycarboxylic acid produced in step (b) or step (c), thereby disinfecting the surface or the inanimate object. It is about the method including.</p><p num="0020"> A further embodiment is a method of treating a garment or textile for bleaching, stain removal, odor reduction, sterilization or disinfection using an enzymatically produced peroxycarboxylic acid composition. (a) (1) (i) Enzyme powders discussed above; and (ii) Carboxylic acid ester With formulations containing; (2) With a peroxygen source With the process of providing a set of reaction components, including; (b) A step of combining the reaction components under suitable aqueous reaction conditions, thereby forming a peroxycarboxylic acid product; (c) With the step of optionally diluting the peroxycarboxylic acid product; (d) The step of contacting the garment or woven fabric with the peroxycarboxylic acid produced in step (b) or step (c). Including Here, it is about a method in which the garment or textile is antifouled, deodorized, disinfected, bleached, sterilized, or a combination thereof.</p><p num="0021">A brief description of the biological sequence The following sequence follows 37C.FR §§1.821-1.825 (Requirements for Patent Applications Containing Nucleotide Sequences and / or Amino Acid Sequence Disclosures-the Sequence Rules) and follows the World Intellectual Property Organization (WIPO) Standard ST.25 (WIPO) Standard ST.25 ( 1998) and the European Patent Convention (EPC) and Patent Cooperation Treaty (PCT) Rules 5.2 and 49.5 (a-bis) sequence specifications, as well as Section 208 and Annex of the Administrative Instructions. Matches C. The symbols and formats used for nucleotide and amino acid sequence data follow the rules described in 37C.FR §1.822.</p><p num="0022"> SEQ ID NO: 1 is Bacillus subtilis ATCC® 31954^TMIt is an estimated amino acid sequence of cephalosporin C deacetylase from.</p><p num="0023"> SEQ ID NO: 2 is B. subtilis ATCC® 6633.^TMIt is an estimated amino acid sequence of cephalosporin C deacetylase from.</p><p num="0024"> SEQ ID NO: 3 is B. licheniformis ATCC® 14580.^TMIt is an estimated amino acid sequence of cephalosporin C deacetylase from.</p><p num="0025"> SEQ ID NO: 4 is the deduced amino acid sequence of acetylxylan esterase from B. pumilus PS213.</p><p num="0026"> SEQ ID NO: 5 is Clostridium thermocellum ATCC® 27405.^TMIt is an estimated amino acid sequence of acetylxylan esterase from.</p><p num="0027"> SEQ ID NO: 6 is the deduced amino acid sequence of acetylxylan esterase from Thermotoga neapolitana.</p><p num="0028"> SEQ ID NO: 7 is the deduced amino acid sequence of acetylxylan esterase from Thermotoga maritima MSB8.</p><p num="0029"> SEQ ID NO: 8 is the deduced amino acid sequence of acetylxylan esterase from Thermoanaerobacterium sp. JW / SL YS485.</p><p num="0030"> SEQ ID NO: 9 is the deduced amino acid sequence of cephalosporin C deacetylase from Bacillus sp. NRRL B-14911. The amino acid sequence encoding cephalosporin C deacetylase from Bacillus sp. NRRL B-14911 as reported in GENBANK® Accession No. ZP_01168674 is a sequence with other cephalosporin C deacetylases. Alignment and reported length (340 amino acids) vs. observed length of other CAH enzymes (typically 318-325 amino acids in length; ENZYMATIC PERACID PRODUCTION USING, incorporated herein by reference. Seems to be wrong based on a comparison of co-owned, co-filed, and co-pending US patent applications under the agent reference number CL4205 US NA entitled "A COSOLVENT" 15 It should be pointed out that it appears to encode the amino acid N-terminal addition. Therefore, Bacillus sp. NRRL The putative amino acid sequence reported herein for the cephalosporin C deacetylase sequence from B-14911 does not contain the N-terminal 15 amino acids as reported under GENBANK® Accession No. ZP_01168674. ..</p><p num="0031"> SEQ ID NO: 10 is the deduced amino acid sequence of cephalosporin C deacetylase from Bacillus halodurans C-125.</p><p num="0032"> SEQ ID NO: 11 is the deduced amino acid sequence of cephalosporin C deacetylase from Bacillus clausii KSM-K16.</p><p num="0033"> SEQ ID NO: 12 is Bacillus subtilis ATCC® 29233.^TMIt is a deduced amino acid sequence of cephalosporin C deacetylase (CAH).</p><p num="0034"> SEQ ID NO: 13 is the deduced amino acid sequence of the Thermoanearobacterium saccharolyticum cephalosporin C deacetylase.</p><p num="0035"> SEQ ID NO: 14 is the deduced amino acid sequence of Thermotoga lettingae acetylxylan esterase.</p><p num="0036"> SEQ ID NO: 15 is the deduced amino acid sequence of Thermotoga petrophila acetylxylan esterase.</p><p num="0037"> SEQ ID NO: 16 is the deduced amino acid sequence of the first acetylxylan esterase from Thermotoga sp. RQ2, referred to herein as "RQ2 (a)".</p><p num="0038"> SEQ ID NO: 17 is the deduced amino acid sequence of the second acetylxylan esterase from Thermotoga sp. RQ2, which is referred to herein as "RQ2 (b)".</p><p num="0039"> SEQ ID NO: 18 is the amino acid sequence of the region containing the amino acid residues 118 to 299 of SEQ ID NO: 1.</p><p num="0040"> SEQ ID NO: 19 is Thermotoga neapolitana from the co-owned, co-filed, and co-pending U.S. Patent Application Agent Reference No. CL4392 US NA (incorporated herein by reference in its entirety). The putative amino acid sequence of the acetylxylan esterase variant, where the Xaa residue at position 277 is Ala, Val, Ser, or Thr.</p><p num="0041"> SEQ ID NO: 20 is the putative amino acid sequence of the Thermotoga maritima MSB8 acetylxylan esterase variant from co-owned, co-filed, and co-pending US Patent Application Agent Reference No. CL4392 US NA. , Where the Xaa residue at position 277 is Ala, Val, Ser, or Thr.</p><p num="0042"> SEQ ID NO: 21 is the putative amino acid sequence of the Thermotoga lettingae acetylxylan esterase variant from co-owned, co-filed, and co-pending US Patent Application Agent Reference No. CL4392 US NA. Here, the Xaa residue at position 277 is Ala, Val, Ser, or Thr.</p><p num="0043"> SEQ ID NO: 22 is the putative amino acid sequence of the Thermotoga petrophila acetylxylan esterase variant from co-owned, co-filed, and co-pending US Patent Application Agent Reference No. CL4392 US NA. Here, the Xaa residue at position 277 is Ala, Val, Ser, or Thr.</p><p num="0044"> SEQ ID NO: 23 is Thermotoga sp. RQ2 Acetylxylan from "RQ2 (a)" from US Patent Application Agent Reference Number CL4392 US NA, co-owned, co-filed, and co-pending. The putative amino acid sequence of the esterase variant, where the Xaa residue at position 277 is Ala, Val, Ser, or Thr.</p><p num="0045"> SEQ ID NO: 24 is from Thermotoga sp. RQ2 Acetylxylan from "RQ2 (b)" from US Patent Application Agent Reference Number CL4392 US NA, co-owned, co-filed, and co-pending. The putative amino acid sequence of the esterase variant, where the Xaa residue at position 278 is Ala, Val, Ser, or Thr.</p><p num="0046"> SEQ ID NO: 25 is the deduced amino acid sequence from Thermoanaerobacterium sp. JW / SL YS485 acetylxylan esterase.</p><p num="0047"> SEQ ID NO: 26 is the coding region for the kanamycin resistance gene (kan) from Streptomyces kanamyceticus.</p><p num="0048"> SEQ ID NO: 27 is the plasmid pKD13 containing the kanamycin resistance gene.</p><p num="0049"> SEQ ID NO: 28 is a forward primer used to clone katG from plasmid pKD13.</p><p num="0050"> SEQ ID NO: 29 is a reverse primer used to clone katG from plasmid pKD13.</p><p num="0051"> SEQ ID NO: 30 is a PCR product of katG amplification from plasmid pKD13 using the primers of SEQ ID NO: 28 and SEQ ID NO: 29.</p><p num="0052"> SEQ ID NO: 31 is the coding region for the catalase-peroxidase gene (katG).</p><p num="0053"> SEQ ID NO: 32 is the deduced amino acid sequence of katG.</p><p num="0054"> SEQ ID NO: 33 is the plasmid pKD46 containing the λ-Red recombinase gene.</p><p num="0055"> SEQ ID NO: 34 is a forward primer used to confirm the decay of katG.</p><p num="0056"> SEQ ID NO: 35 is a reverse primer used to confirm the decay of katG.</p><p num="0057"> SEQ ID NO: 36 is a temperature sensitive plasmid pCP20 containing FLP recombinase.</p><p num="0058"> SEQ ID NO: 37 is a forward primer used to clone katE from plasmid pKD13.</p><p num="0059"> SEQ ID NO: 38 is a reverse primer used to clone katE from plasmid pKD13.</p><p num="0060"> SEQ ID NO: 39 is a PCR product of katE amplification from plasmid pKD13 using the primers of SEQ ID NO: 37 and SEQ ID NO: 38.</p><p num="0061"> SEQ ID NO: 40 is the coding region for the catalase HPII gene (katE).</p><p num="0062"> SEQ ID NO: 41 is the deduced amino acid sequence of katE.</p><p num="0063"> SEQ ID NO: 42 is a forward primer used to confirm the decay of katE.</p><p num="0064"> SEQ ID NO: 43 is a reverse primer used to confirm the decay of katE.</p><p num="0065"> SEQ ID NO: 44 is a coding region for a gene encoding acetylxylan esterase from Thermotoga neapolitana, as reported in GENBANK® (Registration No. AE000512).</p><p num="0066"> SEQ ID NO: 45 is a forward primer used to amplify the acetylxylan esterase gene from Thermotoga neapolitana.</p><p num="0067"> SEQ ID NO: 46 is a reverse primer used to amplify the acetylxylan esterase gene from Thermotoga neapolitana.</p><p num="0068"> SEQ ID NO: 47 is a PCR product of acetylxylan esterase amplification using the primers of SEQ ID NO: 45 and SEQ ID NO: 46.</p><p num="0069"> SEQ ID NO: 48 is a gene encoding acetylxylan esterase from Thermotoga maritima MSB8 as reported in GENBANK® (Registration No. NP_227893.1).</p><p num="0070"> SEQ ID NO: 49 is a forward primer used to amplify the acetylxylan esterase gene from Thermotoga maritima.</p><p num="0071"> SEQ ID NO: 50 is a reverse primer used to amplify the acetylxylan esterase gene from Thermotoga maritima.</p><p num="0072"> SEQ ID NO: 51 is a PCR product of acetylxylan esterase amplification using the primers of SEQ ID NO: 49 and SEQ ID NO: 50.</p>

0073Enzyme powders comprising at least one CE-7 carbohydrate esterase with perhydrolysis activity, at least one oligosaccharide excipient, and optionally a spray-dried formulation of at least one surfactant are disclosed herein. .. A method for producing a peroxycarboxylic acid from a carboxylic acid ester using the enzyme powder described above is also disclosed herein. In addition, disinfectant formulations containing peracids produced by the methods described herein are provided.

0074Many terms and abbreviations are used in this disclosure. The following definitions apply unless otherwise stated.

0075As used herein, the articles "a," "an," and "the" that precede an element or component of the invention are non-limiting with respect to the number of cases (ie, occurrences) of the element or component. Is intended to be. Therefore, "a", "an", and "the" should be read as containing one or at least one, and the singular form of an element or component also makes it clear that the number is singular. Includes plurals unless intended.

0076As used herein, the term "comprising" means that the presence of a feature, integer, process, or component is referred to in the claims, but it is one or more. Does not preclude the existence or addition of other features, integers, processes, components or groups thereof. The term "contains" is intended to include embodiments contained by the terms "consisting of" and "consisting of". Similarly, the term "consisting of essentially" is intended to include embodiments contained by the term "consisting of".

0077As used herein, the term "about" that modifies the amount of raw material or reactant used is, for example, a typical measurement and liquid used in the real world for the production of concentrates or the use of solutions. By handling procedures; by inadvertent errors in these procedures; with respect to variations in quantity that may occur due to differences in the manufacture, source, or purity of the raw materials used to make the composition or to carry out the method. The term "about" also includes different quantities for different equilibrium conditions for compositions resulting from a particular initial mixture. The claims, whether modified or unmodified by the term "about", include their equivalents.

0078If present, the entire range is inclusive and can be coalesced. For example, when a range of "1 to 5" is listed, the listed ranges are "1 to 4", "1 to 3", "1 to 2", "1 to 2 and 4 to 5". , "1-3 and 5", etc. should be interpreted.

0079As used herein, the terms "substrate," "suitable substrate," and "carboxylic acid ester substrate" have the same meaning, specifically: (a) Structure [X]_mR₅(During the ceremony X is the formula R₆It is an ester group of C (O) O; R₆Is a linear, branched or cyclic hydrocarbyl moiety of C1 to C7 optionally substituted with a hydroxyl group or a C1 to C4 alkoxy group, where R₆Is R₆If is C2 to C7, it optionally contains one or more ether bonds; R₅Is a linear, branched or cyclic hydrocarbyl moiety of C1 to C6 optionally substituted with a hydroxyl group, where R₅Each carbon atom in it individually contains one or less hydroxyl groups or one or less ester groups and is R.₅Optionally contains one or more ether bonds; m is from 1 to R₅Up to the number of carbon atoms in it) One or more esters with Esters with a solubility in water of at least 5 ppm at 25 ° C; or (b) Structure<chemistry num="1"><img id="000002" he="31" wi="119" file="JP5777516B2_D0001.tif" img-format="tif" img-content="drawing" /></chemistry>(In the formula, R₁Is a linear or branched alkyl of C1-C7 optionally substituted with a hydroxyl or C1-C4 alkoxy group, R₃And R₄Are individually H or R₁C (O)) One or more glycerides with; Equation (c)<chemistry num="2"><img id="000003" he="22" wi="73" file="JP5777516B2_D0001.tif" img-format="tif" img-content="drawing" /></chemistry>(In the formula, R₁Is a linear or branched alkyl of C1-C7 optionally substituted with a hydroxyl or C1-C4 alkoxy group, R₂C1 to C10 linear or branched alkyl, alkenyl, alkynyl, aryl, alkylaryl, alkyl heteroaryl, heteroaryl, (CH₂CH₂O)_nOr (CH₂CH (CH)₃) -O)_nH, n is 1 ~ 10) One or more esters; or (d) One or more acetylated monosaccharides, acetylated disaccharides, or acetylated polysaccharides; or (e) Any combination of (a) to (d) Means.

0080Examples of the carboxylic acid ester substrates include monoacetin; triacetin; monopropionin; diproopionin; tripropionin; monobutyrin; dibutyrin; tributyrin; glucose pentaacetate; xylose tetraacetate; acetylated xylan; acetylated xylan fragment; β-D-ribofuranose- 1,2,3,5-tetraacetate; tri-O-acetyl-D-galactal; tri-O-acetyl-glucarl; propylene glycol diacetate; ethylene glycol diacetate; 1,2-ethanediol, 1,2- Propylene diol, 1,3-propanediol, 1,2-butanediol, 1,3-butanediol, 2,3-butanediol, 1,4-butanediol, 1,2-pentanediol, 2,5-pentane Monoesters or diesters of diols, 1,6-pentanediols, 1,2-hexanediols, 2,5-hexanediols, 1,6-hexanediols, or any combination thereof may be mentioned.

0081As used herein, the term "peracid" has the same meaning as peroxy acid, peroxycarboxylic acid, peroxyic acid, peroxoic acid and peroxoic acid.

0082As used herein, the term "peracetic acid" is abbreviated as "PAA" and has the same meaning as peroxyacetic acid, ethaneperoxoic acid and all other synonyms for CAS Registry Number 79-21-0. ..

0083As used herein, the term "monoacetin" has the same meaning as glycerol monoacetate, glycerin monoacetate, and glyceryl monoacetate.

0084As used herein, the term "diacetin" has the same meaning as glycerol diacetate, glycerin diacetate, glyceryl diacetate, and all other synonyms for CAS Registry Number 25395-31-7.

0085As used herein, the term "triacetin" is used as glycerin triacetate, glycerol triacetate, glyceryl triacetate, 1,2,3-triacetoxypropane; 1,2,3-propanetriol triacetate and CAS Registry Number 102-76- It has the same meaning as all other synonyms of 1.

0086As used herein, the term "monobutyrin" has the same meaning as glycerol monobutyrate, glycerin monobutyrate, and glyceryl monobutyrate.

0087As used herein, the term "dibutyrin" has the same meaning as glycerol dibutyrate and glyceryl dibutyrate.

0088As used herein, the term "tributyrin" is synonymous with glycerol tributyrate, 1,2,3-tributyrylglycerol, and all other synonyms for CAS Registry Number 60-01-5. is there.

0089As used herein, the term "monopropionin" has the same meaning as glycerol monopropionate, glycerin monopropionate, and glyceryl monopropionate.

0090As used herein, the term "dipropionin" has the same meaning as glycerol dipropionate and glyceryl dipropionate.

0091As used herein, the term "tripropionin" is a synonym for glyceryl tripropionate, glycerol tripropionate, 1,2,3-tripropionylglycerol, and all other synonyms for CAS Registry Number 139-45-7. Has the same meaning as.

0092As used herein, the term "ethyl acetate" refers to all of acetic ether, acetoxietan, ethyl ethaneate, ethyl acetate ester, ethyl ethane acid ester, ethyl acetate ester and CAS registration numbers 141-78-6. It has the same meaning as other synonyms.

