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WO2011071762A1 - Perhydrolase fournissant une stabilité améliorée aux peracides - Google Patents

Perhydrolase fournissant une stabilité améliorée aux peracides Download PDF

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Publication number
WO2011071762A1
WO2011071762A1 PCT/US2010/058844 US2010058844W WO2011071762A1 WO 2011071762 A1 WO2011071762 A1 WO 2011071762A1 US 2010058844 W US2010058844 W US 2010058844W WO 2011071762 A1 WO2011071762 A1 WO 2011071762A1
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Prior art keywords
acid
group
optionally substituted
perhydrolase
hydroxyl
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Robert Dicosimo
John Edward Gavagan
Mark S. Payne
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EIDP Inc
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EI Du Pont de Nemours and Co
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2/00—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor
    • A61L2/16—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor using chemical substances
    • A61L2/18—Liquid substances or solutions comprising solids or dissolved gases
    • A61L2/186—Peroxide solutions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/01—Carboxylic ester hydrolases (3.1.1)
    • C12Y301/01072—Acetylxylan esterase (3.1.1.72)

Definitions

  • the invention relates to the field of peroxycarboxylic acid biosynthesis and enzyme catalysis. More specifically, an enzyme catalyst comprising a variant enzyme having perhydrolytic activity is provided having an increase in the ratio of peracetic acid formation (PAAF) specific activity relative to peracetic acid hydrolysis (PAAH) specific activity (PAAF/PAAH ratio) when compared to the PAAF/PAAH ratio of the Thermotoga maritima wild-type perhydrolase.
  • PAAF peracetic acid formation
  • PAAH peracetic acid hydrolysis
  • PAAF/PAAH ratio peracetic acid hydrolysis
  • Use of the variant enzyme enhances the yield and stability of the peracid produced by enzymatic perhydrolysis.
  • Methods of using the present enzyme catalyst to produce peroxycarboxylic acids are also provided.
  • Peroxycarboxylic acid compositions can be effective antimicrobial agents. Methods of using peroxycarboxylic acids to clean, disinfect, and/or sanitize hard surfaces, textiles, meat products, living plant tissues, and medical devices against undesirable microbial growth have been described (U.S.
  • Peroxycarboxylic acids have also been used in a various bleaching
  • the desired efficacious concentration of peroxycarboxylic acid may vary according to the product application (for example, ca. 500 ppm to 1000 ppm for medical instrument disinfection, ca. 30 ppm to 80 ppm for laundry bleaching or disinfection applications) in 1 min to 5 min reaction time at neutral pH.
  • carbohydrate esterases (CE-7) have been employed as perhydrolases to catalyze the reaction of hydrogen peroxide (or alternative peroxide reagent) with alkyl esters of carboxylic acids in water at a basic to acidic pH range (from ca. pH 10 to ca. pH 5) to produce an efficacious concentration of a
  • peroxycarboxylic acid for such applications as disinfection (such as medical instruments, hard surfaces, textiles), bleaching (such as wood pulp or paper pulp processing/delignification, textile bleaching and laundry care applications), and other laundry care applications such as destaining, deodorizing, and sanitization (Published U.S. Patent Application Nos. 2008/0176783,
  • CE-7 enzymes have been found to have high specific activity for perhydrolysis of esters, particularly acetyl esters of alcohols, diols and glycerols.
  • CE-7 perhydrolases may also hydrolyze the carboxylic acid ester substrate.
  • an enzyme catalyst having high selectivity for perhydrolysis (P) relative to hydrolysis (H) when synthesizing peroxycarboxylic acids from carboxylic acid esters i.e., an enzyme catalyst having a higher "P to H" ratio.
  • CE-7 family of carbohydrate esterases has been identified as a class of perhydrolytic enzymes having desirable specific activities for peroxycarboxylic acid formation (e.g., peracetic acid formation; PAAF) and/or desirable perhydrolysis to hydrolysis (P/H) ratios for carboxylic acid ester substrates, these enzymes may also have an undesirable enzymatic activity for hydrolyzing the peroxycarboxylic acid product (e.g., peracetic acid hydrolysis; PAAH) to the corresponding carboxylic acid and hydrogen peroxide.
  • PAAH peracetic acid hydrolysis
  • an enzyme catalyst comprising a CE-7 perhydrolase characterized by a higher PAAF/PAAH ratio may provide greater peroxycarboxylic acid stability in formulations comprising the enzyme catalyst.
  • the problem to be solved is to provide an enzyme catalyst comprising a CE-7 carbohydrate esterase having perhydrolytic activity and a higher
  • a nucleic acid molecule encoding the Thermotoga maritime acetyl xylan esterase (SEQ ID NO: 2) was mutated by error-prone PCR and/or site-directed mutagenesis to create a library of variant perhydrolases.
  • Several perhydrolase variants were identified exhibiting an increase in the ratio of peracetic acid formation (PAAF) to peracetic acid hydrolysis (PAAH) specific activities when compared to the PAAF/PAAH ratio of the wild-type Thermotoga maritima perhydrolase having amino acid sequence SEQ ID NO: 2 under the same assay conditions.
  • PAAF peracetic acid formation
  • PAAH peracetic acid hydrolysis
  • an isolated nucleic acid molecule encoding a polypeptide having perhydrolytic activity is provided selected from the group consisting of:
  • a vector, a recombinant DNA construct, and a recombinant host cell comprising the present polynucleotide are also provided.
  • a method for transforming a cell comprising transforming a ceil with the above nucleic acid molecule.
  • an isolated polypeptide having perhydrolysis activity comprising amino acid sequence SEQ ID NO: 22.
  • the variant polypeptide having perhydrolytic activity is characterized by at least a 1. -fold increase in the PAAF/PAAH ratio of specific activities when compared to the PAAF/PAAF ratio of specific activities of the Thermotoga maritima wild-type sequence SEQ ID NO: 2.
  • a process for producing a peroxycarboxylic acid comprising:
  • R 6 C1 to C7 linear, branched or cyclic
  • Ri C1 to C7 straight chain or branched chain alkyl optionally substituted with an hydroxyl or a C1 to C4 alkoxy group and R 3 and R 4 are individually H or RiC ⁇ 0);
  • R-i is a C1 to C7 straight chain or branched chain alkyl optionally substituted with an hydroxyl or a C1 to C4 alkoxy group and R 2 is a C1 to C10 straight chain or branched chain alkyl, alkenyl, alkynyl, aryl, alkylaryl, alkylheteroaryl, heteroaryl, (CH 2 CH 2 0) n , or (CH 2 CH(CH 3 )-0) n H and n is 1 to 10;
  • step (c) optionally diluting the peroxycarboxylic acid produced in step (b).
  • a process is provided further comprising a step
  • step (d) wherein the peroxycarboxylic acid produced in step (b) or step (c) is contacted with a hard surface, an article of clothing or an inanimate object whereby the hard surface, article of clothing or inanimate object is disinfected, sanitized, bleached, destained, deodorized or any combination thereof.
  • composition comprising:
  • At least one substrate selected from the group consisting of:
  • Re C1 to C7 linear, branched or cyclic
  • Ri C1 to C7 straight chain or branched chain alkyl optionally substituted with an hydroxyl or a C1 to C4 alkoxy group and R 3 and R4 are individually H or RiC(0);
  • Ri is a C1 to C7 straight chain or branched chain alkyl optionally substituted with an hydroxyl or a C1 to C4 alkoxy group and R 2 is a C1 to C10 straight chain or branched chain alkyl, alkenyl, alkynyl, aryl, alkylaryl, alkylheteroaryl, heteroaryl, (CH 2 CH 2 0) n , or (CH 2 CH(CH 3 )-0) n H and n is 1 to 10;
  • the present process produces the desired peroxycarboxylic acid upon combining the reaction components.
  • the reaction components may remain separated until use.
  • a peracid generation and delivery system comprising:
  • X an ester group of the formula R 6 -C(0)0;
  • R 6 C1 to C7 linear, branched or cyclic
  • C1 to C7 straight chain or branched chain alkyl optionally substituted with an hydroxyl or a C1 to C4 alkoxy group and R 3 and R 4 are individually H or RiC(0);
  • R-i is a C1 to C7 straight chain or branched chain a!kyl optionally substituted with an hydroxyl or a C1 to C4 alkoxy group and R 2 is a C1 to C10 straight chain or branched chain alkyl, alkenyl, alkynyl, aryl, alkylaryl, alkylheteroaryl, heteroaryl, (CH 2 CH 2 0) n , or (CH 2 CH(CH 3 )-0)nH and n is 1 to 10;
  • a second compartment comprising (1) source of peroxygen
  • a laundry care composition comprising a polypeptide comprising amino acid sequence SEQ ID NO: 22.
  • SEQ ID NO: 1 is the nucleic acid sequence of the codon-optimized coding region encoding the wild-type Thermotoga maritima acetyl xylan esterase having perhydrolytic activity.
  • SEQ ID NO: 2 is the amino acid sequence of the wild-type Thermotoga maritima acetyl xylan esterase having perhydrolytic activity .
  • SEQ ID NOs: 3 and 4 are the nucleic acid sequences of primers used to prepare the C277S variant acetyl xylan esterase.
  • SEQ ID NO: 5 is the amino acid sequence of the C277S variant acetyl xylan esterase having perhydrolytic activity (U.S. Patent Application No.
  • SEQ ID NO: 6 is the nucleic acid sequence of the plasmid
  • SEQ ID NOs: 7 and 8 are the nucleic acid sequences of primers used for error-prone PCR.
  • SEQ ID NO: 9 is the nucleic acid sequence encoding the "A3" variant acetyl xylan esterase having the following substitutions relative to the wild-type Thermotoga maritime acetyl xylan esterase amino acid sequence:
  • SEQ [D NO: 10 is the amino acid sequence of the "A3" variant acetyl xylan esterase.
  • SEQ ID NOs: 11 and 12 are the nucleic acid sequences of primers used to construct the N275D/C277S variant acetyl xylan esterase.
  • SEQ ID NO: 13 is the nucleic acid sequence encoding the
  • SEQ ID NO: 14 is the amino acid sequence of the N275D/C277S variant acetyl xylan esterase.
  • SEQ ID NOs: 15 and 16 are the nucleic acid sequences of primers used to construct the C277S/F317S variant acetyl xylan esterase.
  • SEQ ID NO: 17 is the nucleic acid sequence encoding the
  • SEQ ID NO: 18 is the amino acid sequence of the C277S/F317S variant acetyl xylan esterase.
  • SEQ ID NOs: 19 and 20 are the nucleic acid sequences of primers used to construct the S35T/C277S variant acetyl xylan esterase.
  • SEQ ID NO: 21 is the nucleic acid sequence encoding the S35T/C277S variant acetyl xylan esterase.
  • SEQ ID NO: 22 is the amino acid sequence of the S35T/C277S variant acetyl xylan esterase.
  • SEQ ID NOs: 23 and 24 are the nucleic acid sequences of primers used to construct the Q179L/C277S variant acetyl xylan esterase.
  • SEQ ID NO: 25 is the nucleic acid sequence encoding the
  • SEQ ID NO: 26 is the amino acid sequence of the Q179L/C277S variant acetyl xylan esterase.
  • a nucleic acid molecule encoding the Thermotoga maritima acetyl xylan esterase (SEQ ID NO: 2) was mutated by error-prone PCR and/or site-directed mutagenesis to create a library of variant perhydrolases.
  • Several perhydrolase variants were identified exhibiting an increase in the ratio of peracetic acid formation (PAAF) to peracetic acid hydrolysis (PAAH) specific activities when compared to the PAAF/PAAH ratio of the wild-type Thermotoga maritima perhydrolase having amino acid sequence SEQ ID NO: 2.
  • the term "about" modifying the quantity of an ingredient or reactant employed refers to variation in the numerical quantity that can occur, for example, through typical measuring and liquid handling procedures used for making concentrates or use solutions in the real world; through inadvertent error in these procedures; through differences in the manufacture, source, or purity of the ingredients employed to make the compositions or carry out the methods; and the like.
  • the term “about” also encompasses amounts that differ due to different equilibrium conditions for a composition resulting from a particular initial mixture. Whether or not modified by the term “about”, the claims include equivalents to the quantities.
  • multi-component system will refer to a system of enzymatically generating peroxycarboxylic acid wherein the components remain separated until use.
  • the multi-component system will include at least one first component that remains separated from at least one second component. The first and second components are separated in different compartments until use (i.e., using first and second compartments).
  • the design of the multi-component systems will often depend on the physical form of the components to be combined and are described in more detail below.
  • peroxycarboxylic acid is synonymous with peractd, peroxyacid, peroxy acid, percarboxylic acid and peroxoic acid.
  • peracetic acid is abbreviated as "PAA” and is synonymous with peroxyacetic acid, ethaneperoxoic acid and all other synonyms of CAS Registry Number 79-21-0.
  • monoacetin is synonymous with glycerol monoacetate, glycerin monoacetate, and glyceryl monoacetate.
  • diacetin is synonymous with glycerol diacetate; glycerin diacetate, glyceryl diacetate, and all other synonyms of CAS
  • triacetin is synonymous with glycerin triacetate; glycerol triacetate; glyceryl triacetate, 1,2,3-triacetoxypropane, 1 ,2,3- propanetriol triacetate and all other synonyms of CAS Registry Number 102-
  • the term "monobutyrin” is synonymous with glycerol monobutyrate, glycerin monobutyrate, and glyceryl monobutyrate.
  • dibutyrin is synonymous with glycerol dibutyrate and glyceryl dibutyrate.
  • tributyrin is synonymous with glycerol tributyrate, 1 , 2 , 3-tri buty ry Ig ly cerol : and all other synonyms of CAS Registry Number 60-01-5.
  • the term "monopropionin” is synonymous with glycerol monopropionate, glycerin monopropionate, and glyceryl monopropionate.
  • dipropionin is synonymous with glycerol dipropionate and glyceryl dipropionate.
  • tripropionin is synonymous with glyceryl tripropionate, glycerol tripropionate, 1 ,2,3-tripropionylglycerol, and all other synonyms of CAS Registry Number 139-45-7.
  • ethyl acetate is synonymous with acetic ether, acetoxyethane, ethyl ethanoate, acetic acid ethyl ester, ethanoic acid ethyl ester, ethyl acetic ester and all other synonyms of CAS Registry Number 141-78-6.
  • ethyl lactate is synonymous with lactic acid ethyl ester and all other synonyms of CAS Registry Number 97-64-3.
  • saccharide refers to mono-, di- and polysaccharides comprising at least one acetyl group. Examples include, but are not limited to, glucose pentaacetate, xylose tetraacetate, acetylated xylan, acetylated xylan fragments, ⁇ -D- ribofuranose-1 ,2,3,5-tetraacetate, tri-O-acetyl-D-galactal, and tri-O-acetyl- glucal.
  • hydrocarbyl As used herein, the terms “hydrocarbyl”, “hydrocarbyl group”, and “hydrocarbyl moiety” mean a straight chain, branched or cyclic arrangement of carbon atoms connected by single, double, or triple carbon to carbon bonds and/or by ether linkages, and substituted accordingly with hydrogen atoms. Such hydrocarbyl groups may be aliphatic and/or aromatic.
  • hydrocarbyl groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, cyclopropyl, cyclobutyl, pentyl, cyc!opentyl, methylcyclopentyl, hexyl, cyclohexyl, benzyl, and phenyl.
  • the hydrocarbyl moiety is a straight chain, branched or cyclic arrangement of carbon atoms connected by single carbon to carbon bonds and/or by ether linkages, and substituted accordingly with hydrogen atoms.
  • suitable enzymatic reaction formulation As used herein, the terms “suitable enzymatic reaction formulation”, “components suitable for generation of a peroxycarboxylic acid”, “suitable reaction components”, “reaction components”, “reaction formulation”, and “suitable aqueous reaction formulation” refer to the materials and water in which the reactants and the enzyme catalyst comprising the present variant polypeptide having perhydrolytic activity come into contact to form the desired peroxycarboxylic acid.
  • the components of the reaction formulation are provided herein and those skilled in the art appreciate the range of component variations suitable for this process.
  • the enzymatic reaction formulation produces peroxycarboxylic acid in situ upon combining the reaction components.
  • the reaction components may be provided as a multi- component system wherein one or more of the reaction components remains separated until use.
  • multi-component systems used to generate peroxycarboxylic acid may include, but are not limited to, those designed for one or more solid components or combinations of solid-liquid components, such as powders used in many commercially available bleaching compositions (e.g., U.S. Patent 5,116,575), multi-layered tablets (e.g., U.S. Patent 6,210,639), water dissolvable packets having multiple compartments (e.g., U.S. Patent 6,995,125) and solid agglomerates that react upon the addition of water (e.g., U.S. Patent 6,319,888).
  • solid-liquid components such as powders used in many commercially available bleaching compositions (e.g., U.S. Patent 5,116,575), multi-layered tablets (e.g., U.S. Patent 6,210,639), water dissolvable packets having multiple compartments (e.g., U.S. Patent 6,995,125) and solid agglomerates that react upon the addition of water (e.g., U
  • the term "substrate” or “carboxylic acid ester substrate” will refer to the reaction components enzymaticaliy perhydrolyzed using the present enzyme catalyst in the presence of a suitable source of peroxygen, such as hydrogen peroxide.
  • the substrate comprises at least one ester group capable of being enzymaticaliy perhydrolyzed using the enzyme catalyst, whereby a peroxycarboxylic acid is produced.
  • perhydrolysis is defined as the reaction of a selected substrate with a source of hydrogen peroxide to form a
  • inorganic peroxide is reacted with the selected substrate in the presence of a catalyst to produce the
  • chemical perhydrolysis includes perhydrolysis reactions in which a substrate (such as a
  • peroxycarboxylic acid precursor is combined with a source of hydrogen peroxide wherein peroxycarboxylic acid is formed in the absence of an enzyme catalyst.
  • enzyme perhydrolysis refers a reaction of a selected substrate with a source of hydrogen peroxide to form a peroxycarboxylic acid, wherein the reaction is catalyzed by an enzyme catalyst having perhydrolysis activity.
  • perhydrolase activity refers to the enzyme catalyst activity per unit mass (for example, milligram) of protein, dry cell weight, or immobilized catalyst weight.
  • one unit of enzyme activity or “one unit of activity” or “U” is defined as the amount of perhydrolase activity required for the production of 1 ⁇ of peroxycarboxylic acid product (such as peracetic acid) per minute at a specified temperature.