0093As used herein, the use "ethyl lactate" has the same meaning as all other synonyms for ethyl lactate ester and CAS Registry Number 97-64-3.

0094As used herein, the terms "acetylated sugar" and "acetylated sugar" mean mono-, 2- and polysaccharides containing at least one acetyl group. Examples include glucose pentaacetate; xylose tetraacetate; acetylated xylan; acetylated xylan fragment; β-D-ribofuranose-1,2,3,5-tetraacetate; tri-O-acetyl-D-galactal; and Examples include, but are not limited to, tri-O-acetyl-glucal.

0095As used herein, the terms "hydrocarbyl", "hydrocarbyl group", and "hydrocarbyl moiety" are linked by a single, double, or triple carbon-carbon bond and / or an ether bond, and accordingly. It means a linear, branched or cyclic arrangement of carbon atoms substituted with hydrogen atoms. Such hydrocarbyl groups may be aliphatic and / or aromatic. Examples of hydrocarbyl groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, cyclopropyl, cyclobutyl, pentyl, cyclopentyl, methylcyclopentyl, hexyl, cyclohexyl, benzyl, and phenyl. In a preferred embodiment, the hydrocarbyl moiety is a linear, branched or cyclic arrangement of carbon atoms linked by carbon-carbon single bonds and / or ether bonds and correspondingly substituted with hydrogen atoms.

0096As used herein, the terms 1,2-ethanediol, 1,2-propanediol, 1,3-propanediol, 1,2-butanediol, 1,3-butanediol, 2,3-butanediol , 1,4-Butanediol, 1,2-Pentanediol, 2,5-Pentanediol, 1,6-Pentanediol, 1,2-Hexanediol, 2,5-Hexanediol, 1,6-Hexanediol, And "monoesters" and "diesters" of their mixtures mean said compounds containing at least one ester group of formula RC (O) O (where R is the linear hydrocarbyl moiety of C1-C7). To do. In one embodiment, the carboxylic acid ester substrate is selected from the group consisting of propylene glycol diacetate (PGDA), ethylene glycol diacetate (EDGA), and mixtures thereof.

0097As used herein, the term "propylene glycol diacetate" refers to all of 1,2-diacetoxypropane, propylene diacetate, 1,2-propanediol diacetate, and CAS Registry Number 623-84-7. It has the same meaning as other synonyms.

0098As used herein, the term "ethylene glycol diacetate" is used with 1,2-diacetoxyethane, ethylene diacetate, glycol diacetate, and all other synonyms for CAS Registry Number 111-55-7. It has the same meaning.

0099As used herein, the terms "suitable enzyme reaction mixture", "suitable component for in-situ generation of peracid", "suitable reaction component", and "suitable aqueous reaction mixture" are used herein. It means the material and water in which the reactant and the enzyme catalyst come into contact. The components of a suitable aqueous reaction mixture are provided herein and one of ordinary skill in the art will fully understand the range of component variations suitable for this method. In one embodiment, a suitable enzymatic reaction mixture produces peracid in situ when the reaction components are combined. Thus, the reaction components may be provided as a multi-component system in which one or more of the reaction components remain separate until use. In another embodiment, the reaction components are first combined to form an aqueous solution of peracid, which is then contacted with a surface to be disinfected and / or bleached. The design of systems and means for separating and combining multiple active ingredients will be known in the art and will generally depend on the physical form of the individual reaction ingredients. For example, a large number of active fluid (liquid-liquid) systems are typically found in multi-chamber distribution bottles or two-phase systems, such as those found in several bleaching applications where the desired bleaching agent is produced when the reactive fluid is mixed. (For example, US Patent Application Publication No. 2005/0139608; US Patent No. 5,398,846; US Patent No. 5,624,634; US Patent Application Publication No. 6,391,840; European Patent No. 0807156B1; US Patent Application Publication No. 2005/0008526; and PCT Publication No. 00/61713 pamphlet). Other forms of the multi-component system used to generate peracid include powders (eg, US Pat. No. 5,116,575), multilayer tablets (eg, US Pat. No. 6,210,639), multi-partitions. Has a water-soluble packet (eg, US Pat. No. 6,995,125) and a solid mass that reacts upon addition of water (eg, US Pat. No. 6,319, 888) may include, but are not limited to, those designed for one or more solid component or solid-liquid component combinations. In one embodiment, the multi-component formulation is provided as two individual components, whereby an aqueous solution containing a peroxycarboxylic acid is generated by combining the two components. In another embodiment a) i) Enzyme powders as disclosed herein, and ii) Carboxylic acid ester With the first component comprising, optionally further raw materials selected from the group consisting of inorganic or organic buffers, corrosion inhibitors, wetting agents, and combinations thereof; b) With the second component containing a peroxygen source and water, which optionally contains a hydrogen peroxide stabilizer. A multi-component formulation containing the above is provided.

0100In another embodiment, the carboxylic acid ester in the first component is selected from the group consisting of monoacetin, diacetin, triacetin, and combinations thereof. In another embodiment, the carboxylic acid ester in the first component is an acetylated saccharide. In another embodiment, the enzyme catalyst in the first component is a granular solid. In another embodiment, the first reaction component is a solid tablet or powder.

0101As used herein, the term "perhydrolysis" is defined as the reaction of a peroxide with a selected substrate to form a peracid. Typically, the inorganic peroxide is reacted with the substrate of choice in the presence of a catalyst to produce the peracid. As used herein, the term "chemical perhydrolysis" refers to a perhydrolysis reaction in which a substrate (peracid precursor) is combined with a hydrogen peroxide source, in which the peracid is formed in the absence of an enzyme catalyst. Reaction is included.

0102As used herein, the term "perhydrolase activity" means catalytic activity per unit mass of protein, dry cell mass, or immobilized catalytic mass (eg, milligram).

0103As used herein, "1 unit of enzymatic activity" or "1 unit of activity" or "U" is the perhydrolase required to produce 1 μmol per second of peracid product at a specified temperature. Defined as the amount of activity.

0104As used herein, the terms "enzyme catalyst" and "perhydrolase catalyst" mean catalysts containing enzymes with perhydrolase activity, one or more of all microbial cells, permeabilized microbial cells, microbial cell extracts. It may be in the form of a cellular component, a partially purified enzyme, or a purified enzyme. The enzyme catalyst may also be chemically modified (eg, by pegging or by reacting with a cross-linking reactant). Perhydrolase catalysts may also be immobilized on soluble or insoluble carriers using methods well known to those of skill in the art; for example, Immobilization of Enzymes and Cells; Gordon F. Bickerstaff, Editor; Humana Press, Totowa, NJ, USA. See 1997. As described herein, all of the enzymes with perhydrolysis activity are structurally members of the Carbohydrate Esterase Group 7 (CE-7 Group) (Coutinho, PM, Henrissat, B. "Carbohydrate-active". enzymes: an integrated database approach in Recent Advances in Carbohydrate Bioengineering, HJ Gilbert, G. Davies, B. Henrissat and B. Svensson eds., (1999) The Royal Society of Chemistry, Cambridge, pp. 3-12). Group CE-7 enzymes have been demonstrated to be particularly effective in producing peracids from various carboxylic acid ester substrates when combined with a peroxygen source (each of which is hereby referenced in its entirety). International Publication No. 2007/070609 and US Patent Application Publication No. 2008/0176299, 2008/176783, and 2009/0005590, which are incorporated in the document. Please refer to the book).

0105Members of the CE-7 family include cephalosporin C deacetylase (CAH; EC3.1.1.41) and acetylxylan esterase (AXE; EC3.1.1.72). Members of the CE-7 esterase family share a conserved signature motif (Vincent et. al., J. Mol. Biol., 330: 593-606 (2003)). Perhydrolases containing CE-7 signature motifs and / or substantially similar structures are suitable for use in the present invention. Means for identifying substantially similar biological molecules are well known in the art (eg, sequence alignment protocols, nucleic acid hybridization and / or the presence of conserved signature motifs). In one aspect, the perhydrolase comprises at least 30%, preferably at least 33%, more preferably at least 40%, even more preferably at least 42%, with the CE-7 signature motif and the sequences provided herein. Even more preferably at least 50%, even more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90%, most preferably at least 90%, 91%, Includes enzymes containing 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity. In a further aspect, the perhydrolase contains CE-7 signature motif and SEQ ID NO: 1 at least 30%, preferably at least 33%, more preferably at least 40%, even more preferably at least 42%, even more preferably. At least 50%, even more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90%, most preferably at least 90%, 91%, 92%, 93 Includes enzymes containing%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity.

0106As used herein, the term "enzyme powder" is used to (1) at least one enzyme structurally classified as CE-7 with perhydrolysis activity, (2) at least one oligosaccharide excipient, and optionally. Means a spray-dried product of an aqueous formulation that selectively comprises at least one surfactant. In some embodiments, the at least one oligosaccharide excipient has a number average molecular weight of at least about 1250 and a mass average molecular weight of at least about 9000. In one embodiment, the aqueous formulation further comprises at least one buffer.

0107As used herein, the terms "cephalosporin C deacetylase" and "cephalosporin C acetylhydrolase" catalyze the deacetylation of cephalosporins such as cephalosporin C and 7-aminocephalosporin acid. It means an enzyme (EC 3.1.1.41) (Mitsushima et al., (1995) Appl.Env. Microbiol.61 (6): 2224-2229)). Several cephalospoly C deacetylases with significant perhydrolysis activity are provided herein.

0108As used herein, "acetylxylan esterase" means an enzyme (EC 3.1.1.72; AX) that catalyzes the deacetylation of acetylated xylan and other acetylated saccharides. As illustrated herein, several enzymes classified as acetylxylan esterases are provided that have significant perhydrolysis activity.

0109As used herein, the term Bacillus subtilis ATCC® 31954^TMIs the International Deposit Registration Number ATCC® 31954^TMBacterial cells deposited in the American Type Culture Collection (ATCC) with. Bacillus subtilis ATCC® 31954^TMHas been reported to have ester hydrolase ("diasetinase") activity capable of hydrolyzing glycerol esters with 2-8 carbon acyl groups (incorporated herein by reference in its entirety, USA). Patent No. 4,444,886). As described herein, enzymes with significant perhydrolase activity are Bacillus subtilis (B. subtilis) ATCC® 31954.^TMIsolated from, provided as SEQ ID NO: 1. The amino acid sequence of the isolating enzyme has 100% amino acid identity with the cephalosporin C deacetylase provided by GENBANK® Accession No. BAA01729.1 (Mitsushima et al., See above).

0110As used herein, the term Bacillus subtilis ATCC® 29233^TMIs the International Deposit Registration Number ATCC® 29233^TMIt means a strain of Bacillus subtilis deposited in the American Type Culture Collection (ATCC). As described herein, enzymes with significant perhydrolase activity are Bacillus subtilis (B. subtilis) ATCC® 29233.^TMIsolated from, sequenced and provided as SEQ ID NO: 12.

0111As used herein, the term "Clostridium thermocellum ATCC® 27405"^TMIs the International Deposit Registration Number ATCC® 27405^TMAmerican Type with It means a strain of Clostridium thermocellum deposited in the Culture Collection (ATCC). C. thermocellum ATCC® 27405^TMThe amino acid sequence of the enzyme having perhydrolase activity from is provided as SEQ ID NO: 5.

0112As used herein, the term Bacillus subtilis ATCC® 6633^TMIs the International Deposit Registration Number ATCC® 6633^TMBacterial cells deposited in the American Type Culture Collection (ATCC) with. Bacillus subtilis ATCC® 6633^TMIs reported to have cephalosporin acetylhydrolase activity (US Pat. No. 6,465,233). Bacillus subtilis (B. subtilis) ATCC® 6633^TMThe amino acid sequence of the enzyme having perhydrolase activity from is provided as SEQ ID NO: 2.

0113As used herein, the term "Bacillus licheniformis ATCC® 14580"^TMIs the International Deposit Registration Number ATCC® 14580^TMBacterial cells deposited in the American Type Culture Collection (ATCC) with. Bacillus licheniformis ATCC® 14580^TMHas been reported to have cephalosporin acetylhydrolase activity. B. licheniformis ATCC® 14580^TMThe amino acid sequence of the enzyme having perhydrolase activity from is provided as SEQ ID NO: 3.

0114As used herein, the term "Bacillus pumilus PS213" means bacterial cells that have been reported to have acetylxylan esterase activity (GENBANK® AJ249957). The amino acid sequence of the enzyme with perhydrolase activity from Bacillus pumilus PS213 is provided as SEQ ID NO: 4.

0115As used herein, the term "Thermotoga neapolitana" means a strain of Thermotoga neapolitana reported to have acetylxylan esterase activity (GENBANK® AAB70869). ). The amino acid sequence of the enzyme with perhydrolase activity from Thermotoga neapolitana is provided as SEQ ID NO: 6.

0116As used herein, the term "Thermotoga maritima MSB8" means bacterial cells that have been reported to have acetylxylan esterase activity (GENBANK® NP_227893.1). The amino acid sequence of the enzyme with perhydrolase activity from Thermotoga maritima MSB8 is provided as SEQ ID NO: 7.

0117As used herein, "Bacillus clausii KSM-K16" means a bacterial cell reported to have cephalosporin C deacetylase activity (GENBANK® YP_175265). The amino acid sequence of the enzyme with perhydrolase activity from Bacillus clausii KSM-K16 is provided as SEQ ID NO: 11.

0118As used herein, "Thermoanearobacterium saccharolyticum means a bacterial strain reported to have acetylxylan esterase activity (GENBANK® S41858). The amino acid sequence of the enzyme with perhydrolase activity from Thermoanearobacterium saccharolyticum is provided as SEQ ID NO: 13.

0119As used herein, the term "Thermotoga lettingae" means a bacterial cell that has been reported to have acetylxylan esterase activity (GENBANK® CP000812). The putative amino acid sequence of the enzyme with perhydrolase activity from Thermotoga lettingae is provided as SEQ ID NO: 14.

0120As used herein, the term "Thermotoga petrophila" means a bacterial cell that has been reported to have acetylxylan esterase activity (GENBANK® CP000702). The putative amino acid sequence of the enzyme with perhydrolase activity from Thermotoga lettingae is provided as SEQ ID NO: 15.

0121As used herein, the term "Thermotoga sp. RQ2" means bacterial cells that have been reported to have acetylxylan esterase activity (GENBANK® CP000969). Two different acetylxylan esterases have been identified from the Thermotoga sp. RQ2, "RQ2 (a)" (estimated amino acid sequence provided as SEQ ID NO: 16) and "RQ2 (b)" herein. (Estimated amino acid sequence provided as SEQ ID NO: 17).

0122As used herein, "isolated nucleic acid molecule" and "isolated nucleic acid fragment" may be used interchangeably and are single-stranded or double-stranded, optionally synthesized, unnatural. Alternatively, it means a polymer of RNA or DNA which is a modified nucleotide base. An isolated nucleic acid molecule in the form of a polymer of DNA may consist of one or more segments of cDNA, genomic DNA or synthetic DNA.

0123The term "amino acid" means the basic chemical structural unit of a protein or polypeptide. The following abbreviations are used herein to identify unique amino acids.

0124<tables num="1"><img id="000004" he="145" wi="137" file="JP5777516B2_D0001.tif" img-format="tif" img-content="drawing" /></tables>

0125As used herein, "substantially similar" means that a change in one or more nucleotide bases results in the addition, substitution, or deletion of one or more amino acids, but in a protein encoded by a DNA sequence. For nucleic acid molecules that do not affect functional properties (ie, perhydrolysis activity). As used herein, "substantially similar" also means that the resulting enzyme retains this functional property (ie, perhydrolysis activity), at least 30%, preferably at least 33%, more preferably. At least 40%, more preferably at least 50%, even more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, and even more preferably at least 90%, 91%, 92%. , 93%, 94%, 95%, 96%, 97%, 98%, or 99% relate to enzymes having amino acid sequences that are identical to the sequences reported herein. A "substantially similar" may also relate to an enzyme having perhydrolysis activity encoded by a nucleic acid molecule that hybridizes to the nucleic acid molecule reported herein under severe conditions. Therefore, the present invention is understood to include more than the unique exemplary sequences.

0126For example, it is well known in the art that genetic alterations that result in the production of chemically equivalent amino acids at a given site but do not affect the functional properties of the encoded protein are common. .. For the purposes of the present invention, substitution is defined as an exchange within one of the following five groups: 1. Small aliphatic, non-polar or slightly polar residues: Ala, Ser, Thr (Pro, Gly); 2. Polar, negatively charged residues and their amides: Asp, Asn, Glu, Gln; 3. Polar, positively charged residues: His, Arg, Lys; 4. Large aliphatic, non-polar residues: Met, Leu, Ile, Val (Cys); and 5. Large aromatic residues: Phe, Tyr, and Trp.

0127Thus, the amino acid alanine, a codon for hydrophobic amino acids, is a codon that encodes another less hydrophobic residue (such as glycine) or more hydrophobic residue (such as valine, leucine, or isoleucine). May be replaced with. Similarly, the substitution of one negatively charged residue in place of another (such as aspartic acid in place of glutamic acid) or the substitution of one positively charged residue in place of another (instead of arginine). Changes that result in (such as lysine) can also be expected to produce functionally equivalent products. In many cases, nucleotide changes that result in alterations in the N-terminal and C-terminal moieties of a protein molecule would also not be expected to alter the activity of the protein.

0128Each of the proposed denaturations is well within a given skill in the art, as is the measurement of retention of biological activity of the encoded product. Moreover, one of ordinary skill in the art will appreciate that substantially similar sequences are included by the present invention. In one embodiment, substantially similar sequences are washed under harsh conditions (0.1XSSC, 0.1% SDS, 65 ° C and 2XSSC, 0.1% SDS, followed by 0.1XSSC, 0.1% SDS, 65 ° C). , Defined by their ability to hybridize with the sequences exemplified herein.

0129As used herein, a nucleic acid molecule, such as cDNA, genomic DNA, or RNA, when the single strand of the first molecule can be annealed to another molecule under the appropriate conditions of temperature and solution ion intensity. Can be "hybridized" into another nucleic acid molecule. Hybridization and cleaning conditions are well known, Sambrook, J. and Russell, DT, Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, Cold Spring Illustrated in Harbor (2001). Temperature and ionic strength conditions determine the "severity" of hybridization. Severity conditions select moderately similar molecules, such as homologous sequences from distantly related organisms, into highly similar molecules, such as genes that replicate functional enzymes from closely related organisms. Can be adjusted to. Post-hybrid cleaning typically determines rigorous conditions. A set of preferred conditions starts with 6XSSC, 0.5% SDS for 15 minutes at room temperature, then repeats at 2XSSC, 0.5% SDS for 30 minutes at 45 ° C, then 0.2 for 30 minutes at 50 ° C. Use a series of washes repeated twice with XSSC, 0.5% SDS. A more preferred set of conditions is to use a higher temperature at which the wash is identical to the above, except that the temperature of the last two 30-minute washes at 0.2XSSC, 0.5 SDS% is raised to 60 ° C. .. Another preferred set of stringent hybridization conditions is 0.1XSSC, 0.1% SDS, 65 ° C for the sequences exemplified herein, 2XSSC, 0.1% SDS, followed by 0.1XSSC, 0.1% SDS, 65 ° C. It is washed in the final wash of. In a further embodiment, the compositions and methods are enzymes having perhydrolase activity encoded by an isolated nucleic acid molecule that hybridizes to a nucleic acid molecule encoding a polypeptide having perhydrolysis activity under harsh conditions. The polypeptides are SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 , SEQ ID NO: 13; SEQ ID NO: 14; SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, and An enzyme having an amino acid sequence selected from the group consisting of SEQ ID NO: 25 is used.

0130Hybridization, depending on the severity of the hybridization, requires the two nucleic acids to contain complementary sequences and allows for base-to-base mismatches. Appropriate rigor for hybridization of nucleic acids depends on the length and degree of complementarity of the nucleic acids, variables well known in the art. The greater the degree of similarity or homology between two nucleotide sequences, the greater the value of Tm for a hybrid of nucleic acids having those sequences. The relative stability of nucleic acid hybridization (corresponding to higher Tm) decreases in the following order: RNA: RNA, DNA: RNA, DNA: DNA. For hybrids of nucleotides over 100 lengths, an equation was derived to calculate Tm (Sambrook and Russell, see above). For shorter nucleic acids, ie, hybridization with oligonucleotides, the location of the mismatch becomes more important and the length of the oligonucleotide determines its specificity (Sambrook and Russell, see above). In one aspect, the length for a hybridizable nucleic acid is at least about 10 nucleotides. Preferably, the minimum length for a hybridizable nucleic acid is at least about 15 nucleotides in length, more preferably at least about 20 nucleotides in length, even more preferably at least 30 nucleotides in length, even more preferably length. It is at least 300 nucleotides, most preferably at least 800 nucleotides in length. In addition, one of ordinary skill in the art will recognize that temperature and wash solution salt concentration may be adjusted as needed according to factors such as probe length.

0131As used herein, the term "percent identity" is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as measured by comparing sequences. In the art, "identity" also means, in some cases, the degree of sequence association between polypeptide or polynucleotide sequences, as measured by matching between strings of such sequences. "Identity" and "similarity" are referred to as Computational Molecular Biology (Lesk, AM, ed.) Oxford University Press, NY (1988); Biocomputing: Informatics and Genome Projects (Smith, DW, ed.) Academic Press, NY ( 1993); Computer Analysis of Sequence Data, Part I (Griffin, AM, and Griffin, HG, eds.) Humana Press, NJ (1994); Sequence Analysis in Molecular Biology (von Heinje, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Stockton Press, NY (1991) Can be easily calculated by known methods, including but not limited to. Methods for measuring identity and similarity are systematized in publicly available computer programs. Sequence alignment and percent identity calculations are available at LASERGENE Bioinformatics Computer Computational Suit Megalign Program (DNASTAR Inc., Madison, WI), the AlignX program of Vector NTI v.7.0 (Informax, Inc., Bethesda, MD), or EMBOSS Open Software Suite (EMBL-EBI; Rice et al., Trends in Genetics 16, (6): 276-277 (2000)) may be used. Multiple alignment of sequences is available from the European Molecular Biology Laboratory via the European Bioinformatics Institute with default parameters, the CLUSTAL method of alignment (eg CLUSTAL W; eg version 1.83) (Higgins and Sharp, CABIOS, 5: 151-). 153 (1989); Higgins et al., Nucleic Acids Res. 22: 4673-4680 (1994); and Chenna et al., Nucleic Acids Res. 31 (13): 3497-500 (2003)) Can be done. Suitable parameters for CLUSTALW protein alignment are GAP Includes Existence penalty = 15, GAP extension = 0.2, matrix = Gonnet (eg Gonnet250), protein ENDGAP = -1, protein GAPDIST = 4, and KTUPLE = 1. In one embodiment, fast or slow alignment is used with default settings, where slow alignment is preferred. Alternatively, parameters using the CLUSTALW method (version 1.83) also use KTUPLE = 1, GAP PENALTY = 10, GAP extension = 1, matrix = BLOSUM (eg BLOSUM64), WINDOW = 5, and TOP DIAGONALS SAVED = 5. May be modified for

0132In one aspect, a suitable isolated nucleic acid molecule is at least about 30%, preferably at least 33%, preferably at least 40%, preferably at least 50%, preferably at least 60% of the amino acid sequence reported herein. , More preferably at least 70%, more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 Encode a polypeptide having an amino acid sequence that is%, 98%, or 99% identical. Suitable nucleic acid molecules are typically polypeptides having not only the above homology, but also about 300 to about 340 amino acids, more preferably about 310 to about 330 amino acids, most preferably about 318 amino acids. To encode.

0133As used herein, the terms "signature motif", "CE-7 signature motif", and "diagnostic motif" mean a conservative structure shared among a family of enzymes with defined activity. Signature motifs can be used to identify and / or identify a family of structurally related enzymes that have similar enzymatic activity to a defined family of substrates. A signature motif can be a single flanking amino acid sequence or a collection of non-contact conserved motifs that together form a signature motif. Typically, the conserved motif is represented by an amino acid sequence. As described herein, the enzyme having perhydrolase activity (perhydrolase) belongs to the CE-7 hydrocarbon esterase family (DiCosimo et al., Supra). As used herein, the phrase "enzyme is structurally classified as a CE-7 enzyme", or "CE-perhydrolase" is structurally classified as a CE-7 carbohydrate esterase, an enzyme with perhydrolesis activity. Will be used to mean. Enzymes of this group can be defined by the presence of signature motifs (see Vincent et al., Above). As defined herein, the signature motif for CE-7 esterase contains three conserved motifs (residue position numbers associated with reference sequence SEQ ID NO: 1): a) Arg118-Gly119-Gln120; b) Gly179-Xaa180-Ser181-Gln182-Gly183; and c) His298-Glu299.

0134Typically, Xaa at amino acid residue position 180 is glycine, alanine, proline, tryptophan, or threonine. Two of the three amino acid residues belonging to the catalyst triplet are fleshy. In one embodiment, Xaa at amino acid residue position 180 is selected from the group consisting of glycine, alanine, proline, tryptophan, and threonine.

0135Further analysis of conservative motifs within the CE-7 carbohydrate esterase family may be used to further define perhydrolases belonging to the CE-7 carbohydrate esterase family of additional conservative motifs (amino acid positions 267-269 of SEQ ID NO: 1). Suggests the existence of LXD) in. In a further embodiment, the signature motif defined above Leu267-Xaa268-Asp269 Contains a fourth preserved motif defined as.

0136Xaa at amino acid residue position 268 is typically isoleucine, valine, or methionine. The fourth motif includes aspartic acid residues (thick) belonging to the catalyst triplet (Ser181-Asp269-His298).

0137Many well-known global alignment algorithms may be used to align two or more amino acid sequences representing an enzyme with perhydrolase activity to determine if the enzyme consists of this signature motif. The aligned sequence is compared with the reference sequence (SEQ ID NO: 1) to determine the presence of this signature motif. In one embodiment, the reference amino acid sequence, Bacillus subtilis ATCC® 31954 as used herein.^TMCLUSTAL alignments (such as CLUSTALW) using the perhydrolase sequence from (SEQ ID NO: 1)) are used to identify perhydrolases belonging to the CE-7 esterase family. The relative numbering of conserved amino acid residues is based on the residue numbering of the reference amino acid sequence to account for small insertions or deletions (eg, 5 amino acids or less) within the aligned sequence. There is.

0138Examples of other suitable algorithms that may be used to identify sequences containing this signature motif (when compared to reference sequences) include Needleman and Wunsch (J. Mol. Biol. 48, 443-). 453 (1970); Global Alignment Tool) and Smith-Waterman (J.Mol.Biol. 147: 195-197 (1981); Local Alignment Tool), but not limited to them. In one embodiment, Smith-Waterman alignment is performed using default parameters. Examples of suitable default parameters include the use of the BLOSUM62 scoring matrix with a GAP open penalty of 10 and a GAP extension penalty of 0.5.

0139A comparison of the overall percent identity between the perhydrolases exemplified herein shows that enzymes with as little as 33% identity with SEQ ID NO: 1 (while retaining this signature motif) have significant perhydrolase activity. It is shown that it is structurally classified as CE-7 carbohydrate esterase. In one embodiment, suitable perhydrolases include CE-7 signature motif and SEQ ID NO: 1 and at least 30%, preferably at least 33%, more preferably at least 40%, even more preferably at least 42%, even more preferably. Is at least 50%, even more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90%, most preferably at least 90%, 91%, 92%, Includes enzymes containing 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity.

0140Alternatively, adjacent amino acid sequences containing regions containing this conserved motif may also be used to identify Group 7 CE-7 members.

0141As used herein, "codon degeneracy" relates to the nature of the genetic code that allows nucleotide sequence variation without affecting the amino acid sequence of the encoded polypeptide. Accordingly, the present invention relates to any nucleic acid molecule that encodes all or a significant portion of the amino acid sequence that encodes the microbial polypeptide. Those skilled in the art are familiar with the "codon bias" exhibited by host cells specific in the use of nucleotide codons to identify a given amino acid. Therefore, when synthesizing a gene for enhanced expression in a host cell, it is desirable to design the gene so that its frequency of codon utilization approaches the preferred frequency of codon utilization in the host cell.

0142As used herein, the term "optimized codon" refers to the gene or coding region of a nucleic acid molecule for the transformation of various hosts, without altering the polypeptide encoded by the DNA. It means a change of codons in a gene or coding region of a nucleic acid molecule to reflect the typical codon utilization of a host organism.

0143As used herein, a "synthetic gene" can be assembled from oligonucleotide components that are chemically synthesized using procedures known to those of skill in the art. These components are combined and annealed to form the gene segment, which is then enzymatically assembled to build the entire gene. As with respect to DNA sequences, "chemically synthesized" means that the component nucleotides were assembled in vitro. Manual chemical synthesis of DNA may be accomplished using well-established procedures, or automated chemical synthesis can be performed using one of many commercially available machines. Thus, genes can be tuned for optimal gene expression based on nucleotide sequence optimization to reflect host cell codon bias. One of skill in the art fully understands the potential for successful gene expression when codon utilization is biased towards those codons that are favored by the host. Determining the preferred codon can be based on a study of genes derived from the host cell for which sequence information is available.

0144As used herein, a "gene" expresses a specific protein, including a regulatory sequence preceding the coding sequence (5'non-coding sequence) and a regulatory sequence after the coding sequence (3'non-coding sequence). Means a nucleic acid molecule. "Wild gene" means a gene that is found in nature in its own regulatory sequence. "Chimeric gene" means any gene that is not a wild gene, including regulatory and coding sequences that are not found together in nature. Thus, chimeric genes may contain regulatory and coding sequences from different sources, or regulatory and coding sequences from the same source but arranged in a manner different from those found in nature. "Intrinsic gene" means a wild gene at its natural position in the genome of an organism. A "foreign" gene is a gene that is not normally found in a host organism but is introduced into the host organism by gene transfer. The foreign gene can include a wild gene inserted into a foreign species organism or a chimeric gene. A "trans gene" is a gene introduced into the genome by a transformation method.

0145As used herein, "coding sequence" means a DNA sequence that encodes for a unique amino acid sequence. A "suitable regulatory sequence" is located upstream (5'-non-coding sequence), within, or downstream (3'-non-coding sequence) of the coding sequence, and transcription, RNA processing or stability, or associated coding. Means a nucleotide sequence that affects the translation of a sequence. Regulatory sequences may include promoters, translation leader sequences, RNA processing sites, effector binding sites and stem-loop structures.

0146As used herein, "promoter" means a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. Generally, the coding sequence is located at the 3'position on the promoter sequence. The promoter may be entirely derived from a wild gene, or may consist of different elements derived from different promoters found in nature, or may further comprise a synthetic DNA segment. It will be appreciated by those skilled in the art that different promoters may direct gene expression at different stages of growth or in response to different environmental or physiological conditions. Promoters that express genes most of the time are commonly referred to as "constitutive promoters." In most cases the exact boundaries of the regulatory sequences were not completely demarcated, further recognizing that DNA fragments of different lengths may have the same promoter activity.

0147As used herein, "3'non-coding sequence" means a DNA sequence located downstream of the coding sequence, a polyadenylation recognition sequence (usually limited to eukaryotes) and mRNA processing or Includes other sequences that encode regulatory signals that can affect gene expression. The polyadenylation signal is usually characterized by affecting the addition of polyadenylate tracts (usually limited to eukaryotes) to the 3'end of the pre-mRNA.

0148As used herein, the term "manipulatively linked" means the linking of nucleic acid sequences on a single nucleic acid molecule such that one function is influenced by the other. For example, a promoter binds to a coding sequence operably when it can affect the expression of that coding sequence, i.e., when the coding sequence is under transcriptional control of the promoter. The coding sequences can be combined so that they can be manipulated into regulatory sequences in the sense or antisense direction.

0149As used herein, the term "expression" means transcription and stable accumulation of sense RNA (mRNA) or antisense RNA derived from the nucleic acid molecules of the invention. Expression may also mean translation of the mRNA into a polypeptide.

0150As used herein, "transformation" means the introduction of a nucleic acid molecule into the genome of a host organism that results in a genetically stable genetic trait. In the present invention, the genome of a host cell comprises chromosomal and extrachromosomal (eg, plasmid) genes. A host organism containing a transformed nucleic acid molecule is referred to as a "transgenic" or "recombinant" or "transformed" organism.

0151As used herein, "plasmids," "vectors," and "cassettes" are chromosomes that often carry genes, usually in the form of circular double-stranded DNA molecules, rather than parts of the cell's central metabolism. Means an external element. Such elements are single-stranded or double-stranded, derived from any source, with self-replicating sequences, genomic integration sequences, and numerous nucleotide sequences bound or recombined into a unique structure into which a promoter fragment can be introduced. It may be a DNA sequence for a gene product selected along with a linear or circular, phage or nucleotide sequence of DNA or RNA as well as a suitable 3'untranslated sequence to the cell. "Transformation cassette" means a unique vector containing a foreign gene and having an element that facilitates transformation of a specific host cell in addition to the foreign gene. "Expression cassette" means a unique vector containing a foreign gene and having, in addition to the foreign gene, an element that allows enhanced expression of the gene in a foreign host.

0152As used herein, the term "sequence analysis software" means any computer algorithm or software program that is useful for the analysis of nucleotide or amino acid sequences. The "sequence analysis software" may be commercially available or developed independently. Typical sequence analysis software includes GCG suit programs (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, WI), BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol. Biol. 215: 403-410 (1990)), and DNASTAR (DNASTAR, Inc. 1228 S. Park St. Madison, WI 53715 USA), CLUSTAL W (eg, version 1.83; Thompson et al., Nucleic Acids). Research, 22 (22): 4673-4680 (1994)), and FASTA programs incorporating the Smith-Waterman algorithm (WR Pearson, Comput.Methods Genome Res., [Proc.Int.Symp.] (1994), Meeting Date 1992. , 111-20.Editor (s): Suhai, Sandor.Publisher: Plenum, New York, NY), Vector NTI (Informax, Bethesda, MD) and Sequencher v.4.05 will be included, but not limited to them. .. In the context of this application, when sequence analysis software is used for analysis, it is understood that the results of the analysis are based on the "default values" of the reference program, unless otherwise stated. Let's go. As used herein, "default value" will mean any set of values or parameter sets by the software manufacturer that originally loads the software when it is first initialized.

0153As used herein, the term "biological pollutant" includes, but is not limited to, microorganisms, spores, viruses, prions, and mixtures thereof. Means the biological entity of. The method produces at least one percarboxylic acid in an effective concentration that is useful for reducing and / or eliminating the presence of viable biological contaminants. In a preferred embodiment, the biological contaminant is a viable pathogenic microorganism.

0154As used herein, the term "disinfect" means the process of destroying or preventing the growth of biological contaminants. As used herein, the term "disinfectant" means a reagent that disinfects by destroying, neutralizing, or suppressing the growth of biological contaminants. Typically, disinfectants are used to treat inanimate objects or surfaces. As used herein, the term "disinfection" means the act or process of disinfection. As used herein, the term "preservative" means a chemical agent that suppresses the growth of disease-carrying microorganisms. In one aspect, the biological contaminant is a pathogenic microorganism.