  • One unit of enzyme activity may also be used herein to refer to the amount of peroxycarboxylic acid hydrolysis activity required for the hydrolysis of 1 pmol of peroxycarboxylic acid (e.g., peracetic acid) per minute at a specified temperature.
  • PAAF peracetic acid formation
  • PAAH peracetic acid hydrolysis
  • PAAF/PAAH ratio refers to the ratio of the specific activities of the variant enzyme catalyst for producing peracetic acid from a carboxylic acid ester substrate and for hydrolyzing peracetic acid into acetic acid and hydrogen peroxide, respectively.
  • Enzymatically-produced peracids in reaction formulations comprising an enzyme catalyst having a perhydrolytic enzyme having an increased PAAF/PAAH ratio of specific activities are typically more stable as the peracid is less likely to be hydrolyzed to the corresponding carboxylic acid and hydrogen peroxide when the peracid formulation comprises the perhydrolytic enzyme.
  • reactions to measure peracetic acid formation (PAAF) specific activity are run at ca.
  • the reactions to measure peracetic acid hydrolysis (PAAH) specific activity are run at ca. 25 °C, phosphate buffer (50 mM, pH 7.2) containing approximately 2000 ppm peracetic acid (ca. 26.3 mM) and 25 pg/mL of heat-treated extract supernatant total protein from E. coli strain KLP18 expressing wild-type or variant perhydrolase (see Example 14).
  • PAAH peracetic acid hydrolysis
  • the fold increase in PAAF/PAAH ratio of specific activities is measured relative to the PAAF/PAAH ratio of the Thermotoga maritime wild-type perhydrolase (SEQ ID NO: 2) under the same reaction conditions.
  • the fold increase in the PAAF/PAAH ratio of the variant polypeptide (i.e., variant perhydrolase) relative to the Thermotoga maritima wild-type perhydrolase is at least 1.1 , 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9., 2.0, 3,0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, or 10-fold when compared under identical reaction/assay conditions.
  • the fold increase in the PAAF/PAAH ratio of the variant enzyme may be measured relative to the C277S variant perhydrolase of SEQ ID NO: 5 (U.S. Patent Application No. 2010/0087529) and is at least 1.1 , 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9., 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, or 10-fold when compared under identical reaction conditions.
  • identification assay conditions or “same assay conditions” refer to the conditions used to measure the peracid formation (i.e.,
  • perhydrolysis of a carboxylic acid ester substrate specific activity or the peroxycarboxylic acid hydrolysis specific activity of the variant polypeptide relative to the respective specific activities of the polypeptide from which is was derived (i.e., Thermotoga maritime wild-type acetyl xylan esterase of SEQ ID NO: 2).
  • the assay conditions used to measure the respective specific activities should be as close to identical as possible such that only the structure of the polypeptide having perhydrolytic activity varies.
  • the carboxylic acid ester substrate and the corresponding peroxycarboxylic acid used to measure the respective perhydrolytic specific activity and peracid hydrolysis specific activities may vary depending upon the desired substrate/product combination.
  • the perhydrolytic specific activity is measured using triacetin as a substrate and the peracid hydrolysis specific activity is measured using peracetic acid as the respective peracid.
  • reactions used to measure peracetic acid formation (PAAF) specific activity are run at ca. 25 °C in phosphate buffer (50 mM, pH 7.2) containing 100 mM triacetin, 100 mM hydrogen peroxide and approximately 2.5 ⁇ g/mL of heat-treated extract supernatant total protein from E. coli strain KLP18 expressing wild-type or variant perhydrolase (see example 13).
  • the reactions to measure peracetic acid hydrolysis (PAAH) specific activity are run at ca.
  • enzyme catalyst and “perhydrolase catalyst” refer to a catalyst comprising an enzyme (i.e., a polypeptide) having
  • perhydrolysis activity may be in the form of a whole microbial cell, permeabilized microbial cell(s), one or more cell components of a microbial cell extract, partially purified enzyme, or purified enzyme.
  • the enzyme catalyst may also be chemically modified (for example, by pegylation or by reaction with cross-linking reagents).
  • the perhydrolase catalyst may also be immobilized on a soluble or insoluble support using methods well-known to those skilled in the art; see for example, Immobilization of Enzymes and Cells; Gordon F. Bickerstaff, Editor; Humana Press, Totowa, NJ, USA; 1997.
  • the present enzyme catalyst comprises a variant polypeptide having perhydrolytic activity and is structurally classified as a member of the carbohydrate family esterase family 7 (CE-7 family) of enzymes (see Coutinho, P.M., Henrissat, B. "Carbohydrate-active enzymes: an integrated database approach" in Recent Advances in Carbohydrate Bioengineering, H.J. Gilbert, G. Davies, B. Henrissat and B. Svensson eds., (1999) The Royal Society of Chemistry, Cambridge, pp. 3-12.).
  • CE-7 family of enzymes has been demonstrated to be particularly effective for producing peroxycarboxylic acids from a variety of carboxylic acid ester substrates when combined with a source of peroxygen (See PCT publication No.
  • the CE-7 enzyme family includes cephalosporin C deacetylases (CAHs; E.C. 3.1.1.4 ) and acetyl xylan esterases (AXEs; E.C. 3.1.1.72).
  • CAHs cephalosporin C deacetylases
  • AXEs acetyl xylan esterases
  • Members of the CE-7 enzyme family share a conserved signature motif (Vincent et a/., J. Mol. Biol., 330:593-606 (2003)).
  • signature motif and "CE-7 signature motif, refer to conserved structures shared among a family of enzymes having a perhydrolytic activity.
  • structurally classified as a CE-7 enzyme As used herein, "structurally classified as a CE-7 enzyme”, “structurally classified as a carbohydrate esterase family 7 enzyme”, “structurally classified as a CE-7 carbohydrate esterase”, and “CE-7 perhydrolase” wilt be used to refer to enzymes having perhydrolysis activity that are structurally classified as a CE-7 carbohydrate esterase based on the presence of the CE-7 signature motif (Vincent et al., supra).
  • the "signature motif for CE-7 esterases comprises three conserved motifs (residue position numbering relative to reference sequence SEQ ID NO; 2; the wild-type Thermotoga maritima acetyl xylan esterase): a) Arg118-Gly119-Gln120;
  • the Xaa at amino acid residue position 187 is glycine, alanine, proline, tryptophan, or threonine. Two of the three amino acid residues belonging to the catalytic triad are in bold. In one embodiment, the Xaa at amino acid residue position 187 is selected from the group consisting of glycine, alanine, proline, tryptophan, and threonine.
  • the signature motif defined above includes a fourth conserved motif defined as: Leu272-Xaa273-Asp274.
  • the Xaa at amino acid residue position 273 is typically isoleucine, valine, or methionine.
  • the fourth motif includes the aspartic acid residue (bold) belonging to the catalytic triad (Ser188-Asp274-His303).
  • cephalosporin C deacetylase As used herein, the terms "cephalosporin C deacetylase” and
  • cephalosporin C acetyl hydrolase refers to an enzyme (E.C. 3.1.1.41) that catalyzes the deacetylation of cephalosporins such as cephalosporin C and 7- aminocephalosporanic acid (Mitsushima er a/., Appl. Environ. Microbiol., 61 (6): 2224-2229 (1995); U.S. 5,528,152; and U.S. 5,338,676). Enzymes classified as cephalosporin C deacetylases have been shown to often have significant perhydrolytic activity (U.S. Patent Application Publication Nos. 2008-0176783 and 2008-0176299 to DiCosimo ef a/.). As used herein, “acetyl xylan esterase” refers to an enzyme (E.C.
  • Thermotoga maritima refers to a bacterial cell reported to have acetyl xylan esterase activity (GENBANK ® NP_227893.1).
  • the Thermotoga maritima strain is Thermotoga maritima MSB8.
  • the amino acid sequence of the wild-type enzyme having perhydrolase activity from Thermotoga maritima is provided as SEQ ID NO: 2.
  • variant refers to an enzyme catalyst comprising at least one polypeptide (i.e., a perhydrolase) having perhydrolytic activity wherein the polypeptide comprises at least one amino acid change relative to the enzyme/polypeptide from which it was derived (typically the wild-type perhydrolase).
  • polypeptide i.e., a perhydrolase
  • variant polypeptides are provided herein having perhydrolytic activity and are characterized by an increase in the PAAF/PAAH ratio relative to the Thermotoga maritima wild-type acetyl xylan esterase having amino acid sequence SEQ ID NO: 2.
  • amino acid substitutions are specified with reference to the Thermotoga maritima amino acid sequence (SEQ ID NO: 2).
  • the wild-type amino acid (denoted by the standard single letter abbreviation) is followed by the amino acid residue position of SEQ ID NO: 2 followed by the amino acid of the variant (also denoted by the standard single letter abbreviation).
  • C277S describes a change in SEQ ID NO: 2 at amino acid residue position 277 where cysteine was changed to serine.
  • the variant polypeptide may be comprised of multiple point
  • N275D/C277S refers to a variant polypeptide having two point substitutions: 1) a change at amino acid residue position 275 where an asparagine was changed to aspartic acid, and 2) a change at residue position 277 wherein a cysteine was changed to a serine.