0155As used herein, the term "hygiene" means the restoration or maintenance of health, typically by removing, preventing or controlling agents that may be harmful to health. Related to them. As used herein, the term "sterilize" means hygienic. As used herein, the term "bactericidal agent" means a bactericidal reagent. As used herein, the term "sterilization" means the act or process of sterilization.

0156As used herein, the term "virucide" means a reagent that suppresses or destroys a virus and has the same meaning as "viricide." A reagent that exhibits the ability to suppress or destroy a virus is described as having "virus-killing" activity. Peracids can have viral killing activity. Typical alternative virus-killing agents known in the art that may be suitable for use in the present invention include, for example, alcohols, ethers, chloroform, formaldehyde, phenols, beta-propiolactone, iodine, etc. Includes chlorine, mercury salts, hydroxylamine, ethylene oxide, ethylene glycol, quaternary ammonium compounds, enzymes, and detergents.

0157As used herein, the term "biocide" means a chemical agent that inactivates or destroys a microorganism, typically a broad spectrum. Chemical agents that exhibit the ability to inactivate or destroy microorganisms are described as having "biological" activity. Peracids can have biokilling activity. Typical alternative biocides known in the art that may be suitable for use in the present invention include, for example, chlorine, chlorine dioxide, chloroisocyanurates, hypochlorites, ozone, etc. Includes achlorine, amines, chlorinated phenolic compounds, copper salts, organosulfur compounds, and quaternary ammonium salts.

0158As used herein, the phrase "minimum killing concentration" means the lowest concentration of killing agent that will result in an irreversible reduction in the desired lethality of the surviving population of the target microorganism at a particular contact time. To do. Efficacy is log of surviving microorganisms after treatment₁₀It can be measured by reduction. In one aspect, the target reduction of viable microorganisms after treatment is at least 3-log reduction, more preferably at least 4-log reduction, and most preferably at least 5-log reduction. In another aspect, the lowest killing concentration is at least a 6-log reduction in viable microbial cells.

0159As used herein, the terms "peroxy source" and "source of peroxy" are hydrogen peroxide, hydrogen peroxide adduct (eg, urea-hydrogen peroxide adduct (carbamide peroxide)), perborate. It means a compound capable of providing hydrogen peroxide at a concentration of about 1 mM or more in an aqueous solution containing, but not limited to, a borate, and a peroxide. As described herein, the concentration of hydrogen peroxide provided by the peroxygen compound in the aqueous reaction formulation is initially at least 1 mM or more when the reaction components are combined. In one embodiment, the hydrogen peroxide concentration in the aqueous reaction formulation is at least 10 mM. In another embodiment, the hydrogen peroxide concentration in the aqueous reaction formulation is at least 100 mM. In another embodiment, the hydrogen peroxide concentration in the aqueous reaction formulation is at least 200 mM. In another embodiment, the hydrogen peroxide concentration in the aqueous reaction formulation is greater than or equal to 500 mM. In yet another embodiment, the hydrogen peroxide concentration in the aqueous reaction formulation is 1000 mM or higher. Hydrogen peroxide vs. enzyme substrates in aqueous reaction formulations, such as triglycerides (H)₂O₂The molar ratio of: substrate) may be about 0.002 to 20, preferably about 0.1 to 10, and most preferably about 0.5 to 5.

0160"Ori The Gore-sugar" refers to a compound containing from 2 to at least 24 monosaccharide units have been linked by glycosidic bonds. The term "monosaccharide" is an empirical formula (CH)₂O)_nIt means a compound (in the formula, n 3), the carbon skeleton is non-branched, each carbon atom contains a hydroxyl group except one, and the remaining carbon atoms are aldehydes or aldehydes with 2 carbon atoms. It is a ketone. The term "monosaccharide" also means an intracellular cyclic hemiacetal or hemicetal form.

0161As used herein, the term "excipient" means an inert substance used to stabilize the active ingredient in a formulation. Excipients are also sometimes used to bulken formulations containing active ingredients. As described herein, an "active ingredient" is an enzyme catalyst comprising at least one enzyme having perhydrolysis activity. In one embodiment, the active ingredient is at least one CE-7 carbohydrate esterase with perhydrolysis activity.

0162As used herein, the term "oligosaccharide shaping" improves the recovery / retention of active enzyme (ie, perhydrolase activity) after spray drying when added to an aqueous enzyme solution and / Or means an oligosaccharide that improves the storage stability of the resulting spray-dried enzyme powder or formulation of the enzyme powder with a carboxylic acid ester. In one embodiment, the addition of oligosaccharide excipients prior to spray drying improves the storage stability of the enzyme when stored in a carboxylic acid ester (ie, a storage mixture that is substantially free of water). Carboxylic acid esters may contain very low concentrations of water, for example triacetin typically has 180 ppm to 300 ppm water. As used herein, the phrase "substantially water-free" is used in a mixture of enzyme powder and carboxylic acid ester that does not adversely affect the storage stability of the enzyme powder when present in the carboxylic acid ester. It would mean the concentration of water. In a further embodiment, "substantially water-free" means less than 2000 ppm, preferably less than 1000 ppm, more preferably less than 500 ppm, even more preferably less than 250 ppm of water in the formulation comprising the enzyme powder and the carboxylic acid ester. May mean.

0163Enzyme powder One embodiment is structurally classified as a CE-7 enzyme and comprises at least one enzyme having perhydrolysis activity, at least one oligosaccharide excipient, and optionally a spray formulation of at least one surfactant. About powder. In some embodiments, the at least one oligosaccharide excipient has a number average molecular weight of at least about 1250 and a mass average molecular weight of at least about 9000.

0164At least one enzyme can be any of the CE-7 carbohydrate esterases described herein, or is co-owned, co-pending U.S. Patent Application Publication No. 2008/0176299 and the same. It can be any of the CE-7 carbohydrate esterases described in No. 2009/0005590, each of which is incorporated herein by reference in its entirety. In some embodiments, at least one enzyme is SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 19 , 20, 21, 22, 23, 24, and 25.

0165At least one enzyme is present in the spray-dried formulation in an amount ranging from about 5% by weight to about 75% by weight based on the dry mass of the spray-dried formulation. The preferred mass% range of the enzyme in the spray-dried formulation is from about 10% to 50% by weight, and the more preferred mass% range of the enzyme in the spray-dried formulation is from about 20% to 33% by weight. ..

0166The spray-dried formulation further comprises at least one oligosaccharide excipient. In some embodiments, the at least one oligosaccharide excipient has a number average molecular weight of at least about 1250 and a mass average molecular weight of at least about 9000. In some embodiments, the at least one oligosaccharide excipient has a number average molecular weight of at least about 1700 and a mass average molecular weight of at least about 15,000. Specific oligosaccharides useful in the present invention include maltodextrin, xylan, mannan, fucoidan, galactomannan, chitosan, raffinose, stachyose, pectin, inulin, levan, graminan, amylopectin, saccharose, lactose, lactose, maltose, trehalose. , Cerobiose, Nigerotriose, Maltotriose, Melezitose, Malttrehalose, Raffinose, Kestose, and mixtures thereof, including, but not limited to. Oligosaccharide-based excipients useful in the present invention include, but are not limited to, water-soluble nonionic cellulose ethers such as, but not limited to, hydroxymethyl cellulose and hydroxypropyl methyl cellulose, and mixtures thereof.

0167Excipients are present in the formulation in an amount ranging from about 95% to about 25% by weight based on the dry mass of the spray-dried formulation. The preferred mass% range of the excipient in the spray-dried formulation is from about 90% by weight to about 50% by weight, and the more preferred mass% range of the excipient in the spray-dried formulation is from about 80% by weight. It is about 67% by mass.

0168In some embodiments, the formulation further comprises at least one surfactant. Useful surfactants include ethoxylated castor oil, polyglycolated glycerides, acetylated monoglycerides, sorbitan fatty acid esters, poroxamers, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene derivatives, their monoglycerides or ethoxylated derivatives, Ionic and non-ionic and non-ionic, such as diglycerides or polyoxyethylene derivatives, sodium docusate, sodium lauryl sulfate, cholic acid or derivatives thereof, lecithin, phospholipids, block copolymers of ethylene glycol and propylene glycol, and nonionic organosilicone. Includes, but is not limited to, ionic surfactants or wetting agents. Preferably, the surfactant is polyoxyethylene sorbitan fatty ester, with polysorbate 80 being more preferred.

0169When part of the formulation, the surfactant is about 5% to 0.1% by weight, preferably the mass of protein present in the spray-dried formulation, relative to the mass of protein present in the spray-dried formulation. As a reference, it exists in an amount in the range of about 2% by mass to 0.5% by mass.

0170Spray-dry formulations include one or more buffers (eg, bicarbonate, citric acid, acetic acid, phosphoric acid, pyrophosphate, methylphosphonic acid, succinic acid, malic acid, fumaric acid, tartrate acid, or sodium maleate and / / Alternatively, it may further contain an enzyme stabilizer (such as ethylenediamine tetraacetic acid, (1-hydroxyethylidene) bisphosphonic acid).

0171Spray drying of a formulation of at least one enzyme, at least one oligosaccharide oligosaccharide excipient and optionally at least one surfactant can be described, for example, in the Spray Drying Handbook, 5^th PCT Patent Publication International Publication No. 97/41833 Pamphlet (1997) and No. ed., K. Masters, John Wiley &amp; Sons, Inc., NY, NY (1991) and Platz, R. et al. Implemented as generally described in Pamphlet 96/32149 (1996).

0172In general, spray drying consists of combining a highly dispersed liquid with a volume of hot air sufficient to allow evaporation and drying of the liquid droplets. Typically, the feed is sprayed into a stream of warm filtered air that evaporates the solvent and transports the drying product to the collector. The used air is then discharged with the solvent. Those skilled in the art will fully understand that several different types of equipment may be used to provide the desired product. For example, Buchi Ltd. (Postfach, Switzerland) or GEA Niro Commercially available spray dryers manufactured by Corp. (Copenhagen, Denmark) will effectively produce particles of the desired size. It is further understood that these spray dryers, and in particular their sprayers, may be modified or custom made for special applications, such as simultaneous spraying of two solutions using the double nozzle technique. There will be. More specifically, the water-in-oil emulsion can be sprayed from one nozzle, and the solution containing a non-adhesive such as mannitol can be co-sprayed from the second nozzle. In other cases, it may be desirable to push the feed solution through a custom designed nozzle that uses a high performance liquid chromatography (HPLC) pump. The choice of device is not critical, provided that microstructures and / or compositions containing the correct morphology are produced, and will be apparent to those skilled in the art in light of the teachings herein. Let's do it.

0173The temperature of both the inlet and outlet of the gas used to dry the spray material is such that it does not cause the decomposition of enzymes in the spray material. Such temperatures generally range from about 50 ° C to about 225 ° C for inlet temperatures, while ranges from about 30 ° C to about 150 ° C for outlet temperatures, but are typically determined experimentally. Will be done. Preferred parameters include spray pressures ranging from about 20 to 150 psi (0.14 MPa to 1.03 MPa), preferably from about 30 to 40 to 100 psi (0.21 to 0.28 MPa to 0.69 MPa). Typically, the spray pressure used will be one of the following (MPa) 0.14, 0.21, 0.28, 0.34, 0.41, 0.48, 0.55, 0.62, 0.69, 0.76, 0.83 and above.

0174Formulations of spray-dried enzyme powders or spray-dried enzyme powders in carboxylic acid esters substantially retain their enzymatic activity for extended periods of time when stored at ambient temperature. Formulations of spray-dried enzyme powders or spray-dried enzyme powders in carboxylic acid esters substantially retain their enzymatic activity at elevated temperatures for short periods of time. In one embodiment, "substantially retaining its enzyme activity" is a spray-dried enzyme powder or carboxylic acid as compared to the initial enzyme activity of the enzyme powder prior to preparation of the formulation consisting of the carboxylic acid ester and the enzyme powder. A formulation of spray-dried enzyme powder in an ester is stored in a formulation consisting of a carboxylic acid ester and enzyme powder after an extended storage period at ambient temperature and / or at an increased temperature (above ambient temperature) for a short period of time. It means retaining at least about 75% of the enzyme activity of the enzyme in the enzyme powder or formulation of the enzyme powder after a period of time. The extended storage period is about 1 to about 2 years at ambient temperature. In one embodiment, the short storage period is from the time the formulation consisting of the carboxylic acid ester and the enzyme powder is produced at 40 ° C to about 8 weeks at 40 ° C at elevated temperatures. .. In another embodiment, the elevated temperature is in the range of about 30 ° C to about 52 ° C. In a preferred embodiment, the elevated temperature is in the range of about 30 ° C to about 40 ° C.

0175In some embodiments, the spray-dried enzyme powder is from the carboxylic acid ester and the enzyme powder as compared to the initial enzymatic activity of the enzyme powder before the preparation of the formulation consisting of the carboxylic acid ester and the enzyme powder at 40 ° C. Has at least 75% of the enzymatic activity of at least one enzyme after 8 weeks of storage at 40 ° C. In another embodiment, the enzyme powder is a formulation consisting of a carboxylic acid ester and an enzyme powder as compared to the initial enzymatic activity of the enzyme powder prior to the preparation of the formulation consisting of the carboxylic acid ester and the enzyme powder at 40 ° C. At least 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95 after 8 weeks of storage at 40 ° C. , 96, 97, 98, 99, or 100%. Preferably, the perhydrolysis activity is measured as described in Examples 8-13 below, but any method of measuring perhydrolysis activity can be used in the practice of the present invention.

0176In some embodiments, further improvements in enzyme activity over the stated period include a buffer having a buffering capacity in the pH range of about 5.5 to about 9.5 into a formulation consisting of a carboxylic acid ester and a spray-dried enzyme powder. It can be achieved by adding. Suitable buffers for use in this formulation include bicarbonate, pyrophosphate, phosphoric acid, methylphosphonic acid, citric acid, acetic acid, malic acid, fumaric acid, tartaric acid, maleic acid or sodium salts of succinic acid, Potassium salts, or mixtures of sodium or potassium salts may be included, but are not limited thereto. Preferred buffers for use in this formulation consisting of carboxylic acid esters and spray-dried enzyme powders are sodium bicarbonate, phosphoric acid, methylphosphonic acid, or succinic acid sodium, potassium, or sodium or potassium salts. Contains a mixture of.

0177In embodiments where the buffer is present in the carboxylic acid ester and enzyme powder formulation, the buffer is from about 0.01% by mass to about 0.01 mass% based on the mass of the carboxylic acid ester in the formulation consisting of the carboxylic acid ester and the enzyme powder. It may be present in an amount in the range of 50% by mass. The buffer may be present in a more preferable range of about 0.10% by mass to about 10% by mass based on the mass of the carboxylic acid ester in the formulation consisting of the carboxylic acid ester and the enzyme powder. Further, in these embodiments, a comparison between the perhydrolysis activity of the enzyme is made in a formulation consisting of (a) a carboxylic acid ester, a buffer having a buffering capacity in the pH range of about 5.5 to about 9.5, and the enzyme powder. Enzyme powder that retains at least 75% of the perhydrolysis activity of at least one enzyme after 8 weeks of storage at 40 ° C, (b) carboxylic acid ester, and a buffer capable of buffering in the pH range of about 5.5 to about 9.5. And the initial perhydrolysis activity of the enzyme powder prior to preparation of the formulation consisting of the enzyme powder.

0178The spray-dried enzyme powder is intended to be stored as a formulation in an organic compound that is a substrate for at least one enzyme, such as triacetin. In the absence of added hydrogen peroxide, triacetin is usually hydrolyzed in aqueous solution by CE-7 carbohydrate esterase to produce diacetin and acetic acid, which produces a decrease in the pH of the reaction mixture. One requirement for long-term storage stability of the enzyme in triacetin is that there is no significant reaction between triacetin and any water that may be present in triacetin; one commercial triacetin (Tessenderlo). The standard for water content in (supplied by Group, Brussels, Belgium) is 0.03% by weight water (300ppm). Any hydrolysis of triacetin that occurs during storage of the enzyme in triacetin will produce acetic acid, which can result in diminished or inactivated perhydrolysis of CE-7 carbohydrate esterase; CE. -7 The perhydrolase activity of carbohydrate esterase is typically inactivated below pH 5.0 (see US Patent Application No. 12 / 539,025 granted to DiCosimo, R. et al.). .. The oligosaccharide excipients selected for use in this application are enzymes in organic substrates for enzymes under conditions where acetic acid may occur due to the presence of low concentrations of water in the formulation. Must provide stability.

0179Suitable reaction conditions for enzyme-catalyzed preparation of peracids from carboxylic acid esters and hydrogen peroxide In one aspect of the invention, one or more carboxylic acid esters are reacted by reacting one or more carboxylic acid esters with a peroxygen source (hydrogen peroxide, sodium perborate or sodium percarbonate) in the presence of an enzyme catalyst with perhydrolysis activity. A method for producing an aqueous formulation containing an acid is provided. In one embodiment, the enzyme catalyst is at least one enzyme with perhydrolysis activity and structurally as a member of the CE-7 carbohydrate esterase family (CE-7; Coutinho, PM, Henrissat, B., see above). Includes enzymes that are classified. In another embodiment, the perhydrolase catalyst is structurally classified as a cephalosporin C deacetylase. In another embodiment, the perhydrolase catalyst is structurally classified as acetylxylan esterase.

0180In one embodiment, the perhydrolase catalyst has perhydrolase activity and a) RGQ motif as amino acid residues 118-120; b) GXSQG motif at amino acid residues 179-183; and c) HE motif as amino acid residues 298-299 when aligned to SEQ ID NO: 1 of the reference sequence using CLUSTALW Includes enzymes with signature motifs including.