  • amino acid refers to the basic chemical structural unit of a protein or polypeptide. The following abbreviations are used herein to identify specific amino acids:
  • biological contaminants refers to one or more unwanted and/or pathogenic biological entities including, but not limited to, microorganisms, spores, viruses, prions, and mixtures thereof.
  • the present enzyme can be used to produce an efficacious concentration of at least one peroxycarboxylic acid useful to reduce and/or eliminate the presence of the viable biological contaminants.
  • the biological contaminant is a viable pathogenic microorganism.
  • the term "disinfect” refers to the process of destruction of or prevention of the growth of biological contaminants.
  • disinfectant refers to an agent that disinfects by destroying, neutralizing, or inhibiting the growth of biological contaminants. Typically, disinfectants are used to treat inanimate objects or surfaces.
  • antiseptic refers to a chemical agent that inhibits the growth of disease- carrying microorganisms.
  • the biological contaminants are pathogenic microorganisms.
  • sanitary means of or relating to the restoration or preservation of health, typically by removing, preventing or controlling an agent that may be injurious to health.
  • cleaning means to make sanitary.
  • sanitizer refers to a sanitizing agent.
  • sanitization refers to the act or process of sanitizing.
  • virucide refers to an agent that inhibits or destroys viruses, and is synonymous with "viricide”.
  • An agent that exhibits the ability to inhibit or destroy viruses is described as having "virucidal” activity.
  • Peroxycarboxylic acids can have virucidal activity.
  • Typical alternative virucides known in the art which may be suitable for use with the present invention include, for example, alcohols, ethers, chloroform, formaldehyde, phenols, beta propiolactone, iodine, chlorine, mercury salts, hydroxylamine, ethylene oxide, ethylene glycol, quaternary ammonium compounds, enzymes, and detergents.
  • biocide refers to a chemical agent, typically broad spectrum, which inactivates or destroys microorganisms.
  • a chemical agent that exhibits the ability to inactivate or destroy microorganisms is described as having "biocidal” activity.
  • Peroxycarboxylic acids can have biocidal activity.
  • Typical alternative biocides known in the art, which may be suitable for use in the present invention include, for example, chlorine, chlorine dioxide, chloroisocyanurates, hypochlorites, ozone, acrolein, amines, chlorinated phenolics, copper salts, organo-su!phur compounds, and quaternary ammonium salts.
  • the phrase "minimum biocidal concentration” refers to the minimum concentration of a biocidal agent that, for a specific contact time, will produce a desired lethal, irreversible reduction in the viable population of the targeted microorganisms.
  • the effectiveness can be measured by the logio reduction in viable microorganisms after treatment.
  • the targeted reduction in viable microorganisms after treatment is at least a 3-log reduction, more preferably at least a 4-log reduction, and most preferably at least a 5-log reduction.
  • the minimum biocidal concentration is at least a 6-log reduction in viable microbial cells.
  • peroxygen source and “source of peroxygen” refer to compounds capable of providing hydrogen peroxide at a concentration of about 1 mM or more when in an aqueous solution including, but not limited to, hydrogen peroxide, hydrogen peroxide adducts (e.g., urea- hydrogen peroxide adduct (carbamide peroxide)), perborates, and
  • the concentration of the hydrogen peroxide provided by the peroxygen compound in the aqueous reaction formulation is initially at least 1 mM or more upon combining the reaction components.
  • the hydrogen peroxide concentration in the aqueous reaction formulation is at least 10 mM.
  • the hydrogen peroxide concentration in the aqueous reaction formulation is at least 100 mM.
  • the hydrogen peroxide concentration in the aqueous reaction formulation is at least 200 mM.
  • the hydrogen peroxide concentration in the aqueous reaction formulation is 500 mM or more.
  • the hydrogen peroxide concentration in the aqueous reaction formulation is 1000 mM or more.
  • (H 2 0 2 :substrate) in the aqueous reaction formulation may be from about 0.002 to 20, preferably about 0.1 to 10, and most preferably about 0.5 to 5.
  • the term "benefit agent” refers to a material that promotes or enhances a useful advantage, a favorable/desirable effect or benefit.
  • a process is provided whereby a benefit agent, such as a composition comprising a peroxycarboxylic acid, is applied to a textile or article of clothing to achieve a desired benefit, such as disinfecting, bleaching, destaining, deodorizing, and any combination thereof.
  • a benefit agent such as a composition comprising a peroxycarboxylic acid
  • the present variant polypeptides were derived from the Thermotoga maritime wild-type acetyl xylan esterase that has been previously
  • peroxycarboxylic acids from carboxylic acid esters and a source of peroxygen, such as hydrogen peroxide (U.S. Patent Application Publication No. 2008- 0176299 to DiCosimo et a/.).
  • a source of peroxygen such as hydrogen peroxide
  • the Thermotoga maritima wild-type acetyl xylan esterase also has the ability to hydrolyze the peroxycarboxylic acid product into the corresponding carboxylic acid and hydrogen peroxide.
  • the desired peroxycarboxylic acid-based bleaching or disinfecting formulation comprising enzymatically-produced peroxycarboxylic acid will likely contain at least some of the active enzyme catalyst, there is a need to identify at least one variant polypeptide having a higher PAAF/PAAH ratio when compared to the PAAF/PAAH ratio of the Thermotoga maritima wild-type enzyme under the same (or as reasonably identical as possible) assay conditions.
  • the increase in the PAAF/PAAH ratio preferably occurs across the pH range where the enzyme is typically active.
  • the increase in the PAAF/PAAH ratio preferably occurs without a substantial drop in the perhydrolysis to hydrolysis reaction (P/H) ratio of the enzyme for the carboxylic acid ester substrate (i.e., the perhydrolytic reactions typically occur in an aqueous reaction formulation where the carboxylic acid ester may be hydrolyzed by the enzyme catalyst).
  • P/H perhydrolysis to hydrolysis reaction
  • a library of variant polypeptides was created from the wild-type
  • Thermotoga maritima perhydrolase (SEQ ID NO: 2) and assayed for an increase in the ratio of perhydrolytic specific activity relative to the
  • a process is provided to produce an aqueous formulation comprising at least one peroxycarboxylic acid by reacting carboxylic acid esters and an inorganic peroxide (such as hydrogen peroxide, sodium perborate or sodium percarbonate) in the presence of an enzyme catalyst having perhydrolysis activity, wherein the enzyme catalyst comprises an enzyme having amino acid sequence SEQ ID NO: 22.
  • an enzyme catalyst having perhydrolysis activity such as hydrogen peroxide, sodium perborate or sodium percarbonate
  • the enzyme catalyst comprises an enzyme having amino acid sequence SEQ ID NO: 22.
  • R 5 a C1 to C6 linear, branched, or cyclic hydrocarbyl moiety optionally substituted with hydroxy! groups; wherein each carbon atom in R 5 individually comprises no more than one hydroxyl group or no more than one ester group; wherein R 5 optionally comprises one or more ether linkages;
  • R 6 is C1 to C7 linear hydrocarbyl moiety, optionally substituted with hydroxyl groups or C1 to C4 alkoxy groups, optionally comprising one or more ether linkages.
  • R 6 is C2 to C7 linear hydrocarbyl moiety, optionally substituted with hydroxyl groups, and/or optionally comprising one or more ether linkages.
  • suitable substrates also include one or more glycerides of the formula:
  • Ri C1 to C7 straight chain or branched chain aikyl optionally substituted with an hydroxyl or a C1 to C4 alkoxy group and R3 and R 4 are individually H or R-iC(O).
  • suitable substrates may also include one or more esters of the formula:
  • Ri is a C1 to C7 straight chain or branched chain alkyl optionally substituted with an hydroxyl or a C1 to C4 alkoxy group and R 2 is a C1 to C10 straight chain or branched chain alkyl, alkenyl, alkynyl, aryl, alkylaryl, alkylheteroaryl, heteroaryl, (CH 2 CH 2 0) n , or (CH 2 CH(CH 3 )-0) n H and n is 1 to 10.
  • Suitable substrates may also include one or more acetylated
  • the acetylated saccharides are selected from the group consisting of acetylated mono-, di-, and polysaccharides.
  • the acetylated saccharides are selected from the group consisting of acetylated xylan, fragments of acetylated xylan, acetylated xylose (such as xylose tetraacetate), acetylated glucose (such as glucose pentaacetate), ⁇ -D-ribofuranose-l ,2,3,5-tetraacetate, tri-O- acetyl-D-galactal, tri-O-acetyl-D-giucal, and acetylated cellulose.
  • the acetylated saccharide is selected from the group consisting of ⁇ -D-ribofuranose-l ,2,3,5-tetraacetate, tri-O-acetyl-D-galactal, tri-O-acetyl-D- giucal , and acetylated cellulose.
  • suitable substrates are selected from the group consisting of: monoacetin; diacetin; triacetin; monopropionin; dipropionin;
  • the carboxylic acid ester is selected from the group consisting of monoacetin, diacetin, triacetin, and combinations thereof.
  • the substrate is a C1 to C6 polyol comprising one or more ester groups.
  • one or more of the hydroxyl groups on the C1 to C6 polyol are substituted with one or more acetoxy groups (such as 1 ,3-propanediol diacetate, 1 ,4-butanediol diacetate, etc.).
  • the substrate is propylene glycol diacetate (PGDA), ethylene glycol diacetate (EGDA), or a mixture thereof.
  • suitable substrates are selected from the group consisting of ethyl acetate; methyl lactate; ethyl lactate; methyl glycolate; ethyl glycolate; methyl methoxyacetate; ethyl methoxyacetate; methyl 3- hydroxybutyrate; ethyl 3-hydroxybutyrate; triethyl 2-acetyl citrate; glucose pentaacetate; gluconolactone; glycerides (mono-, di-, and triglycerides) such as monoacetin, diacetin, triacetin, monopropionin, dipropionin (glyceryl dipropionate), tripropionin (1,2,3-tripropionylglycerol), monobutyrin, dibutyrin (glyceryl dibutyrate), tributyrin ( ,2,3-tributyrylglycerol); acetylated saccharides; and mixtures thereof.
  • glycerides mono-
  • suitable substrates are selected from the group consisting of monoacetin, diacetin, triacetin, monopropionin, dipropionin, tripropionin, monobutyrin, dibutyrin, tributyrin, ethyl acetate, and ethyl lactate.
  • the substrate is selected from the group consisting of diacetin, triacetin, ethyl acetate, and ethyl lactate.
  • the suitable substrate comprises triacetin.
  • the carboxylic acid ester is present in the aqueous reaction formulation at a concentration sufficient to produce the desired concentration of
  • the carboxylic acid ester need not be completely soluble in the aqueous reaction formulation, but has sufficient solubility to permit conversion of the ester by the
  • the carboxylic acid ester is present in the aqueous reaction formulation at a concentration of 0.0005 wt % to 40 wt % of the aqueous reaction formulation, preferably at a concentration of 0.01 wt % to 20 wt % of the aqueous reaction formulation, and more preferably at a concentration of 0.05 wt % to 10 wt % of the aqueous reaction formulation.
  • the wt % of carboxylic acid ester may optionally be greater than the solubility limit of the carboxylic acid ester, such that the concentration of the carboxylic acid ester is at least 0.0005 wt % in the aqueous reaction formulation that is comprised of water, enzyme catalyst, and source of peroxide, where the remainder of the carboxylic acid ester remains as a second separate phase of a two-phase aqueous/organic reaction formulation. Not all of the added carboxylic acid ester must immediately dissolve in the aqueous reaction formulation, and after an initial mixing of all reaction components, additional continuous or discontinuous mixing is optional.
  • the peroxycarboxylic acid produced is peracetic acid, perpropionic acid, perbutyric acid, perlactic acid, perglycolic acid, permethoxyacetic acid, per-p-hydroxybutyric acid, or mixtures thereof.
  • the peroxygen source may include, but is not limited to, hydrogen peroxide, hydrogen peroxide adducts (e.g., urea-hydrogen peroxide adduct (carbamide peroxide)), perborate salts and percarbonate salts.
  • hydrogen peroxide hydrogen peroxide adducts
  • urea-hydrogen peroxide adduct carbamide peroxide
  • perborate salts percarbonate salts.
  • concentration of peroxygen compound in the aqueous reaction formulation may range from 0.0033 wt % to about 50 wt %, preferably from 0.033 wt % to about 40 wt %, more preferably from 0.33 wt % to about 30 wt %.
  • perhydrolase catalysts such as whole cells, permeabilized whole cells, and partially purified whole cell extracts
  • catalase activity EC 111.1.6
  • Catalases catalyze the conversion of hydrogen peroxide into oxygen and water.
  • the enzyme catalyst having perhydrolase activity lacks catalase activity.
  • a catalase inhibitor is added to the aqueous reaction formulation. Examples of catalase inhibitors include, but are not limited to, sodium azide and hydroxylamine sulfate.
  • One of skill in the art can adjust the concentration of catalase inhibitor as needed.
  • the concentration of the catalase inhibitor typically ranges from 0.1 mM to about 1 M; preferably about 1 mM to about 50 mM; more preferably from about 1 mM to about 20 mM.
  • sodium azide concentration typically ranges from about 20 mM to about 60 mM while hydroxylamine sulfate is concentration is typically about 0.5 mM to about 30 mM, preferably about 10 mM.
  • the catalase activity in a host cell can be down-regulated or eliminated by disrupting expression of the gene(s) responsible for the catalase activity using well known techniques including, but not limited to, transposon mutagenesis, RNA antisense expression, targeted mutagenesis, and random mutagenesis.
  • the gene(s) encoding the endogenous catalase activity are down-regulated or disrupted (i.e., "knocked- out”).
  • a "disrupted" gene is one where the activity and/or function of the protein encoded by the modified gene is no longer present.
  • the production host is an E. colt production host comprising a disrupted catalase gene selected from the group consisting of katG and aiE (see U.S. Patent Application Publication No. 2008-0176783 to DiCosimo et a/.).
  • the production host is an E. coli strain comprising a down-regulation and/or disruption in both katG and katE catalase genes.
  • An E. coli strain comprising a double-knockout of katG and katE has been prepared and is described as E. coli strain KLP18 (U.S. Patent Application Publication No. 2008-0176783 to DiCosimo et ai).
  • the concentration of the catalyst in the aqueous reaction formulation depends on the specific catalytic activity of the catalyst, and is chosen to obtain the desired rate of reaction.
  • the weight of catalyst in perhydrolysis reactions typically ranges from 0.0001 mg to 50 mg per mL of total reaction volume, preferably from 0.0005 mg to 10. mg per mL, more preferably from 0.00 0 mg to 2.0 mg per mL.
  • the catalyst may also be immobilized on a soluble or insoluble support using methods well-known to those skilled in the art; see for example, Immobilization of Enzymes and Cells; Gordon F. Bickerstaff, Editor; Humana Press, Totowa, NJ, USA; 1997. The use of immobilized catalysts permits the recovery and reuse of the catalyst in subsequent reactions.
  • the enzyme catalyst may be in the form of whole microbial cells, permeabilized microbial cells, microbial cell extracts, partia!ly-puhfied or purified enzymes, and mixtures thereof.
  • the concentration of peroxycarboxylic acid generated by the combination of chemical perhydrolysis and enzymatic perhydrolysis of the carboxylic acid ester is sufficient to provide an effective concentration of peroxycarboxylic acid for disinfection, bleaching, sanitization, deodorizing or destaining at a desired pH.
  • the present methods provide combinations of enzymes and enzyme substrates to produce the desired effective concentration of peroxycarboxylic acid, where, in the absence of added enzyme, there is a significantly lower concentration of peroxycarboxylic acid produced.
  • peroxycarboxylic acid generated to provide an effective concentration of peroxycarboxylic acid in the desired applications, and a significant increase in total peroxycarboxylic acid concentration is achieved by the addition of an appropriate perhydrolase catalyst to the aqueous reaction formulation.
  • concentration of peroxycarboxylic acid generated e.g.
  • the concentration of peroxycarboxylic acid generated (e.g.
  • peracetic acid) by the enzymatic perhydrolysis is at least about 2 ppm, preferably at least 20 ppm, preferably at least 30 ppm, more preferably at least about 40 ppm peroxycarboxylic acid, more preferably at least 50 ppm, more preferably at least 60 ppm, more preferably at least 70 ppm, more preferably at least about 80 ppm peroxycarboxylic acid, most preferably at least 100 ppm peroxycarboxylic acid within 5 minutes, more preferably within 1 minute, of initiating the enzymatic perhydrolysis reaction ⁇ i.e., time measured from combining the reaction components to form the formulation).
  • the aqueous formulation comprising the peroxycarboxylic acid may be optionally diluted with diluent comprising water, or a solution predominantly comprised of water, to produce a formulation with the desired lower target concentration of peroxycarboxylic acid.
  • the reaction time required to produce the desired concentration (or concentration range) of peroxycarboxylic acid is about 5 minutes or less, more preferably about 1 minute or less.
  • the surface or inanimate object contaminated with a concentration of a biological contaminant(s) is contacted with the
  • the peroxycarboxylic acid formed in accordance with the processes describe herein is used in a laundry care application wherein the peroxycarboxylic acid is contacted with clothing or a textile to provide a benefit, such as disinfecting, bleaching, destaining, deodorizing and/or a combination thereof.
  • the peroxycarboxylic acid may be used in a variety of laundry care products including, but not limited to, laundry or textile pre-wash treatments, laundry detergents or additives, stain removers, bleaching compositions, deodorizing compositions, and rinsing agents.
  • the present process to produce a peroxycarboxylic acid for a target surface is conducted in situ.
  • the term "contacting an article of clothing or textile” means that the article of clothing or textile is exposed to a formulation disclosed herein.
  • the formulation may be used to treat articles of clothing or textiles including, but not limited to, liquid, solids, gel, paste, bars, tablets, spray, foam, powder, or granules and can be delivered via hand dosing, unit dosing, dosing from a substrate, spraying and automatic dosing from a laundry washing or drying machine.
  • Granular compositions can also be in compact form; liquid compositions can also be in a concentrated form.
  • the formulation can further contain components typical to laundry detergents.
  • typical components included, but are not limited to, surfactants, bleaching agents, bleach activators, additional enzymes, suds suppressors, dispersants, lime-soap dispersants, soil suspension and anti- redeposition agents, softening agents, corrosion inhibitors, tarnish inhibitors, germicides, pH adjusting agents, non-builder alkalinity sources, chelating agents, organic and/or inorganic fillers, solvents, hydrotropes, optical brighteners, dyes, and perfumes.