0181In a further embodiment, the signature motif further comprises a fourth conserved motif defined as the LXD motif at amino acid residues 267-269 when aligned to SEQ ID NO: 1 of the reference sequence using CLUSTALW.

0182In another embodiment, the perhydrolase catalyst comprises an enzyme having at least 30% amino acids conferred on this signature motif and SEQ ID NO: 1.

0183In another embodiment, the perhydrolase catalyst is SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 19, 20. , 21, 22, 23, 24, and 25 include enzymes with perhydrolase activity selected from the group.

0184In another embodiment, the perhydrolase catalyst is an enzyme having at least 40% amino acid identity with the flanking signature motif defined in SEQ ID NO: 18, the conserved motifs described above (ie, RGQ, GXSQG, and HE, and HE, as well. Optionally contains an enzyme in which LXD) is conserved.

0185In another embodiment, the perhydrolase catalyst is SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 19, 20. , 21, 22, 23, 24, and 25, an enzyme having an amino acid sequence selected from the group consisting of one or more additions, deletions, or deletions as long as the signature motif is preserved and perhydrolase activity is retained. Includes enzymes that may have substitutions.

0186Suitable carboxylic acid ester substrates include: [X]_mR₅(In the formula, X = formula R₆It is an ester group of C (O) O; R₆= Linear, branched or cyclic hydrocarbyl moieties of C1 to C7 optionally substituted with hydroxyl groups or C1 to C4 alkoxy groups, where R₆Optionally contains one or more ether bonds for R6 = C2 ~ C7; R₅= Linear, branched, or cyclic hydrocarbyl moieties of C1 to C6 optionally substituted with hydroxyl groups, where R₅Each carbon atom in it individually contains less than one hydroxyl group or less than one ester group, R₅Optionally contains one or more ether bonds; m = 1 to R₅Up to the number of carbon atoms in it) Is an ester provided by It may contain an ester having a solubility in water of at least 5 ppm at 25 ° C.

0187In other embodiments, the preferred substrate also has the formula:<chemistry num="3"><img id="000005" he="21" wi="67" file="JP5777516B2_D0001.tif" img-format="tif" img-content="drawing" /></chemistry>(In the formula, R₁= C1-C7 linear or branched-chain alkyl optionally substituted with hydroxyl or C1-C4 alkoxy groups, R₂= C1 ~ C10 linear or branched alkyl, alkenyl, alkynyl, aryl, alkylaryl, alkyl heteroaryl, heteroaryl, (CH₂CH₂-O)_nH or (CH₂CH (CH)₃) -O)_nH, n is 1 ~ 10) Esters may be included.

0188In other embodiments, suitable carboxylic acid ester substrates include the formula:<chemistry num="4"><img id="000006" he="32" wi="111" file="JP5777516B2_D0001.tif" img-format="tif" img-content="drawing" /></chemistry>(In the formula, R₁= C1-C7 linear or branched-chain alkyl optionally substituted with hydroxyl or C1-C4 alkoxy groups, R₃And R₄Are individually H or R₁C (O)) May contain glycerides.

0189In other embodiments, R₆Is a linear hydrocarbyl moiety of C1 to C7 that optionally contains one or more ether bonds, optionally substituted with a hydroxyl group or a C1 to C4 alkoxy group. In a more preferred embodiment, R₆Is a linear hydrocarbyl moiety of C2 to C7 optionally substituted with a hydroxyl group and / or optionally containing one or more ether bonds.

0190In other embodiments, suitable carboxylic acid ester substrates may also include acetylated saccharides selected from the group consisting of acetylated mono-, 2- and polysaccharides. In a preferred embodiment, the acetylated saccharides include acetylated mono-, 2- and polysaccharides. In other embodiments, the acetylated saccharides are acetylated xylan, fragments of acetylated xylan, acetylated xylose (such as xylose tetraacetate), acetylated glucose (such as glucose pentaacetate), β-D-ribofuranose-. It is selected from the group consisting of 1,2,3,5-tetraacetate, tri-O-acetyl-D-galactal, tri-O-acetyl-D-glucarl, and acetylated cellulose. In a preferred embodiment, the acetylated saccharides are β-D-ribofuranose-1,2,3,5-tetraacetate, tri-O-acetyl-D-galactal, tri-O-acetyl-D-glucal, and acetyl. Selected from the group consisting of converted cellulose. Therefore, acetylated carbohydrates may be a suitable substrate for generating percarboxylic acids using this method and system (ie, in the presence of a peroxygen source).

0191In additional embodiments, the carboxylic acid ester substrate is monoacetin; triacetin; monopropionin; diproopionin; tripropionin; monobutyrin; dibutyrin; tributyrin; glucose pentaacetate; xylose tetraacetate; acetylated xylan; acetylated xylan fragment; β-D-ribo Furanose-1,2,3,5-tetraacetate; tri-O-acetyl-D-galactal; tri-O-acetyl-glucarl; propylene glycol diacetate; ethylene glycol diacetate; 1,2-ethanediol, 1, 2-Propanediol, 1,3-propanediol, 1,2-butanediol, 1,3-butanediol, 2,3-butanediol, 1,4-butanediol, 1,2-pentanediol, 2,5 -It may be a monoester or diester of pentandiol, 1,6-pentanediol, 1,2-hexanediol, 2,5-hexanediol, 1,6-hexanediol; and mixtures thereof. In a preferred embodiment of the method and system, the substrate comprises triacetin.

0192The carboxylic acid ester is present in the reaction formulation at a concentration sufficient to produce the desired concentration of peracid when enzyme-catalyzed perhydrolysis. The carboxylic acid ester does not need to be completely soluble in the reaction formulation, but has sufficient solubility to allow perhydrolase-catalyzed conversion of the ester to the corresponding peracid. The carboxylic acid ester is at a concentration of 0.05% by mass to 40% by mass of the reaction formulation, preferably at a concentration of 0.1% by mass to 20% by mass of the reaction formulation, and more preferably 0.5% by mass to 10% by mass of the reaction formulation. It is present in the reaction mixture at a concentration of%.

0193Peroxygen sources may include, but are limited to, hydrogen peroxide, hydrogen peroxide adducts (eg, urea-hydrogen peroxide adducts (carbamide peroxides)), perborates and percarbonates. Not done. The concentration of the peroxygen compound in the reaction formulation may be in the range of 0.0033% by mass to about 50% by mass, preferably 0.033% by mass to about 40% by mass, and more preferably 0.33% by mass to about 30% by mass. ..

0194Many perhydrolase catalysts (whole cells, permeabilized whole cells, and partially purified whole cell extracts) have been previously reported to have catalase activity (EC 1.11.1.6). Catalase catalyzes the conversion of hydrogen peroxide to oxygen and water. In one aspect, the perhydrolysis catalyst lacks catalase activity. In another aspect, a catalase inhibitor is added to the reaction formulation. Examples of catalase inhibitors include, but are not limited to, sodium azide and hydroxylamine sulfate. One of ordinary skill in the art can adjust the concentration of the catalase inhibitor as needed. Concentrations of catalase inhibitors typically range from 0.1 mM to about 1 M, preferably from about 1 mM to about 50 mM, more preferably from about 1 mM to about 20 mM. In one aspect, the sodium azide concentration typically ranges from about 20 mM to about 60 mM, while the hydroxylamine sulfate concentration typically ranges from about 0.5 mM to about 30 mM, preferably about 10 mM.

0195In another embodiment, the enzyme catalyst lacks significant catalase activity or is engineered to reduce or eliminate catalase activity. Catalase activity in host cells includes, but is not limited to, transposon mutagenesis, RNA antisense expression, target mutagenesis, and random mutagenesis, using well-known techniques to express genes involved in catalase activity. It can be controlled downward or eliminated by collapsing. In a preferred embodiment, the gene encoding endogenous catalase activity is downregulated or disrupted (ie, inactivated). As used herein, a "disintegrated" gene is one in which the activity and / or function of the modified gene-encoded protein no longer exists. Means for disrupting a gene are well known in the art and may include insertions, deletions, or mutations to the gene, as long as the activity and / or function of the corresponding protein is no longer present. Not limited to them. In a more preferred embodiment, the production host is an E. coli production host containing a disrupted catalase gene selected from the group consisting of katG and katE (see US Patent Application Publication No. 2008/0176299). I want). In another embodiment, the production host is an E. coli strain that contains downregulation and / or disruption of both the katG and katg1 catalase genes.

0196The concentration of catalyst in the aqueous formulation depends on the unique catalytic activity of the catalyst and is selected to obtain the desired rate of reaction. The mass of the catalyst in the perhydrolysis reaction typically ranges from 0.0001 mg to 10 mg per mL of total reaction volume, preferably 0.001 mg to 2.0 mg per mL. The catalyst may also be immobilized on a soluble or insoluble carrier using methods well known to those of skill in the art; for example, Immobilization of Enzymes and Cells; Gordon F. Bickerstaff, Editor; Humana Press, Totowa, NJ, USA; See 1997. The use of an immobilized catalyst allows the catalyst to be recovered and reused in subsequent reactions. The enzyme catalyst may be in the form of whole microbial cells, permeabilized microbial cells, microbial cell extracts, partially purified or purified enzymes, and mixtures thereof.

0197In one aspect, the concentration of peracid generated by the combination of chemical perhydrolysis of the carboxylic acid ester and enzymatic perhydrolysis is sufficient to provide an effective concentration of peracid for bleaching or disinfecting at the desired pH. is there. In another aspect, the method provides a combination of an enzyme and an enzyme substrate to produce the desired effective concentration of peroxy acid, wherein in the absence of the added enzyme, the peroxy acid produced. The concentration is significantly lower. In some cases, there is considerable chemical perhydrolysis of the enzyme substrate by a direct chemical reaction of the inorganic peroxide with the enzyme substrate, but at a concentration sufficient to provide an effective concentration of peracid for the desired application. There may be no generation of peracids, and a significant increase in total peracid concentration is achieved by the addition of a suitable perhydrolase catalyst to the reaction formulation.

0198The concentration of peracid (such as peracetic acid) generated by perhydrolysis of at least one carboxylic acid ester is at least about 20 ppm within 10 minutes, preferably within 5 minutes, more preferably within 1 minute of the initiation of the perhydrolysis reaction. At least 100 ppm, more preferably at least about 200 ppm peracid, more preferably at least 300 ppm, more preferably at least 500 ppm, more preferably at least 700 ppm, more preferably at least about 1000 ppm peracid, most preferably at least 2000 ppm peracid. is there. The product formulation containing peracid may be optionally diluted with water, or a solution consisting primarily of water, to produce a formulation of peracid at a lower concentration of desired concentration. In one aspect, the reaction time required to produce the desired concentration of peracid is about 2 hours or less, preferably about 30 minutes or less, more preferably about 10 minutes or less, most preferably about 5 minutes or less. is there. In another aspect, a hard surface or inanimate object contaminated with a biological contaminant is within about 5 minutes to about 168 hours, or about 5 minutes to about 48 hours, or said from the combination of the reaction components. Within about 5 minutes to 2 hours of the combination of reaction components, or at any time interval within it, contact with the peracid formed according to the methods described herein.

0199In another aspect, the peroxycarboxylic acid formed according to the methods described herein is for the peroxycarboxylic acid to provide benefits such as disinfection, bleaching, antifouling, sterilization, deodorization or a combination thereof. Used for laundry care applications that come into contact with clothing or textiles. Peroxycarboxylic acids may be used in a variety of laundry care products, including, but not limited to, textile prewash treatment agents, laundry detergents, stain removers, bleaching compositions, deodorizing compositions, and rinsing agents. In one embodiment, the method for producing a peroxycarboxylic acid for a target surface is performed in situ.

0200In the context of laundry care applications, the term "contacting a garment or fabric" means that the garment or fabric is exposed to the formulations disclosed herein. For this purpose, there are numerous formats, including, but not limited to, liquids, solids, gels, pastes, sticks, tablets, sprays, foams, powders, or granules for clothing or textiles. It may be used for processing and can be delivered by manual metering, device metering, substrate metering, spraying from a laundry washer or dryer and automatic metering. Granular compositions can also be in compact form; liquid compositions can also be in concentrated form.

0201When the formulations disclosed herein are used in a washing machine, the formulations may further contain ingredients typical of laundry detergents. For example, typical ingredients include surfactants, bleaching agents, bleaching activators, additional enzymes, soap foam inhibitors, dispersants, lime soap dispersants, soil suspending agents and anti-redeposition agents, softeners. Includes, anti-corrosion agents, anti-discoloring agents, disinfectants, pH adjusters, non-builder alkaline sources, chelating agents, organic and / or inorganic fillers, solvents, hydrotropes, fluorescent whitening agents, dyes, and fragrances. Not limited to them.

0202The formulations disclosed herein can also be used as detergent additive products in solid or liquid form. Such additive products are intended to supplement or enhance the performance of conventional detergent compositions and can be added at any stage of the cleaning process.

0203Peracids generated by perhydrolysis of at least one carboxylic acid ester in connection with this system and method for laundry care where peracids occur for one or more of bleaching, decontamination, and odor reduction (eg, perhydrolysis). , Peracetic acid) may be at least about 2 ppm, preferably at least 20 ppm, preferably at least 100 ppm, more preferably at least about 200 ppm peracid. Peracids are generated for disinfection or sterilization In connection with this system and method for laundry care, the concentration of peracids (eg, peracetic acids) generated by perhydrolysis of at least one carboxylic acid ester is perhydrolysis. Within 10 minutes, preferably within 5 minutes, most preferably within 1 minute of the start of the reaction, at least about 2 ppm, more preferably at least 20 ppm, more preferably at least 200 ppm, more preferably at least 500 ppm, more preferably at least 700 ppm, more. It may preferably be at least about 1000 ppm peracid, most preferably at least 2000 ppm peracid. The product mixture containing peracid may be optionally diluted with water, or a solution consisting primarily of water, to produce a mixture of peracid at a lower concentration of desired concentration. In one aspect of the method and system, the reaction time required to produce the desired concentration of peracid is about 2 hours or less, preferably about 30 minutes or less, more preferably about 10 minutes or less, even more preferably. Is about 5 minutes or less, most preferably about 1 minute or less.

0204The temperature of the reaction is chosen to control both the reaction rate and the stability of the enzyme catalytic activity. The reaction temperature may be in the more preferred range of reaction temperatures from about 5 ° C to about 55 ° C, in the range of about 95 ° C from just above the freezing point (approximately 0 ° C) of the reaction formulation.

0205The pH of the final reaction formulation containing the peracid is about 2 to about 9, preferably about 3 to about 8, more preferably about 5 to about 8, even more preferably about 5.5 to about 8, and even more. It is preferably about 6.0 to about 7.5. In another embodiment, the pH of the reaction formulation is acidic (pH &lt;7). Although the pH of the reaction solution and of the final reaction formulation includes bicarbonate, pyrophosphate, phosphate, methyl phosphate, citrate, acetate, maleate, fumarate, tartrate maleate or succinate. , And may be optionally adjusted by the addition of suitable buffers, not limited to them. When used, the concentration of buffer is typically 0.1 mM to 1.0 M, preferably 1 mM to 300 mM, most preferably 10 mM to 100 mM.

0206In another aspect, the enzymatic perhydrolysis reaction formulation may contain an organic solvent that acts as a dispersant to increase the rate of dissolution of the carboxylic acid ester in the reaction formulation. Such solvents include propylene glycol methyl ether, acetone, cyclohexanone, diethylene glycol butyl ether, tripropylene glycol methyl ether, diethylene glycol methyl ether, propylene glycol butyl ether, dipropylene glycol methyl ether, cyclohexanol, benzyl alcohol, isopropanol, ethanol, propylene glycol, and the like. And their mixtures are included, but not limited to them.

0207In another aspect, the enzymatic perhydrolysis product may contain additional ingredients that provide the desired functionality. These additional ingredients include buffers, detergent builders, thickeners, emulsifiers, surfactants, wetting agents, corrosion inhibitors (such as benzotriazoles), enzyme stabilizers, and peroxide stabilizers (eg, metals). Ion chelating agents), but not limited to them. Many of the additional ingredients are well known in the detergent industry (see, eg, US Pat. No. 5,932,532, incorporated herein by reference). Examples of emulsifiers include, but are not limited to, polyvinyl alcohol or polyvinylpyrrolidone. Examples of thickeners include LAPONITE® RD, cornstarch, PVP, CARBOWAX®, CARBOPOL®, CABOSIL®, polysorbate. 20, PVA, and lecithin include, but are not limited to. Examples of buffer systems are sodium sodium phosphate monobasic / sodium phosphate dibasic; sulfamic acid / triethanolamine; citric acid / triethanolamine; tartaric acid / triethanolamine; succinic acid / triethanolamine; and acetic acid. / Examples include, but are not limited to, triethanolamine. Examples of surfactants include a) block copolymers of ethylene oxide and propylene oxide, ethoxylated or propoxylated linear or branched primary and secondary alcohols, and nonionic surfactants such as aliphatic phosphine oxides. Agents; b) Tertiary ammonium compounds, especially cationic surfactants such as quaternary ammonium compounds having C8-C20 alkyl groups attached to nitrogen atoms attached to three more C1-C2 alkyl groups; c) Anionic surfactants such as alkanecarboxylic acids (eg C8-C20 fatty acids), alkylphosphonates, alkane sulfonates (eg sodium dodecyl sulfate "SDS") or linear or branched alkylbenzene sulfonates, alken sulfonates; as well as d) amino Examples include, but are not limited to, amphoteric and diionic surfactants such as carboxylic acids, aminodicarboxylic acids, alkylbetaines, and mixtures thereof. Additional ingredients include fragrances, dyes, hydrogen peroxide stabilizers (eg, 1-hydroxyethylidene-1,1-diphosphonic acid (DEQUEST® 2010, Solutia Inc, St. Louis, MO) and ethylenediaminetetraac. Metal chelating agents such as EDTA), TURPINAL® SL (CAS # 2809-21-4), DEQUEST® 0520, DEQUEST® 0531, Stabilizers for enzymatic activity (eg polyethylene) Glycol (PEG), as well as a detergent builder may be included.