  • formulations disclosed herein can also be used as detergent additive products in solid or liquid form. Such additive products are intended to supplement or boost the performance of conventional detergent compositions and can be added at any stage of the cleaning process.
  • the concentration of peracid generated (e.g., peracetic acid) by the perhydrolysis of at least one carboxylic acid ester may be at least about 2 ppm, preferably at least 20 ppm, preferably at least 100 ppm, and more preferably at least about 200 ppm peracid.
  • the concentration of peracid generated (e.g., peracetic acid) by the perhydrolysis of at least one carboxylic acid ester may be 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 preferably at least about 1000 ppm peracid, most preferably at least 2000 ppm peracid within 10 minutes, preferably within 5 minutes, and most preferably within 1 minute of initiating the perhydrolysis reaction.
  • the product formulation comprising the peracid may be optionally diluted with water, or a solution predominantly comprised of water, to produce a formulation with the desired lower concentration of peracid.
  • the reaction time required to produce the desired concentration of peracid is not greater than about two hours, preferably not greater than about 30 minutes, more preferably not greater than about 10 minutes, even more preferably not greater than about 5 minutes, and most preferably in about 1 minute or less.
  • the temperature of the reaction is chosen to control both the reaction rate and the stability of the enzyme catalyst activity.
  • the temperature of the reaction may range from just above the freezing point of the aqueous reaction formulation (approximately 0 °C) to about 85 °C, with a preferred range of reaction temperature of from about 5 °C to about 55 °C.
  • the pH of the aqueous reaction formulation while enzymatically producing peroxycarboxylic acid is maintained at a pH ranging from about 5.0 to about 10.0, preferably about 6.5 to about 8.5, and yet even more preferably about 6.5 to about 7.5. In one embodiment, the pH of the aqueous reaction formulation ranges from about 6.5 to about 8.5 for at least 30 minutes after combining the reaction components.
  • the pH of the aqueous reaction formulation may be adjusted or controlled by the addition or incorporation of a suitable buffer, including, but not limited to, phosphate, pyrophosphate, bicarbonate, acetate, or citrate. In one embodiment, the buffer is selected from a phosphate buffer and a bicarbonate buffer.
  • the concentration of buffer when employed, is typically from 0.1 mM to 1.0 M, preferably from 1 mM to 300 mM, most preferably from 0 mM to 100 mM. In another aspect of the present invention, no buffer is added to the reaction mixture while enzymatically producing peroxycarboxylic acid.
  • the enzymatic perhydrolysis aqueous reaction formulation may contain an organic solvent that acts as a dispersant to enhance the rate of dissolution of the carboxylic acid ester in the aqueous reaction formulation.
  • organic solvents include, but are not limited to, 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, cyclohexanoi, benzyl alcohol, isopropanol, ethanol, propylene glycol, and mixtures thereof.
  • the enzymatic perhydrolysis product may contain additional components that provide desirable functionality.
  • additional components include, but are not limited to, buffers, detergent builders, thickening agents, emulsifiers, surfactants, wetting agents, corrosion inhibitors (e.g., benzotriazole), enzyme stabilizers, and peroxide stabilizers (e.g., metal ion chelating agents).
  • corrosion inhibitors e.g., benzotriazole
  • enzyme stabilizers e.g., enzyme stabilizers
  • peroxide stabilizers e.g., metal ion chelating agents.
  • emulsifiers include, but are not limited to, polyvinyl alcohol or polyvinylpyrrolidone.
  • thickening agents include, but are not limited to, LAPONITE ® RD, corn starch, PVP,
  • buffering systems include, but are not limited to, sodium phosphate monobasic/sodium phosphate dibasic; sulfamic
  • surfactants include, but are not limited to, a) non-ionic surfactants such as block copolymers of ethylene oxide or propylene oxide, ethoxylated or propoxylated linear and branched primary and secondary alcohols, and aliphatic phosphine oxides b) cationic surfactants such as quaternary ammonium compounds, particularly quaternary ammonium compounds having a C8-C20 alkyl group bound to a nitrogen atom additionally bound to three C1 - C2 alkyl groups, c) anionic surfactants such as alkane carboxylic acids (e.g., C8-C20 fatty acids), alkyl phosphonates, alkane sulfonates (e.g., sodium dodecylsulphate "SDS”) or linear or branched alky
  • non-ionic surfactants such as block copolymers of ethylene oxide or propylene oxide, ethoxylated or propoxylated linear and branched primary
  • fragrances may include fragrances, dyes, stabilizers of hydrogen peroxide ⁇ e.g., metal chelators such as 1-hydroxyethylidene-1 ,1- diphosphonic acid (DEQU EST® 20 0, Solutia Inc., St. Louis, MO) and ethylenediaminetetraacetic acid (EDTA)), TURPINAL ® SL, DEQUEST® 0520, DEQUEST® 0531 , stabilizers of enzyme activity (e.g., polyethylene glycol (PEG)), and detergent builders.
  • metal chelators such as 1-hydroxyethylidene-1 ,1- diphosphonic acid (DEQU EST® 20 0, Solutia Inc., St. Louis, MO) and ethylenediaminetetraacetic acid (EDTA)
  • TURPINAL ® SL DEQUEST® 0520, DEQUEST® 0531
  • stabilizers of enzyme activity e.g., polyethylene glycol (PEG)
  • detergent builders e.g
  • the enzymatic perhydrolysis product may be pre- mixed to generate the desired concentration of peroxycarboxylic acid prior to contacting the surface or inanimate object to be disinfected.
  • the enzymatic perhydrolysis product is not pre-mixed to generate the desired concentration of peroxycarboxylic acid prior to contacting the surface or inanimate object to be disinfected, but instead, the components of the aqueous reaction formulation that generate the desired concentration of peroxycarboxylic acid are contacted with the surface or inanimate object to be disinfected and/or bleached or destained, generating the desired concentration of peroxycarboxylic acid.
  • the components of the aqueous reaction formulation combine or mix at the locus.
  • the reaction components are delivered or applied to the locus and subsequently mix or combine to generate the desired concentration of peroxycarboxylic acid.
  • the peroxycarboxylic acids once produced, are quite reactive and may decrease in concentration over extended periods of time, depending on variables that include, but are not limited to, temperature and pH. As such, it may be desirable to keep the various reaction components separated, especially for liquid formulations.
  • the hydrogen peroxide source is separate from either the substrate or the perhydrolase catalyst, preferably from both. This can be accomplished using a variety of techniques including, but not limited to, the use of multicompartment chambered dispensers (U.S. Patent 4,585,150) and at the time of use physically combining the perhydrolase catalyst with a source of peroxygen (such as hydrogen peroxide) and the present substrates to initiate the aqueous enzymatic perhydrolysis reaction.
  • a source of peroxygen such as hydrogen peroxide
  • the perhydrolase catalyst may optionally be immobilized within the body of reaction chamber or separated (e.g., filtered, etc.) from the reaction product comprising the peroxycarboxylic acid prior to contacting the surface and/or object targeted for treatment.
  • the perhydrolase catalyst may be in a liquid matrix or in a solid form (e.g., powder or tablet) or embedded within a solid matrix that is subsequently mixed with the substrates to initiate the enzymatic perhydrolysis reaction.
  • the perhydrolase catalyst may be contained within a dissolvable or porous pouch that may be added to the aqueous substrate matrix to initiate enzymatic perhydrolysis.
  • the perhydrolase catalyst may comprise the contents contained within a separate compartment of a dissolvable or porous pouch that has at least one additional compartment for the containment contents comprising the ester substrate and/or source of peroxide.
  • a powder comprising the enzyme catalyst is suspended in the substrate (e.g., triacetin), and at time of use is mixed with a source of peroxygen in water.
  • a variety of analytical methods can be used in the present method to analyze the reactants and products including, but not limited to, titration, high performance liquid chromatography (HPLC), gas chromatography (GC), mass spectroscopy (MS), capillary electrophoresis (CE), the analytical procedure described by U. Karst et al. (Anal. Chem,, 69(17):3623-3627 (1997)), and the 2,2'-azino-bis (3-ethylbenzothazoline)-6-sulfonate (ABTS) assay (S. Minning, et al., Analytica Chimica Acta 378:293-298 (1999) and WO 2004/058961 A1) as described in U.S. Patent Application Publication No. 2008/0176783. Determination of Minimum Biocidal Concentration of Peroxycarboxylic acids
  • the method described by J. Gabrielson et al. can be employed for determination of the Minimum Biocidal Concentration (MBC) of peroxycarboxylic acids, or of hydrogen peroxide and enzyme substrates.
  • MCC Minimum Biocidal Concentration
  • the assay method is based on XTT reduction inhibition, where XTT (2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-5- [(phenylamino)carbonyl]-2H-tetrazolium, inner salt, monosodium salt) is a redox dye that indicates microbial respiratory activity by a change in optical density (OD) measured at 490 nm or 450 nm.
  • OD optical density
  • the enzyme catalyst-generated peroxycarboxylic acid produced according to the present method can be used in a variety of hard
  • the enzyme-generated peroxycarboxylic acids may be used in formulations designed to inactivate prions (e.g., certain proteases) to additionally provide biocidal activity (see U.S. Patent 7,550,420 to DiCosimo et al.).
  • the peroxycarboxylic acid composition is useful as a disinfecting agent for non-autoclavable medical instruments and food packaging equipment.