0208In-situ formation of peracid using perhydrolase catalyst Cephalosporin C deacethylase (EC 3.1.1.41; strain name cephalosporin C acetylhydrolase; CAH) is a cephalosporin C, 7-aminocephalosporin acid, and 7- (thiophen-2-acetamide) cephalosporin acid. It is an enzyme having the ability to hydrolase acetyl esters bound on cephalosporins such as. Abbott, B.and Fukuda, D., Appl.Microbiol.30 (3): 413-419 (1975)). CAH belongs to a larger group of structurally related enzymes called Carbohydrate Esterases 7 (CE-7; Coutinho, PM, Henrissat, B., see above).

0209The CE-7 carbohydrate esterase family includes both CAH and acetylxylan esterase (AX; EC 3.1.1.72). CE-7 members are completely abnormal in that they share a common structural motif and they typically exhibit ester hydrolyzing activity for both acetylated xylooligosaccharides and acetylated cephalosporin C. It suggests that the CE-7 family represents a single class of proteins with multifunctional deacetylase activity for a variety of small substrates (see Vincent et al., Above). Vincent et al. Describe structural similarities between members of this group and define the signature sequence motif traits of the CE-7 group.

0210Members of the CE-7 family include plants, fungi (eg, Cephalosporidium acremonium), yeast (eg, Rhodosporidium toruloides), Rhodotorula glutinis, And bacteria such as Thermoanaerobacterium sp.; Norcardia lactamdurans, and various members of the genus Bacillus (Politino et al., Appl.Environ.Microbiol., 63 (12) ): 4807-4811 (1997); Sakai et al., J. Ferment. Bioeng. 85: 53-57 (1998); Lorenz, W. and Wiegel, J., J. Bacteriol 179: 5436-5441 (1997) Cardoza et al., Appl.Microbiol.Biotechnol., 54 (3): 406-412 (2000); see Mitsushima et al., Above; Abbott, B. and Fukuda, D., Appl.Microbiol.30 (3): 413- 419 (1975); Vincent et al., See above; Takami et al., NAR, 28 (21): 4317-4331 (2000); Rey et al., Genome Biol., 5 (10): article 77 (2004). Degrassi et al., Microbiology., 146: 1585-1591 (2000); U.S. Pat. No. 6,645,233; U.S. Pat. No. 5,281,525; No. 5,338,676; and WO 99/03984) Found in.

0211International Publication No. 2007/070609 and US Patent Application Publication No. 2008/0176299, and 2008/176783, granted to DiCosimo et al., Have various carboxylic acids when combined with a peroxygen source. Various enzymes structurally classified as CE-7 enzymes with perhydrolysis activity suitable for producing effective concentrations of peracid from acid ester substrates are disclosed. Mutant CE-7 enzyme with improved perhydrolysis activity is also co-filed, co-owned, and co-pending US patent application (incorporated herein by reference in its entirety, proxy reference number). It is described in CL4392 US NA).

0212The method uses the perhydrolase activity of an enzyme belonging to the CE-7 group of carbohydrate esterases to produce an effective concentration of peracid that is industrially useful in situ under aqueous reaction conditions.

0213HPLC Assay Method for Measuring Peroxy Acid and Hydrogen Peroxide Concentrations Titration, High Performance Liquid Chromatography (HPLC), Gas Chromatography (GC), Mass Spectrometry (MS), Capillary Electrophorography (CE), Analytical Procedures (Anal. Chem) described by U. Karst et al. , 69 (17): 3623-3627 (1997)), and 2,2'-azino-bis (3-ethylbenzothiazolin) -6-sulfonate (ABTS) assay (S) as described in this example. Analyzing reactants and products using a variety of analytical methods, including, but not limited to, Minning, et al., Analytica Chimica Acta 378: 293-298 (1999) and WO 2004/058961 A1. It can be used in this method to do so.

0214Measurement of the minimum killing concentration of peracid The method described by J. Gabrielson, et al. (J. Microbiol. Methods 50: 63-73 (2002)) to measure the minimum killing concentration (MBC) of peracid or hydrogen peroxide and enzyme substrates. Can be used for. This assay method is based on XTT reduction inhibition, where XTT ((2,3-bis [2-methoxy-4-nitro-5-sulfophenyl] -5-[(phenylamino) carbonyl] -2H -Tetrazolium, intramolecular salt, monosodium salt) is a redox dye that exhibits microbial respiration activity by changes in optical density (OD) measured at 490 nm or 450 nm, however, survival plate count, direct microscopic count, dry mass. , Turbidity measurement, absorbance, and bioluminescence (eg, Brock, Semour S., Disinfection, Sterilization, and Preservation, 5)^th There are various other methods available for testing the activity of disinfectants and preservatives, including, but not limited to, edition, Lippincott Williams &amp; Wilkins, Philadelphia, PA, USA; 2001). ..

0215Use of enzymatically prepared peroxycarboxylic acid composition Enzyme-catalyzed peroxycarboxylic acids produced according to this method are used for medical devices (eg, endoscopes), textiles (eg, clothes, carpets), food cooking surfaces, food storage and food packaging equipment, food packaging. Various hardeners for reducing the concentration of biological pollutants, such as materials used, chicken hatcheries and chicken farms, animal enclosures, and decontamination of used process wastewater with microbial and / or virus-killing activity. Can be used for surface / inanimate object applications. Enzyme-generated peroxycarboxylic acids may be used in formulations designed to inactivate prions (eg, certain proteases) to further provide biokilling activity. In a preferred embodiment, the peroxycarboxylic acid composition is particularly useful as a disinfectant for medical devices and food packaging equipment that cannot be autoclaved. Since the peroxycarboxylic acid-containing formulation can be prepared using GRAS or food grade ingredients (enzymes, enzyme substrates, hydrogen peroxide, and buffers), the enzyme-generated peroxycarboxylic acid can also be used for carcasses, meat and fruits. And may be used for decontamination of vegetables or for decontamination of cooked foods. The enzyme-generated peroxycarboxylic acid may be incorporated into a product whose final form is a powder, liquid, gel, film, solid or aerosol. The enzyme-generated peroxycarboxylic acid may be diluted to a concentration that still provides effective decontamination.

0216Compositions containing an effective concentration of peroxycarboxylic acid can be contaminated (or suspected to be contaminated) with biological contaminants and / or objects with products that produce surfaces or objects by this method. It can be used for disinfection by contact. As used herein, "contacting" is suspected of being contaminated with biological contaminants for a period sufficient to clean and disinfect the disinfectant composition containing an effective concentration of peroxycarboxylic acid. Means contact with a surface or inanimate object. In contact, a peroxycarboxylic acid solution or composition containing an effective concentration of peroxycarboxylic acid, or a solution or composition forming an effective concentration of peroxycarboxylic acid, is contaminated with a concentration of biological contaminants. Spray, treat, soak, flush, pour on or in, mix, combine, paint, coat, apply, paste and otherwise communicate with suspected surfaces or inanimate objects Is included. The disinfectant composition may be combined with a cleaning composition to provide both cleaning and disinfection. Alternatively, the cleaning agent (eg, surfactant or detergent) may be incorporated into the formulation to provide both cleaning and disinfection in a single composition.

0217Compositions containing an effective concentration of peroxycarboxylic acid can also contain at least one additional antibacterial agent, a combination of prion-degrading proteases, a virus-killing agent, a spore-killing agent, or a biocide. The combination of these reagents and the peroxycarboxylic acid produced by the claimed method cleans and disinfects surfaces and / or objects contaminated (or suspected to be contaminated) with biological contaminants. Can provide increased effects and / or synergistic effects when used to. Suitable antibacterial agents include carboxylic acid esters (eg, p-hydroxyalkylbenzoate and alkyl cinnamates) in sufficient amounts to provide the desired degree of microbial protection; sulfonic acids (eg, dodecylbenzene sulfonic acid). ); Iode compounds or active halogen compounds (eg elemental halogens, halogen oxides (eg NaOCl, HOCl, HOBr, ClO)₂), Iodine, interhalogen compounds (eg iodine monochloride, iodine dichloride, iodine trichloride, iodine tetrachloride, bromine chloride, iodine monobromide, or iodine dibromide), polyhalides, hypochlorites , Hypochlorite, Hypochlorite, Hypochlorite, Chloro- and Bromo-Hydantine, Chloride Dioxide, and Hypochlorite); benzoyl peroxides, organic peroxides including alkylbenzoyl peroxides, ozone, single Oxygen generators, and mixtures thereof; phenol derivatives (o-phenylphenol, o-benzyl-p-chlorophenol, tertiary amylphenol and C₁~ C₆Includes quaternary ammonium compounds (such as alkyldimethylbenzylammonium chloride, dialkyldimethylammonium chloride and mixtures thereof); and mixtures of such antibacterial agents. Effective amounts of antimicrobial agents include from about 0.001% to about 60% by weight antibacterial agents, from about 0.01% to about 15% by weight antibacterial agents, or from about 0.08% to about 2.5% by weight antibacterial agents.

0218In one aspect, the peroxycarboxylic acid formed by the method reduces the concentration of viable biological contaminants (such as surviving microbial populations) when applied on and / or in place. Can be used for. As used herein, "location" includes some or all of the target surfaces suitable for disinfection or bleaching. Target surfaces include all surfaces that can be potentially contaminated with biological contaminants. Non-limiting examples are equipment surfaces found in the food or beverage industry (tanks, conveyors, floors, sewage equipment, coolers, freezer, equipment surfaces, walls, valves, belts, pipes, sewage equipment, joints, clevas, etc. Building surfaces (walls, floors and windows, etc.); non-food industry related pipes and sewage facilities, including water treatment facilities, pools and health facilities, and fermentation tanks; hospital or animal hospital surfaces (walls, etc.) Floors, beds, equipment (endoscope), clothing, surgical gowns, shoes, and clothing used in hospital / animal hospitals or other medical settings, including other hospital or animal hospital surfaces); Restaurant surfaces; Bathroom surfaces; Toilet; Cloths and shoes; Poultry, cattle, dairy cows, goats, horses and pigs, livestock barns or shackles; poultry or shrimp hatcheries; and pharmaceutical or biopharmaceutical surfaces ( For example, pharmaceutical or biopharmaceutical manufacturing equipment, pharmaceutical or biopharmaceutical raw materials, pharmaceuticals or biopharmaceutical excipients). Additional hard surfaces also include foods such as beef, chicken, pork, vegetables, fruits, seafood and combinations thereof. This location may also include contaminated linen or other water absorbing materials such as textiles. This location also includes harvested plants or plant products, including seeds, bulbs, rhizomes, fruits, and vegetables, growing plants, and above all, grains, leafy vegetables and salad crops, root vegetables, legumes, berries. Includes growing crop plants, including fruits, citrus fruits and hard fruits.

0219Non-limiting examples of hard surface materials are metals (eg steel, stainless steel, chromium, titanium, iron, copper, brass, aluminum, and alloys thereof), minerals (eg concrete), polymers and plastics (eg steel). Polyethylene, polystyrene, poly (meth) acrylate, polyacrylonitrile, polybutadiene, poly (acrylonitrile, butadiene, styrene), poly (acrylonitrile, butadiene), acrylonitrile butadiene; polyesters such as polyethylene terephthalate; and nylon, etc. Polyamide). Additional surfaces include brick, tile, ceramic, porcelain, wood, vinyl, linoleum, and carpet.

0220The peroxycarboxylic acid formed by this method may be used to provide the fabric with benefits including, but not limited to, bleaching, sterilization, disinfection, stain removal, odor reduction. Peroxycarboxylic acids formed by this method include, but are not limited to, textile prewash treatment agents, laundry detergents, stain removers, bleaching compositions, deodorizing compositions, and rinsing agents in a significant number of laundry care products. May be used.

0221Recombinant microbial expression Instant sequence genes and gene products may be produced in heterologous host cells, especially cells in microbial hosts. Preferred heterologous host cells for the expression of instant genes and nucleic acid molecules are microbial hosts that can be found within fungal or bacterial families and that grow over a wide range of temperature, pH values, and solvent resistance. For example, it is believed that any of bacteria, yeast, and filamentous fungi may suitably host the expression of this nucleic acid molecule. Perhydrolase may be expressed intracellularly, extracellularly, or in a combination of both intracellular and extracellular, where extracellular expression allows the recovery of the desired protein from the fermentation product, intracellularly. Make it easier than the method for recovering the protein produced by expression. Transcription, translation and protein biosynthesis equipment remain unchanged in relation to the cell feedstock used to generate cellular biomass; The functional gene will be expressed anyway. Examples of host strains include Aspergillus, Trichoderma, Saccharomyces, Pichia, Phaffia, Kluyveromyces, Candida. ), Hansenula, Yarrowia, Salmonella, Bacillus, Acinetobacter, Zymomonas, Agrobacterium, Erythrobacter, Chlorobium Genus Chlorobium, Chromatium, Flavobacterium, Cytophaga, Rhodobacter, Rhodococcus, Streptomyces, Brevibacterium, Corine Corynebacteria, Mycobacterium, Deinococcus, Escherichia, Erwinia, Pantoea, Pseudomonas, Sphingomonas (Methylomonas), Methylobacter, Methylococcus, Methylosinus, Methylomicrobium, Methylocystis, Alcaligenes, Synecocystis Bacterial, fungal or yeast species such as is), Synechococcus, Anabaena, Thiobacillus, Methanobacterium, Klebsiella, and Myxococcus. However, it is not limited to them. In one embodiment, bacterial host strains include Escherichia, Bacillus, Kluyveromyces, and Pseudomonas. In a preferred embodiment, the bacterial host cell is Escherichia coli.

0222Large-scale microbial growth and functional gene expression are carbon dioxide, nitrogen, phosphorus in the case of saturated hydrocarbons or photosynthetic or chemically self-nourishing hosts such as a wide range of simple or complex carbohydrates, organic acids and alcohols or methanes. The form and amount of any trace micronutrient, including sulfur, oxygen, carbon or small inorganic ions, may be used. Regulation of growth rate can be affected by the addition or absence of specific regulatory molecules that are not typically considered nutrients or energy sources.

0223Vectors or cassettes useful for transformation of suitable host cells are well known in the art. Typically, the vector or cassette contains sequences that direct transcription and translation of related genes, selectable markers, and sequences that allow autonomous replication or chromosomal integration. Suitable vectors include region 5'of the gene contained within the control of transcription initiation and region 3'of the DNA fragment that controls transcription termination. It is most preferred when both regulatory regions are derived from genes homologous and / or produced host-born to the transformed host cell, but such regulatory regions need not be so induced.

0224There are numerous initiation control regions or promoters that are useful for promoting expression of the cephalosporin C deacetylase coding region in the desired host cell and are familiar to those of skill in the art. CYC1, HIS3, GAL1, GAL10, ADH1, PGK, PHO5, GAPDH, ADC1, TRP1, URA3, LEU2, ENO, TPI (useful for expression in Saccharomyces); AOX1 (Pichia) Useful for expression in); as well as amy, apr, npr promoters and various phage promoters useful for expression in Bacillus, as well as lac, araB, tet, trp, lP_L, LP_R, T7, tac, and trc (useful for expression in Escherichia coli), but not limited to, substantially any promoter capable of promoting these genes in the present invention. Suitable.

0225The termination control region may also be derived from various genes born in the preferred host cell. In one embodiment, inclusion of the termination control region is optional. In another embodiment, the chimeric gene comprises a termination control region derived from a preferred host cell.

0226Industrial production Various culture methodologies may be applied to produce perhydrolase catalysts. For example, large-scale production of unique gene products overexpressed from recombinant microbial hosts may be produced by batch, fed-batch, and continuous culture methodologies. Batch and fed-batch culture methods are common and well known in the art, for example, Thomas D. Brock in Biotechnology: A Textbook of Industrial Microbiology, Second Edition, Sinauer Associates, Inc., Sunderland, MA. It may be found in (1989) and Deshpande, Mukund V., Appl. Biochem. Biotechnol., 36: 227 (1992).

0227Commercial production of the desired perhydrolase catalyst may also be accomplished in continuous culture. Continuous culture is an open system in which certain mediums are continuously added to the bioreactor and equal amounts of conditioned medium are simultaneously removed for processing. Continuous culture generally maintains a constant high liquidus density in which the cells are predominantly logarithmic. Alternatively, continuous culture may be carried out with an immobilized catalyst in which carbon and nutrients are continuously added and valuable products, by-products or wastes are continuously removed from the cell population. Cell immobilization may be performed using a wide range of solid supports made of natural and / or synthetic materials.

0228Recovery of the desired perhydrolase catalyst from batch fermentation, fed-batch fermentation, or continuous culture may be accomplished by any of the methods known to those of skill in the art. For example, when an enzyme catalyst is produced intracellularly, the cell paste is separated from the medium by centrifugation or membrane filtration, optionally washed with water or an aqueous buffer of the desired pH, and then at the desired pH. The suspension of cell paste in the aqueous buffer of is homogenized to produce a cell extract containing the desired enzyme catalyst. The cell extract may be optionally filtered through a suitable filtration aid such as Celite or silica to remove cell debris prior to the heat treatment step to precipitate the unwanted protein from the enzyme catalytic solution. The solution containing the desired enzyme catalyst may then be separated from the precipitated cell debris and protein by membrane filtration or centrifugation, and the resulting partially purified enzyme catalyst solution concentrated by additional membrane filtration is then suitable. Carriers (eg, maltodextrin, phosphate buffers, citrate buffers, or mixtures thereof) are optionally mixed and spray dried to produce a solid powder containing the desired enzyme catalyst. ..

0229When a quantity, concentration, or other value or parameter is given either as a range, as a preferred range, or as a list of preferred upper and preferred lower values, is this disclosed separately? Regardless of what it is, it should be understood as specifically disclosing the entire range formed from any bear of any upper range limit or preferred value and any lower range limit or preferred value. Where a range of numbers is listed herein, this range is intended to include its endpoints and all integers and fractions within this range, unless otherwise stated. It is not intended that the range be limited to the specific values listed when defining the range.

0230General method The following examples are provided to demonstrate preferred embodiments. Those skilled in the art can believe that the techniques disclosed in the examples according to the exemplary techniques found by the present invention will work well in the practice of the methods disclosed in the present invention and thus establish a preferred mode for the practice. Should be fully understood by. However, those skilled in the art may, in light of this disclosure, make many changes in the disclosed specific embodiments without departing from the spirit and scope of the methods now disclosed, with similar or similar results. It should be fully understood that can still be.

0231All reagents and materials are DIFCO Laboratories (Detroit, MI), GIBCO / BRL (Gaithersburg, MD), TCI America (Portland, OR), Roche Diagnostics Corporation (Indianapolis, IN) or Sigma / Aldrich Chemical, unless otherwise stated. Obtained from Company (St. Louis, MO).

0232The following abbreviations herein correspond to units of measurement, techniques, properties, or compounds as follows: "sec" or "s" means seconds, "min" means minutes, " "h" or "hr" means time, "μL" means microgram, "mL" means milliliter, "L" means liter, "mM" means millmol, " "M" means mol, "mmol" means milligram, "ppm" means parts per million, "wt" means mass, "wt%" means mass percent, "G" means gram, "mg" means milligram, "μg" means microgram, "ng" means nanogram, "g" means gravity, "HPLC" means Means high-performance liquid chromatography, "dd H₂"O" means distilled and deionized water, "dcw" means dry cell mass, "ATCC" or "ATCC®" means American Type Culture Collection (Manassas, VA), "U" "" Means a unit of perhydrolase activity, "rpm" means revolutions per second, "Tg" means glass transition temperature, and "EDTA" means ethylenediamine tetraacetic acid.