  • the peroxycarboxylic acid-containing formulation may be prepared using GRAS or food-grade components (enzyme, enzyme substrate, hydrogen peroxide, and buffer), the enzyme-generated peroxycarboxylic acid may also be used for decontamination of animal carcasses, meat, fruits and vegetables, or for decontamination of prepared 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 an efficacious decontamination.
  • compositions comprising an efficacious concentration of peroxycarboxylic acid can be used to disinfect surfaces and/or objects contaminated (or suspected of being contaminated) with biological
  • contacting refers to placing a disinfecting composition comprising an effective concentration of peroxycarboxylic acid in contact with the surface or inanimate object suspected of contamination with a biological contaminant for a period of time sufficient to clean and disinfect.
  • Contacting includes spraying, treating, immersing, flushing, pouring on or in, mixing, combining, painting, coating, applying, affixing to and otherwise communicating a peroxycarboxylic acid solution or composition comprising an efficacious concentration of peroxycarboxylic acid, or a solution or composition that forms an efficacious concentration of peroxycarboxylic acid, with the surface or inanimate object suspected of being contaminated with a concentration of a biological contaminant.
  • the disinfectant compositions may be combined with a cleaning composition to provide both cleaning and disinfection.
  • a cleaning agent e.g., a surfactant or detergent
  • compositions comprising an efficacious concentration of peroxycarboxylic acid can also contain at least one additional antimicrobial agent, combinations of prion-degrading proteases, a virucide, a sporicide, or a biocide. Combinations of these agents with the peroxycarboxylic acid produced by the claimed processes can provide for increased and/or synergistic effects when used to clean and disinfect surfaces and/or objects contaminated (or suspected of being contaminated) with biological
  • Suitable antimicrobial agents include carboxylic esters (e.g., p- hydroxy alkyl benzoates and alkyl cinnamates), sulfonic acids (e.g., dodecylbenzene sulfonic acid), iodo-compounds or active halogen compounds (e.g., elemental halogens, halogen oxides (e.g., NaOCI, HOCI, HOBr, CI0 2 ), iodine, interhalides (e.g., iodine monochloride, iodine dichloride, iodine trichloride, iodine tetrachloride, bromine chloride, iodine monobromide, or iodine dibromide), polyhalides, hypochlorite salts, hypochlorous acid, hypobromite salts, hypobromous acid, chloro- and bromo-hydantoins
  • Effective amounts of antimicrobial agents include about 0.001 wt% to about 60 wt% antimicrobial agent, about 0.01 wt% to about 15 wt% antimicrobial agent, or about 0.08 wt% to about 2.5 wt% antimicrobial agent.
  • the peroxycarboxylic acids formed by the process can be used to reduce the concentration of viable biological contaminants (such as a microbial population) when applied on and/or at a locus.
  • a locus comprises part or all of a target surface suitable for disinfecting or bleaching.
  • Target surfaces include all surfaces that can potentially be contaminated with biological contaminants.
  • Non-limiting examples include equipment surfaces found in the food or beverage industry (such as tanks, conveyors, floors, drains, coolers, freezers, equipment surfaces, walls, valves, belts, pipes, drains, joints, crevasses, combinations thereof, and the like); building surfaces (such as walls, floors and windows); non-food-industry related pipes and drains, including water treatment facilities, pools and spas, and fermentation tanks; hospital or veterinary surfaces (such as walls, floors, beds, equipment (such as endoscopes), clothing worn in hospital/veterinary or other healthcare settings, including clothing, scrubs, shoes, and other hospital or veterinary surfaces); restaurant surfaces; bathroom surfaces; toilets; clothes and shoes; surfaces of barns or stables for livestock, such as poultry, cattle, dairy cows, goats, horses and pigs; hatcheries for poultry or for shrimp; and pharmaceutical or biopharmaceutical surfaces [e.g., pharmaceutical or biopharmaceutical manufacturing equipment, pharmaceutical or
  • Additional hard surfaces include food products, such as beef, poultry, pork, vegetables, fruits, seafood, combinations thereof, and the like.
  • the locus can also include water absorbent materials such as infected linens or other textiles.
  • the locus also includes harvested plants or plant products including seeds, corms, tubers, fruit, and vegetables, growing plants, and especially crop growing plants, including cereals, leaf vegetables and salad crops, root vegetables, legumes, berried fruits, citrus fruits and hard fruits.
  • Non-limiting examples of hard surface materials are metals (e.g., steel, stainless steel, chrome, titanium, iron, copper, brass, aluminum, and alloys thereof), minerals (e.g., concrete), polymers and plastics (e.g., pol o!efins, such as polyethylene, polypropylene, polystyrene, poly(meth)acrylate, polyacrylonitrile, polybutadiene, poly(acrylonitrile, butadiene, styrene), poly(acrylonitrile, butadiene), acrylonitrile butadiene; polyesters such as polyethylene terephthalate; and polyamides such as nylon).
  • Additional surfaces include brick, tile, ceramic, porcelain, wood, wood pulp, paper, vinyl, linoleum, and carpet.
  • the peroxycarboxylic acids formed by the present process may be used to provide a benefit to an article of clothing or a textile including, but not limited to disinfecting, sanitizing, bleaching, destaining, and deodorizing.
  • the peroxycarboxylic acids formed by the present process may be used in any number of laundry care products including, but not limited to textile pre-wash treatments, laundry detergents, laundry detergents or additives, stain removers, bleaching compositions, deodorizing compositions, and rinsing agents, to name a few.
  • the peroxycarboxylic acids formed by the present process can be used in one or more steps of the wood pulp or paper pulp bleaching/delignification process, particularly where peracetic acid is used (for example, see
  • the genes and gene products of the instant sequences may be produced in heterologous host cells, particularly in the cells of microbial hosts.
  • Preferred heterologous host cells for expression of the instant genes and nucleic acid molecules are microbial hosts that can be found within the fungal or bacterial families and which grow over a wide range of temperature, pH values, and solvent tolerances.
  • any of bacteria, yeast, and filamentous fungi may suitably host the expression of the present nucleic acid molecules.
  • the perhydrolase may be expressed intracellular ⁇ , extracel!ularly, or a combination of both intracellular ⁇ and extracellularly, where extracellular expression renders recovery of the desired protein from a fermentation product more facile than methods for recovery of protein produced by intracellular expression.
  • host strains include, but are not limited to, bacterial, fungal or yeast species such as Aspergillus, Trichoderma, Saccharomyces, Pichia, Phaffia, Kluyveromyces, Candida, Hansenula, Yarrowia, Salmonella, Bacillus, Acinetobacter, Zymomonas, Agrobacterium, Erythrobacter,
  • Rhodococcus Streptomyces, Brevibacterium, Corynebacteria, Mycobacterium, Deinococcus, Escherichia, Erwinia, Pantoea, Pseudomonas, Sphingomonas, Methylomonas, Methylobacter, Methylococcus, Methylosinus,
  • Methylomicrobium Methylocystis, Alcaiigenes, Synechocystis,
  • bacterial host strains include Escherichia, Bacillus, and Pseudomonas.
  • the bacterial host cell is Escherichia coll.
  • a variety of culture methodologies may be applied to produce the perhydrolase catalyst.
  • Large-scale production of a specific gene product overexpressed from a recombinant microbial host may be produced by batch, fed-batch or continuous culture methodologies.
  • Batch and fed-batch culturing methods are common and well known in the art and examples may be found in Thomas D. Brock in Biotechnology: A Textbook of Industrial Microbiology, Second Edition, Sinauer Associates, Inc., Sunderland, MA (1989) and
  • commercial production of the desired perhydrolase catalyst is accomplished with a continuous culture.
  • Continuous cultures are an open system where a defined culture media is added continuously to a bioreactor and an equal amount of conditioned media is removed
  • Continuous cultures generally maintain the cells at a constant high liquid phase density where cells are primarily in log phase growth.
  • continuous culture may be practiced with immobilized cells where carbon and nutrients are continuously added and valuable products, by-products or waste products are continuously removed from the cell mass.
  • Cell immobilization may be performed using a wide range of solid supports composed of natural and/or synthetic materials.
  • the cell paste is separated from the culture medium by centrifugation or membrane filtration, optionally washed with water or an aqueous buffer at a desired pH, then a suspension of the cell paste in an aqueous buffer at a desired pH is homogenized to produce a cell extract containing the desired enzyme catalyst.
  • the cell extract may optionally be filtered through an appropriate filter aid such as celite or silica to remove cell debris prior to a heat-treatment step to precipitate undesired protein from the enzyme catalyst solution.
  • the solution containing the desired enzyme catalyst may then be separated from the precipitated cell debris and protein produced during the heat-treatment step by membrane filtration or centrifugation, and the resulting partially-purified enzyme catalyst solution concentrated by additional membrane filtration, then optionally mixed with an appropriate excipient (for example, maltodextrin, trehalose, sucrose, lactose, sorbitol, mannitol, phosphate buffer, citrate buffer, or mixtures thereof) and spray-dried to produce a solid powder comprising the desired enzyme catalyst.