<p num="0233">Example 1 Construction of katG catalase-disrupted E. coli strain PCR (94 °) the coding region of the canamycin resistance gene (kan; SEQ ID NO: 26) using primers identified as SEQ ID NO: 28 and SEQ ID NO: 29 to generate the PCR product identified as SEQ ID NO: 30. Amplified from plasmid pKD13 (SEQ ID NO: 27) by 0.5 min at C, 0.5 min at 55 ° C, 1 min at 70 ° C, 30 cycles). The katG nucleic acid sequence is provided as SEQ ID NO: 31 and the corresponding amino acid sequence is SEQ ID NO: 32. E. coli MG1655 (ATCC® 47076)^TM) Was transformed with the temperature-sensitive plasmid pKD46 (SEQ ID NO: 33) containing the λ-Red recombinase gene (Datsenko and Wanner, (2000), PNAS USA 97: 6640-6645) and LB at 30 ° C for 24 hours. -Selected on the amp plate. MG1655 / pKD46 was transformed with 50-500 ng PCR product by electroporation (BioRad Gene Pulser, 0.2 cm cuvette, 2.5 kV, 200 W, 25 μF) and selected on LB-kan plate at 37 ° C for 24 hours. did. Several colonies were streaked onto LB-kan plates and cultured overnight at 42 ° C to cure the pKD46 plasmid. Colonies were checked to confirm the kanR / ampS phenotype. Genome DNA, PUREGENE® DNA Purification System (Gentra) Isolated from several colonies using Systems, Minneapolis, MN) and checked by PCR to confirm disruption of the katG gene using primers identified as SEQ ID NO: 34 and SEQ ID NO: 35. Several katG-disintegrated strains were transformed with the temperature sensitive plasmid pCP20 (SEQ ID NO: 36) containing FLP recombinase and used to delete the kan gene, on LB-amp plates at 37 ° C for 24 hours. Selected in. Several colonies were stroked onto LB plates and cultured overnight at 42 ° C to cure the pCP20 plasmid. The two colonies are checked to confirm the kanS / ampS phenotype and are called MG1655 KatG1 and MG1655 KatG2.</p><p num="0234">Example 2 Construction of katE catalase-disrupted E. coli strain PCR the canamycin resistance gene (SEQ ID NO: 26) using primers identified as SEQ ID NO: 37 and SEQ ID NO: 38 to generate the PCR product identified as SEQ ID NO: 39 (0.5 minutes at 94 ° C, Amplified from plasmid pKD13 (SEQ ID NO: 27) by (55 ° C for 0.5 minutes, 70 ° C for 1 minute, 30 cycles). The katE nucleic acid sequence is provided as SEQ ID NO: 40 and the corresponding amino acid sequence is SEQ ID NO: 41. E. coli MG1655 (ATCC® 47076)^TM) Was transformed with the temperature-sensitive plasmid pKD46 (SEQ ID NO: 33) containing the λ-Red recombinase gene and selected on an LB-amp plate at 30 ° C. for 24 hours. MG1655 / pKD46, electroporation (Bio Rad Gene) Transformed with 50-500 ng PCR product with Pulser, 0.2 cm cuvette, 2.5 kV, 200 W, 25 μF) and selected on LB-kan plates at 37 ° C for 24 hours. Several colonies were stroked onto LB-kan plates and cultured overnight at 42 ° C to cure the pKD46 plasmid. Colonies were checked to confirm the kanR / ampS phenotype. Genomic DNA is isolated from several colonies using the PUREGENE® DNA purification system and by PCR to confirm disruption of the katE gene using primers identified as SEQ ID NO: 42 and SEQ ID NO: 43. Checked. Several katE-disintegrated strains were transformed with the temperature sensitive plasmid pCP20 (SEQ ID NO: 36) containing FLP recombinase and used to delete the kan gene, on LB-amp plates at 37 ° C for 24 hours. Selected in. Several colonies were stroked onto LB plates and cultured overnight at 42 ° C to cure the pCP20 plasmid. Two colonies were checked to confirm the kanS / ampS phenotype and MG1655 Called KatE1 and MG1655 KatE2.</p><p num="0235">Example 3 Construction of katG catalase and katE catalase-disrupted E. coli strain (KLP18) PCR the canamycin resistance gene (SEQ ID NO: 26) using primers identified as SEQ ID NO: 37 and SEQ ID NO: 38 to generate the PCR product identified as SEQ ID NO: 39 (0.5 minutes at 94 ° C, Amplified from plasmid pKD13 (SEQ ID NO: 27) by (55 ° C for 0.5 minutes, 70 ° C for 1 minute, 30 cycles). E. coli MG1655 KatG1 (Example 1) was transformed with the temperature-sensitive plasmid pKD46 (SEQ ID NO: 33) containing the λ-Red recombinase gene and placed on an LB-amp plate at 30 ° C for 24 hours. Selected. MG1655 KatG1 / pKD46, electroporation (BioRad Gene) Transformed with 50-500 ng PCR product with Pulser, 0.2 cm cuvette, 2.5 kV, 200 W, 25 μF) and selected on LB-kan plates at 37 ° C for 24 hours. Several colonies were stroked onto LB-kan plates and cultured overnight at 42 ° C to cure the pKD46 plasmid. Colonies were checked to confirm the kanR / ampS phenotype. Genomic DNA is isolated from several colonies using the PUREGENE® DNA purification system and PCR is performed to confirm disruption of the katE gene using primers identified as SEQ ID NO: 42 and SEQ ID NO: 43. Checked. Several katE-disintegrated strains (ΔkatE) were transformed with the temperature sensitive plasmid pCP20 (SEQ ID NO: 36) containing FLP recombinase and used to delete the kan gene, LB-at 37 ° C for 24 hours. Selected on the amp plate. Several colonies were streaked onto LB plates and cultured overnight at 42 ° C to cure the pCP20 plasmid. Two colonies were checked to confirm the kanS / ampS phenotype and MG1655 Called KatG1KatE18.1 and MG1655 KatG1KatE23. MG1655 KatG1KatE18.1 is called E. coli KLP18.</p><p num="0236">Example 4 Cloning and expression of perhydrolase from Thermotoga neapolitana The coding region for the gene encoding acetylxylan esterase from Thermotoga neapolitana, as reported in GENBANK® (Registration No. AE000512; Region 80481-81458; SEQ ID NO: 44), was defined in E. coli (E. coli coli) (DNA 2.0, Menlo Synthesized using codons optimized for expression in Park, CA). The coding region of the gene is then subjected to PCR (0.5 minutes at 94 ° C, 0.5 minutes at 55 ° C, 1 minute at 70 ° C, 30 cycles) using primers identified as SEQ ID NO: 45 and SEQ ID NO: 46. Amplified. The resulting nucleic acid product (SEQ ID NO: 47) was subcloned into pTrcHis2-TOPO® to generate a plasmid identified as pSW196. Escherichia coli (E. coli) KLP18 (Example 3) was transformed with the plasmid pSW196 to generate the strain KLP18 / pSW196. KLP18 / pSW196, OD_600nmIt was grown in LB medium at 37 ° C with shaking to = 0.4-0.5, at which point IPTG was added to a final concentration of 1 mM and the culture was continued for 2-3 hours. Cells were harvested by centrifugation and SDS-PAGE was performed to confirm expression of perhydrolase in 20-40% of total soluble protein.</p><p num="0237">Example 5 Cloning and expression of perhydrolase from Thermotoga maritima MSB8 Coding regions for genes encoding acetylxylan esterase from Thermotoga maritima MSB8 as reported in GENBANK® (Registration # NP_227893.1; SEQ ID NO: 48) were synthesized (DNA 2.0, Menlo). Park.CA). The coding region of the gene is then subjected to PCR (0.5 min at 94 ° C, 0.5 min at 55 ° C, 1 min at 70 ° C, 30 cycles) using primers identified as SEQ ID NO: 49 and SEQ ID NO: 50. Amplified. The resulting nucleic acid product (SEQ ID NO: 51) was cut with restriction enzymes PstI and XbaI and subcloned between the PstI and XbaI sites in pUC19 to generate a plasmid identified as pSW207. E. coli KLP18 (Example 3) was transformed using the plasmid pSW207 to generate a strain identified as KLP18 / pSW207. KLP18 / pSW207, OD_600nmIt was grown in LB medium at 37 ° C with shaking to = 0.4-0.5, at which point IPTG was added to a final concentration of 1 mM and the culture was continued for 2-3 hours. Cells were harvested by centrifugation and SDS-PAGE was performed to confirm expression of perhydrolase enzyme in 20-40% of total soluble protein.</p><p num="0238">Example 6 Fermentation of E. coli KLP18 transformant expressing perhydrolase Fermenter seed culture, yeast extract (Amberex 695, 5.0 g / L), K₂HPO₄(10.0g / L), KH₂PO₄(7.0g / L), sodium citrate dihydrate (1.0g / L), (NH₄)₂SO₄(4.0g / L), DDL₄It was prepared by charging 0.5 L of seed medium containing heptahydrate (1.0 g / L) and ammonium ferric citrate (0.10 g / L) into a 2 L shaking flask. The pH of the medium was adjusted to 6.8 and the medium was sterilized in a flask. Post-sterile additives included glucose (50% by weight, 10.0 mL) and 1 mL ampicillin (25 mg / mL) stock solution. Seed medium was inoculated with 1 mL cultures of E. coli KLP18 / pSW196 or E.coli KLP18 / pSW207 in 20% glycerol and cultured at 35 ° C and 300 rpm. Seed culture, KH₂PO₄(3.50g / L), FeSO₄Hexahydrate (0.05g / L), DDL₄Hexahydrate (2.0 g / L), sodium citrate dihydrate (1.90 g / L), yeast extract (Amberex 695, 5.0 g / L), Biospumex 153K defoamer (0.25 mL / L, Cognis Corporation) , Monheim, Germany), NaCl (1.0g / L), CaCl₂Approximately 1-2 OD with 8 L medium at 35 ° C containing dihydrate (10 g / L) and NIT trace element solution (10 mL / L)_550nmTransferred to a 14L fermenter (Braun Biotech, Allentown, PA). Trace element solution is citric acid monohydrate (10 g / L), MnSO₄Hydrate (2g / L), NaCl (2g / L), FeSO₄Hexahydrate (0.5g / L), ZnSO₄Hexahydrate (0.2 g / L), CuSO₄Pentahydrate (0.02g / L) and NaMoO₄It contained dihydrate (0.02 g / L). Post-sterilization additives included glucose solution (50% w / w, 80.0 g) and ampicillin (25 mg / mL) stock solution (16.00 mL). Glucose solution (50% w / w) was used for feeding. Glucose supply started when the glucose concentration dropped to 0.5 g / L, started at 0.31 g supply / minute, and gradually increased to 0.36, 0.42, 0.49, 0.57, 0.66, 0.77, 0.90, 1.04, 1.21, 1.41 per hour, respectively. , And increased to 1.63 g / min; the speed remained constant thereafter. The glucose concentration in the medium was monitored, and if the concentration exceeded 0.1 g / L, the supply rate was reduced or temporarily stopped. Induction is OD with the addition of 16 mL of IPTG (0.5 M) for various strains._550nm= 56 and OD_550nmStarted with = 80. Dissolved oxygen (DO) concentration was controlled at 25% of air saturation. The DO was first controlled by the impeller stirring speed (400 to 1400 rpm) and then by the aeration speed (2 to 10 slpm). The pH was adjusted to 6.8. NH₄OH (29% w / w) and H₂SO₄(20% w / v) was used for pH adjustment. Head pressure was 0.5 bar. Cells were harvested by centrifugation 16 hours after IPTG addition.</p><p num="0239">Example 7 Preparation of CE-7 Esterase / Perhydrolase Heat Treated Cell Extract Cellular extracts of E. coli transformants expressing perhydrolase from Thermotoga neapolitana (KLP18 / pSW196) or Thermotoga maritima MSB8 (KLP18 / pSW207) Suspension of cell paste (20 mass% wet cell mass) in 0.05 M potassium phosphate buffer (pH 7.0) containing tall (1 mM) twice, with an operating pressure of 16,000 psi (approximately 110 MPa) Prepared by passing through a French press. The crude extract was then centrifuged at 20,000 xg to remove cell debris to produce a purified cell extract, which was assayed for total soluble protein (Bicinchoninic Acid Kit for Protein Determination, Sigma Aldrich Catalog # BCA1). -KT). Purification Thermotoga maritima) MSB8 or Thermotoga neapolitana perhydrolase-containing extract was heated at 75 ° C for 20 minutes, followed by immediate cooling to 5 ° C in an ice / water bath. The resulting mixture was centrifuged to remove the precipitated protein, the supernatant was collected and the total soluble protein was analyzed as described above. SDS-PAGE of the heat treated supernatant showed that perhydrolase constitutes at least about 90% of the total soluble protein present in the supernatant.</p><p num="0240">Example 8 Temperature stability of T. neapolitana perhydrolase / trehalose spray-dried enzyme powder Heat-treated cell extract proteins of various concentrations of E. coli KLP18 / pSW196 in sodium bicarbonate buffer (50 mM, pH = 8.1) ( 90% T. neapolitana perhydrolase by PAGE) , Trehalose, and optionally 10 sets of aqueous mixtures containing polysorbate 80 (p80) as a surfactant were prepared (Table 1). These solutions were spray-dried using a Buchi B-290 glass chamber spray dryer (inlet temperature = 170 ° C, outlet temperature = 90 ° C, supply rate = 3 mL / min to 10 mL / min), and 10 pieces were spray-dried. Spray-dried enzyme powders are produced; mass percent proteins in the powders are measured using the BCA (bisinconic acid) protein assay and the glass transition temperature (Tg) of these powders is measured using the modulated differential scanning calorific value measurement method. Measured (Table 1).</p><p num="0241"><tables num="2"><img id="000007" he="92" wi="159" file="JP5777516B2_D0001.tif" img-format="tif" img-content="drawing" /></tables></p><p num="0242"> Spray-dried enzyme powder was stored in sealed vials at 40 ° C, sampled at weekly intervals, and samples were sampled at 25 ° C in sodium bicarbonate buffer (50 mM, pH 7.2) with T. neapolitana (T. neapolitana) Perhydrolase (50 μg protein / mL), H₂O₂Assayed for the concentration of peracetic acid produced in 5 minutes in a reaction containing (100 mM), triacetin (100 mM) and TURPINAL® SL (500 ppm), of the analytical method reported by Karst et al. (below). The modification was used to analyze the production of peracetic acid.</p><p num="0243"> A sample of the reaction mixture (0.040 mL) was removed at the scheduled time (5 minutes) and immediately mixed with 0.960 mL of 5 mM phosphate in water and the reaction was stopped by adjusting the pH of the diluted sample to less than pH 4. .. The resulting solution is passed through the ULTRAFREE® MC-filter device (30,000 Normal Molecular Weight Limit (NMWL), Millipore Corp, Billerica, MA; Catalog # UFC3LKT 00) at 12,000 rpm for 2 minutes. Was filtered by centrifugation. An aliquot (0.100 mL) of the resulting filtrate was added to a 1.5 mL screw cap HPLC vial (Agilent Technologies, Palo) containing 0.300 mL of deionized water. Transfer to Alto, CA; # 5182-0715), then add 20 mM MTS (methyl-p-tolylsulfide) in 0.100 mL of acetonitrile, cover the vial and about 25 ° C in the absence of light. The contents were briefly mixed before leaving at a constant temperature for 10 minutes. The vial is then added with 0.400 mL of acetonitrile and a solution of triphenylphosphine in 0.100 mL of acetonitrile (TPP, 40 mM), the vial is re-covered, the resulting solution is mixed and about in the absence of light. It was left at a constant temperature of 25 ° C for 30 minutes. Next, 0.100 mL of 10 mM N, N-diethyl-m-toluamide (DEET; HPLC internal standard) was added to the vial, and the resulting solution was mixed with MTSO (methyl-p-tolyl sulfoxide), MTS and peracetic acid. The stoichiometric oxidation products produced by the reaction were analyzed by HPLC. Control reaction in the absence of added extract protein or triacetin to measure the rate of MTS oxidation in the assay mixture with hydrogen peroxide to correct the rate of peracetic acid formation with respect to background MTS oxidation. I went to. HPLC method: Supelco Supelguard Supelco Discovery C8 column with Discovery C8 pre-column (Sigma-Aldrich; Catalog # 59590-U) (10 cm x 4.0 mm, 5 μm) (Catalog # 569422-U); 10 microliter injection volume; CH at 1.0 mL / min and ambient temperature₃Gradient method with CN (Sigma-Aldrich; Catalog # 270717) and deionized water.</p><p num="0244"><tables num="3"><img id="000008" he="56" wi="101" file="JP5777516B2_D0001.tif" img-format="tif" img-content="drawing" /></tables></p><p num="0245"> The perhydrolase activity of the T. neapolitana perhydrolase / trehalose spray-dried powder was stable over 8 weeks of storage at 40 ° C (Table 3).</p><p num="0246"><tables num="4"><img id="000009" he="86" wi="158" file="JP5777516B2_D0001.tif" img-format="tif" img-content="drawing" /></tables></p><p num="0247">Example 9 T. neapolitana Perhydrolase / trehalose spray-dried enzyme powder temperature stability in a mixture of enzyme powder and triacetin The spray-dried enzyme powder prepared as described in Example 8 was evaluated for stability when stored at 40 ° C. for 8 weeks as a mixture of spray-dried powders in triacetin. Spray-dried enzyme powder was added to triacetin to produce a mixture containing 0.200 g protein in 87.2 g triacetin. The resulting mixture was stored at 40 ° C. and a well-stirred 2.19 g sample of the mixture was buffered with 50 mM sodium bicarbonate buffer at pH 7.2, where the resulting concentrations of triacetin and protein were 100 mM and 50 μg / mL, respectively. Weekly assay was performed at 25 ° C. in a 100 mL reaction containing 100 mM hydrogen peroxide and TURPINAL® SL (500 ppm) in the solution. Comparison of the data in Table 4 with the data in Examples 8 and 3 demonstrates the instability of the T. neapolitana perhydrolase / trehalose spray-dried enzyme powder when stored as a mixture with triacetin. ..</p><p num="0248"><tables num="5"><img id="000010" he="90" wi="159" file="JP5777516B2_D0001.tif" img-format="tif" img-content="drawing" /></tables></p><p num="0249">Example 10 Temperature stability of T. neapolitana perhydrolase / maltodextrin spray-dried enzyme powder Heat-treated cell extract protein of E. coli KLP18 / pSW196 in 50 mM sodium bicarbonate (pH = 8.1) protein (34 g protein / L, 90% T. neapolitana perhydrolase by PAGE) and Maltodextrin as a form (66.7 g / L MALTRIN® M100 Maltdextrin, 14.7 g / L MALTRIN® M250, 14.7 g / L MALTRIN® M040, Grain Processing Corporation, Muscatine An aqueous mixture containing, IA) was prepared. Spray this solution with a spray dryer (GEA) Spray-dry using Niro, 3-foot diameter, inlet temperature = 226 ° C, outlet temperature = 76 ° C, feed rate = 60 g / min) to produce a spray-dried enzyme powder; mass percent protein in the powder ( 20.3% by mass) was measured using the BCA (bisinconic acid) protein assay, and the glass transition temperature (Tg = 54 ° C) of this powder was measured using the modulated differential scanning calorimetry. The solution was spray dried to produce a powder, which was then tested for stability during storage at 40 ° C. for 9 weeks. Spray-dried enzyme powder (stored at 40 ° C) was sampled at weekly intervals and at 25 ° C in 50 mM bicarbonate buffer (pH 7.2) at 50 μg protein / mL T. neapolitana. ) Perhydrolase, H₂O₂Assay for activity using (100 mM), triacetin (100 mM) and TURPINAL® SL (500 ppm) and for the production of peracetic acid using the modification of the analytical method reported by Karst et al. (See above). analyzed. The perhydrolase activity of the T. neapolitana perhydrolase / maltodextrin spray-dried powder was stable over 8 weeks of storage at 40 ° C (Table 5).</p><p num="0250"><tables num="6"><img id="000011" he="86" wi="158" file="JP5777516B2_D0001.tif" img-format="tif" img-content="drawing" /></tables></p><p num="0251">Example 11 Temperature stability of T. neapolitana perhydrolase / maltodextrin spray-dried enzyme powder stored in a mixture of enzyme powder and triacetin The spray-dried enzyme powder prepared as described in Example 10 was evaluated for stability when stored at 40 ° C. for 21 weeks as a mixture of spray-dried powders in triacetin. Spray-dried enzyme powder (1.235 g, 20.3 mass% protein) was added to 109 g of triacetin. The resulting mixture was stored at 40 ° C. and a well-stirred 2.19 g sample of the mixture was buffered with 50 mM sodium bicarbonate buffer at pH 7.2, where the resulting concentrations of triacetin and protein were 100 mM and 50 μg / mL, respectively. The assay was performed in a double repeat test at 25 ° C. in a 100 mL reaction solution containing hydrogen peroxide (100 mM) and TURPINAL® SL (500 ppm) in the solution. Comparison of the data in Table 6 with the data in Examples 10 and 5 demonstrates the stability of the T. neapolitana perhydrolase / maltodextrin spray-dried enzyme powder when stored as a mixture with triacetin. To do.</p><p num="0252"><tables num="7"><img id="000012" he="104" wi="158" file="JP5777516B2_D0001.tif" img-format="tif" img-content="drawing" /></tables></p><p num="0253">Example 12 Temperature stability of T. maritima perhydrolase / maltodextrin spray-dried enzyme powder Heat-treated cell extract protein of E. coli KLP18 / pSW207 in 50 mM sodium bicarbonate (pH = 8.1) (approximately 21 g protein / L, 90% T. maritima perhydrolase by PAGE) and An aqueous mixture containing maltodextrin as an excipient (31 g / L maltodextrin DE 13-17 and 31 g / L maltodextrin DE 4-7, Aldrich) was prepared. Buchi this solution B-290 Glass chamber spray dryer (inlet temperature = 170 ° C, outlet temperature = 90 ° C, supply rate = 4.5 mL / min) is used to spray dry to produce spray-dried enzyme powder; mass in powder Percentage protein (18.0% by mass) was measured using the BCA (bisinconic acid) protein assay and the glass transition temperature (Tg = 90 ° C) of this powder was measured using the modulated differential scanning calorimetry. The powder was then tested for stability during storage at 40 ° C for 7 weeks. Spray-dried enzyme powder (stored at 40 ° C) was sampled at weekly intervals and at 25 ° C in 50 mM bicarbonate buffer (pH 7.2) at 50 μg protein / mL T. maritima. ) Perhydrolase, H₂O₂Assayed for activity by addition to a reaction mixture containing (100 mM), triacetin (100 mM) and TURPINAL® SL (500 ppm) and analyzed for peracetic acid production using a modification of the analytical method reported by Karst et al. did. The perhydrolase activity of the T. maritima perhydrolase / maltodextrin spray-dried powder was stable over 7 weeks of storage at 40 ° C (Table 7).</p><p num="0254"><tables num="8"><img id="000013" he="81" wi="159" file="JP5777516B2_D0001.tif" img-format="tif" img-content="drawing" /></tables></p><p num="0255">Example 13 Temperature stability of T. maritima perhydrolase / maltodextrin spray-dried enzyme powder stored in a mixture of enzyme powder and triacetin The spray-dried enzyme powder prepared as described in Example 12 was evaluated for stability when stored at 40 ° C. for 7 weeks as a mixture of spray-dried powders in triacetin. Spray-dried enzyme powder (0.556 g, 18.0 mass% protein) was added to 43.6 g of triacetin. The resulting mixture was stored at 40 ° C. and a well-stirred 2.21 g sample of the mixture was subjected to 50 mM hydrogen peroxide at pH 7.2, where the concentration of triacetin and protein produced was 100 mM and 50 μg / mL, respectively. Assayed in a double repeat test at 25 ° C. in a 100 mL reaction containing hydrogen peroxide (100 mM) and TURPINAL® SL (500 ppm) in sodium buffer. Comparison of the data in Table 8 with the data in Examples 12 and 7 demonstrates the stability of the T. maritima perhydrolase / maltodextrin spray-dried enzyme powder when stored as a mixture with triacetin. To do.</p><p num="0256"><tables num="9"><img id="000014" he="84" wi="155" file="JP5777516B2_D0001.tif" img-format="tif" img-content="drawing" /></tables></p><p num="0257">Example 14 Bacillus subtilis ATCC® 31954^TMPerhydrolysis of propylene glycol diacetate or ethylene glycol diacetate using perhydrolase Bacillus subtilis ATCC® 31954^TMA homogenate of a transformant expressing wild-type perhydrolase from (KLP18 / pSW194) was added to a cell paste (20% by weight) in 0.05 M potassium phosphate buffer (pH 7.0) containing dithiothreitol (1 mM). Wet cell mass) prepared from suspension. The crude homogenate was centrifuged to remove cell debris to produce a purified cell extract, which was heat treated at 65 ° C. for 30 minutes. The resulting mixture was centrifuged and the heat-treated supernatant was concentrated to a concentration of 32 mg / mL total dissolved solids with a 30 K MWCO (molecular weight cutoff) membrane; SDS-PAGE of the purified heat-treated cell extract was perhydrolase. Was shown to be at least 85-90% pure. This concentrate is then given an approximately 3: 1 ratio (wt / wt) of phosphate buffer vs. heat treated cell extract protein to provide 2.06 grams of NaH per gram of solids.₂PO₄And 1.17 grams Na₂HPO₄Was added. This solution was diluted 30% by weight with deionized water and then spray dried using a Buchi B-290 laboratory spray dryer (180 ° C inlet temperature, 70 ° C outlet temperature); resulting spray. The dry powder contained 25.5% by weight protein (Bradford protein assay) and had a 94.3% by weight dry solids content.</p><p num="0258"> The reaction (10 mL total volume) was 123 μg / mL heat-treated extract protein from propylene glycol (PGDA) or ethylene glycol diacetate (EGDA), hydrogen peroxide (100 mM) and spray-dried E. coli KLP18 / pSW194. (Bacillus subtilis ATCC® 31954^TMIt was performed at 23 ° C. in 50 mM sodium bicarbonate buffer (initial pH 7.2) containing wild-type perhydrolase (expressing wild-type perhydrolase) (prepared as described above). A control reaction for each reaction condition was performed to measure the concentration of peracetic acid produced by the chemical perhydrolysis of triacetin with hydrogen peroxide in the absence of the added heat treated extract protein. Reactions were sampled at 1, 5, and 30 minutes and samples were analyzed for peracetic acid using the Karst derivatization procedure (Karst et al., See above); aliquots (0.040 mL) of the reaction mixture were removed and 0.960. Mix with 5 mM phosphate in mL of water; adjusting the pH of the sample diluted below pH 4 immediately stopped the reaction. ULTRAFREE® MC-filter device (30,000 Normal Molecular Weight Limit) (NMWL), Millipore Catalog # UFC3LKT It was filtered by centrifugation at 12,000 rpm for 2 minutes using 00). An aliquot (0.100 mL) of the resulting filtrate was transferred to a 1.5 mL screw cap HPLC vial (Agilent Technologies, Palo Alto, CA; # 5182-0715) containing 0.300 mL of deionized water, then 0.100 mL of acetonitrile. Medium 20 mM MTS (methyl-p-tolylsulfide) was added, the vials were capped and the contents were briefly mixed before culturing at about 25 ° C. in the absence of light for 10 minutes. Each vial is then added with 0.400 mL of acetonitrile and 0.100 mL of a solution of triphenylphosphine in acetonitrile (TPP, 40 mM), the vials are re-covered, the resulting solution is mixed and in the absence of light. The cells were cultured at about 25 ° C for 30 minutes. Each vial was then added with 0.100 mL of 10 mM N, N-diethyl-m-toluamide (DEET; HPLC internal standard) and the resulting solution was analyzed by HPLC. Table 9 lists the peracetic acid concentrations produced at 1, 5 and 30 minutes.</p><p num="0259"><tables num="10"><img id="000015" he="69" wi="159" file="JP5777516B2_D0001.tif" img-format="tif" img-content="drawing" /></tables></p><p num="0260">Example 15 Perhydrolysis of propylene glycol diacetate or ethylene glycol diacetate using T. maritima and T. neapolitana wild-type and mutant perhydrolase Thermotoga neapolitana Wild-type perhydrolase (KLP18 / pSW196), Thermotoga neapolitana C277S mutant Perhydrolase (KLP18 / pSW196 / C277S), Thermotoga neapolitana (Thermotoga neapolitana) KLP18 / pSW196 / C277T), Thermotoga maritima wild-type perhydrolase (KLP18 / pSW228), Thermotoga maritima C277S mutant perhydrolase (KLP18 / pSW228 / C277S), and Thermotoga maritima maritima) C277T mutant cell extracts of transformants expressing perhydrolase (KLP18 / pSW228 / C277T) in 0.05 M potassium phosphate buffer (pH 7.0) containing dithiothreitol (1 mM), respectively. A suspension of cell paste (20% by mass wet cell mass) was prepared by passing it through a French press with an operating pressure of 16,000 psi (about 110 MPa) twice. The lysed cells were centrifuged at 12,000 xg for 30 minutes to produce a purified cell extract, which was assayed for total soluble protein (Bradford assay). The supernatant was heated at 75 ° C for 20 minutes and then rapidly cooled in an ice bath for 2 minutes. Precipitated protein was removed by centrifugation at 11,000 xg for 10 minutes. SDS-PAGE of the resulting heat-treated extract protein supernatant showed that the CE-7 enzyme accounted for approximately 85-90% of the total protein in the preparation. The heat-treated extract protein supernatant was frozen on dry ice and stored at -80 ° C until use.</p><p num="0261"> The first set of reactions (10 mL total volume) were propylene glycol diacetate (PGDA) or ethylene glycol diacetate (EGDA) (100 mM), hydrogen peroxide (100 mM) and E. coli KLP18 / pSW196 (Thermotoga. Neapolitana (Thermotoga neapolitana) wild-type perhydrolase), E. coli (E. coli) KLP18 / pSW196 / C277S (Thermotoga neapolitana C277S mutant perhydrolase), E. coli (E. coli) KLP18 / pSW196 / C277T (thermotoga) Neapolitana (Thermotoga neapolitana) C277T mutant perhydrolase), E. coli (E. coli) KLP18 / pSW228 (Thermotoga maritima wild type perhydrolase), E. coli (E. coli) KLP18 / pSW228 / C277S (Thermotoga) E. coli (Thermotoga) 25 μg / from one of maritima) C277S mutant perhydrolase) and E. coli KLP18 / pSW228 / C277T (Thermotoga maritima C277T mutant perhydrolase) (prepared as described above) The procedure was performed at 20 ° C. in 10 mM sodium bicarbonate buffer (initial pH 8.1) containing mL of the heat-treated extract protein. A control reaction for each reaction condition was performed to measure the concentration of peracetic acid produced by the chemical perhydrolysis of triacetin with hydrogen peroxide in the absence of the added extract protein. Reactions were sampled at 1, 5, and 30 minutes and samples were analyzed for peracetic acid using the Karst derivatization procedure (Karst et al., See above) and HPLC analytical methods (see above). Table 10 lists the peracetic acid concentrations produced at 1, 5 and 30 minutes.</p><p num="0262"><tables num="11"><img id="000016" he="124" wi="159" file="JP5777516B2_D0001.tif" img-format="tif" img-content="drawing" /></tables></p><p num="0263"> The second set of reactions (10 mL total volume) were propylene glycol diacetate (PGDA) or ethylene glycol diacetate (EGDA) (2 mM), hydrogen peroxide (10 mM) and E. coli KLP18 / pSW196 (Thermotoga. Neapolitana (Thermotoga neapolitana) wild-type perhydrolase), E. coli (E. coli) KLP18 / pSW196 / C277S (Thermotoga neapolitana C277S mutant perhydrolase), E. coli (E. coli) KLP18 / pSW196 / C277T (thermotoga) Neapolitana (Thermotoga neapolitana) C277T mutant perhydrolase), E. coli (E. coli) KLP18 / pSW228 (Thermotoga maritima wild type perhydrolase), E. coli (E. coli) KLP18 / pSW228 / C277S (Thermotoga) E. coli (Thermotoga) 10 μg / from one of maritima) C277S mutant perhydrolase) and E. coli KLP18 / pSW228 / C277T (Thermotoga maritima C277T mutant perhydrolase) (prepared as described above) The procedure was performed at 20 ° C. in 10 mM sodium bicarbonate buffer (initial pH 8.1) containing mL of the heat-treated extract protein. A control reaction for each reaction condition was performed to measure the concentration of peracetic acid produced by the chemical perhydrolysis of triacetin with hydrogen peroxide in the absence of the added extract protein. The reaction was sampled at 5 minutes and the sample was analyzed for peracetic acid using the Karst derivatization procedure (Karst et al., See above) and the HPLC analysis method (see above). Table 11 lists the peracetic acid concentrations produced in 5 minutes.</p><p num="0264"><tables num="12"><img id="000017" he="126" wi="159" file="JP5777516B2_D0001.tif" img-format="tif" img-content="drawing" /></tables></p>