  • an appropriate excipient for example, maltodextrin, trehalose, sucrose, lactose, sorbitol, mannitol, phosphate buffer, citrate buffer, or mixtures thereof
  • the resulting partially-purified enzyme catalyst solution prepared as described above may be optionally concentrated by additional membrane filtration, and the partially-purified enzyme catalyst solution or resulting enzyme concentrate is then optionally mixed with one or more stabilizing agents (e.g., glycerol, sorbitol, propylene glycol, 1 ,3-propanediol, polyols, polymeric polyols, polyvinyla!cohol), one or more salts (e.g., sodium chloride, sodium sulfate, potassium chloride, potassium sulfate, or mixtures thereof), and one or more biocides, and maintained as an aqueous solution until used.
  • stabilizing agents e.g., glycerol, sorbitol, propylene glycol, 1 ,3-propanediol, polyols, polymeric polyols, polyvinyla!cohol
  • salts e.g., sodium chloride, sodium sulfate, potassium chloride, potassium
  • Thermotopa maritime Acetyl Xylan Esterase C277S Variant The coding sequence of a Thermotoga maritima acetyl xylan esterase (GENBANK ® accession # NP_227893.1) was synthesized using codons optimized for expression in E. coli (DNA 2.0, Menlo Park, Calif.), and cloned into pUC19 between Psi1 and Xba1 to create the plasmid known as pSW202 (U.S. Patent Application Publication 2008-0176299).
  • the codon-optimized sequence is provided as SEQ ID NO:1 encoding the wild-type T. maritima acetyl xylan esterase provided as SEQ ID NO: 2.
  • a codon change was made in the gene using primer pairs identified as SEQ ID NO: 3 and SEQ ID NO: 4, and the QUIKCHANGE ® site-directed mutagenesis kit (Stratagene, La Jolla, CA), according to the manufacturer's instructions, resulting in the amino acid change C277S (SEQ ID NO: 5), to create the plasmid known as pSW202/C277S (SEQ ID NO: 6).
  • Plasmid pSW202/C277S served as a template for error-prone PCR using primers identified as SEQ ID NO: 7 and SEQ ID NO: 8, and the GENEMORPH ® II random mutagenesis kit (Stratagene), according to the manufacturer's recommendations.
  • E. coli KLP18 (see U.S. 2008-0176299; herein incorporated by reference in its entirety) was transformed with the ligation mixture and plated onto LB plates supplemented with 0.1 mg ampicillin/mL. Nucleotide sequencing of a random sample indicated a mutation frequency of 2-8 changes per PCR product
  • Colonies were picked (automated) and placed into 96-well "master plates" containing 0.1 mL LB media supplemented with 0.1 mg ampicillin/mL and grown 16-18 h at 37 °C and 80% humidity. From each well of the master plates, 0.003 mL of culture was transferred to 96-well "induction plates" containing 0.3 mL LB media supplemented with 0.1 mg ampicillin/mL and 0.5 mM IPTG, which were incubated for 16-18 h with shaking at 37 °C and 80% humidity. Separately, 0.1 mL of 50% glycerol was added to each well of the master plates, which were stored at -80 °C as stocks.
  • T. maritima WT codon optimized gene encoding the wild type enzyme
  • T. maritima C277S were incorporated into each plate as controls. Screening of approximately 5000 colonies resulted in the identification of one variant (labeled as "A3") demonstrating activity approximately 3-fold greater than T. maritima C277S.
  • the "A3" strain will also be referred to herein as the "F24I/S35T/Q179L/N275D/C277S/S308G/F317S variant”.
  • the nucleic acid sequence of the A3 variant is provided as SEQ ID NO: 9 and the corresponding amino acid sequence of the A3 variant is provided as SEQ ID NO: 10.
  • N275D/C277S Variant of Thermotoqa maritima Perhydrolase Using plasmid pSW202/C277S as starting template (see Example 1 ), the N275D mutation was added using the primer pair identified as SEQ ID NOs: 11 and 12, and QUIKCHANGE ® (Stratagene) according to the primer pair identified as SEQ ID NOs: 11 and 12, and QUIKCHANGE ® (Stratagene) according to the
  • the plasmid (pSW202/N275D/C277S) was transformed into E. coli KLP18 to generate the strain KLP18/pSW202/N275D/C277S.
  • the nucleic acid sequence encoding N275D/C277S is provided as SEQ ID NO: 13.
  • the amino acid sequence of the N275D/C277S variant is provided as SEQ ID NO: 14.
  • S35T/C277S Variant of Thermotoga maritima Perhydrolase Using plasmid pSW202/C277S as starting template, the S35T mutation was added using the primer pair identified as SEQ ID NOs: 19 and 20, and QUIKCHANGE® (Stratagene) according to the manufacturer's instructions. Mutations were confirmed by nucleotide sequencing, and the plasmid
  • S35T/C277S is provided as SEQ ID NO: 21.
  • the amino acid sequence of the S25T/C277S variant is provided as SEQ ID NO: 22.
  • Q179L/C2775 Variant of Thermotoga maritima Perhydrolase Using plasmid pSW202/C277S as starting template, the Q179L mutation was added using the primer pair identified as SEQ ID NOs: 23 and 24, and QUIKCHANGE ® (Stratagene) according to the manufacturer's instructions. Mutations were confirmed by nucleotide sequencing, and the plasmid (pSW202/Q179L/C277S) was transformed into E. coli KLP18 to generate the strain KLP18/pSW202/Q179L/C277S. The nucleic acid sequence encoding Q179L/C277S is provided as SEQ ID NO: 25. The amino acid sequence of the Q179L/C277S variant is provided as SEQ ID NO: 26. EXAMPLE 1 1
  • KLP18/pSW202/N275D/C277S, KLP18/pSW202/C277S/F317S, KLP18/pSW202/S35TYC277S, and KLP18/pSW202/Q179L/C277S were each grown as described in Examples 3, 5, 7, 9 and 1 , respectively.
  • the resulting cell pastes were re-suspended (20% w/v) in 50 rtiM phosphate buffer (pH 7.0) supplemented with 1.0 mM dithiothreitol (DTT). Re-suspended cells were passed through a French pressure cell twice to ensure >95% cell lysis.
  • PAAF specific activity Perhydrolase specific activity
  • maritima C277S/F317S perhydrolase 25 pg/mL of heat-treated extract supernatant total protein from E. coli KLP18/pSW202/C277S/F317S), T.
  • maritima S35T/C277S perhydrolase 25 pg/mL of heat-treated extract supernatant total protein from E. coli KLP18/pSW202/S35T/C277S), T.
  • maritima Q179L/C277S perhydrolase 25 pg/mL of heat-treated extract supernatant total protein from E. coli KLP18/pSW202/Q179LJC277S (all prepared as described in Example 12). Reactions were stirred for only the first 30 seconds of reaction to initially mix the reactants and enzyme. A sample from each of the reaction mixtures described above was withdrawn after the first minute of each reaction, and every two minutes thereafter for fifteen minutes, and each sample was analyzed for peracetic acid using a modification of the method described by Karst et a/., supra.
  • T. maritima wild-type perhydrolase 50 pg/mL of heat-treated extract supernatant total protein from E. coti KLP18/pSW202
  • T. maritima C277S variant perhydrolase 10 pg/mL of heat-treated extract supernatant total protein from E. coli KLP18/pSW202/C277S
  • the peracid hydrolysis reaction rate and the peracid hydrolysis specific activity for hydrolysis of peracetic acid to acetic acid and hydrogen peroxide for each perhydrolase are reported in Table 6.
  • Thermotoga maritima wild-type and variant perhydrolases are Thermotoga maritima wild-type and variant perhydrolases.
  • Table 7 reports the perhydrolase specific activity (Example 13), the peracid hydrolysis specific activity (Example 14), and the ratio of perhydrolase specific activity to peracid hydrolysis specific activity for each of the listed T, maritima wild-type and variant acetyl xylan esterases.
  • N275D/C277S, F24I/S35T/Q179L/N275D/S308G/F317S/C277S, C277S/F317S, S35T/C277S, and Q179L/C277S acetyl xylan esterases each had an improved ratio of perhydrolase specific activity for peracetic acid formation (PAAF) to peracetic acid hydrolysis specific activity (PAAH) when compared to either the T. maritima wild-type or the C277S variant perhydrolase.
  • PAAF peracetic acid formation
  • PAAH peracetic acid hydrolysis specific activity
  • Thermotoga maritima (U/mg (U/mg specific vs. vs.

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Abstract

L'invention concerne un variant d'acétyl xylan estérase ayant une activité perhydrolytique, qui est proposé pour produire des acides peroxycarboxyliques à partir d'esters d'acides carboxyliques et d'une source de peroxygène. De façon plus spécifique, un gène d'acétyl xylan estérase de Thermotoga maritima a été modifié à l'aide d'une PCR sujette à l'erreur et d'une mutagenèse dirigée sur le site pour créer un catalyseur enzymatique caractérisé par une augmentation du rapport des activités spécifiques de formation d'acide peracétique à hydrolyse d'acide peracétique (rapport PAAF/PAAH). Le variant d'acétyl xylan estérase peut être utilisé pour produire des acides peroxycarboxyliques appropriés pour être utilisés dans une diversité d'applications telles que les applications de nettoyage, de désinfection, d'assainissement, de blanchiment, de traitement de pâte de bois et de traitement de pâte à papier.
PCT/US2010/058844 2009-12-07 2010-12-03 Perhydrolase fournissant une stabilité améliorée aux peracides Ceased WO2011071762A1 (fr)

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