WO2011071761A1 - Perhydrolase fournissant une stabilité améliorée aux peracides - Google Patents

Perhydrolase fournissant une stabilité améliorée aux peracides Download PDF

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WO2011071761A1
WO2011071761A1 PCT/US2010/058843 US2010058843W WO2011071761A1 WO 2011071761 A1 WO2011071761 A1 WO 2011071761A1 US 2010058843 W US2010058843 W US 2010058843W WO 2011071761 A1 WO2011071761 A1 WO 2011071761A1
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acid
group
optionally substituted
perhydrolase
hydroxyl
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PCT/US2010/058843
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English (en)
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Robert Dicosimo
John Edward Gavagan
Mark S. Payne
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E.I. Dupont De Nemours And Company
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Publication of WO2011071761A1 publication Critical patent/WO2011071761A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
    • C11D3/16—Organic compounds
    • C11D3/38—Products with no well-defined composition, e.g. natural products
    • C11D3/386—Preparations containing enzymes, e.g. protease or amylase
    • C11D3/38636—Preparations containing enzymes, e.g. protease or amylase containing enzymes other than protease, amylase, lipase, cellulase, oxidase or reductase
    • 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
    • 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)
    • 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)
    • 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
    • A61L2/22—Phase substances, e.g. smokes, aerosols or sprayed or atomised substances
    • 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
    • A61L2/23—Solid substances, e.g. granules, powders, blocks, tablets
    • 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
    • A61L2202/00—Aspects relating to methods or apparatus for disinfecting or sterilising materials or objects
    • A61L2202/10—Apparatus features
    • A61L2202/17—Combination with washing or cleaning means
    • 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
    • A61L2202/00—Aspects relating to methods or apparatus for disinfecting or sterilising materials or objects
    • A61L2202/20—Targets to be treated
    • A61L2202/24—Medical instruments, e.g. endoscopes, catheters, sharps

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, As such, it is often preferable to employ 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).
  • P perhydrolysis
  • H hydrolysis
  • 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 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 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 cell with the above nucleic acid molecule.
  • the variant polypeptide having perhydrolytic activity is characterized by at least a 1.1-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:
  • X an ester group of the formula R 6 -C(0)0;
  • Ri C1 to C7 straight chain or branched chain alkyl optionally substituted with an hydroxyl 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, alky!aryl, 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
  • At least one substrate selected from the group consisting of:
  • R 6 C1 to C7 linear, branched or cyclic
  • R-i 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);
  • Ri is a C1 to C7 straight chain or branched chain alky! 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;
  • Re 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);
  • 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 (CHaChKCHaK ⁇ H and n is 1 to 10;
  • SEQ ID NO: 5 is the amino acid sequence of the C277S variant acetyl xylan esterase having perhydrolytic activity ⁇ U.S. Patent Application No.
  • 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.
  • the term "substrate” or “carboxylic acid ester substrate” will refer to the reaction components enzymatically 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 enzymatically 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
  • 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 pmol 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
  • 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.
  • identification assay conditions or “same assay conditions” refer to the conditions used to measure the peracid formation (i.e.,
  • 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 00 mM triacetin, 100 mM hydrogen peroxide and approximately 2.5 ⁇ g/mL of heat-treated extract supernatant total protein from E. coli strain KLP 8 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
  • 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 Bioengineerinq, 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 carboxyiic 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. 3.1.1.41) 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 al., 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” will 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 maritime acetyl xylan esterase): a) Arg1 18-Gly119-Gln120;
  • variant polypeptide As used herein, the terms “variant” , “variant polypeptide”, and “variant enzyme catalyst” refer 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).
  • a perhydrolase 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
  • 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, hydroxyl amine, 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-sulphur 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 log 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 m 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 isubstrate) 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 maritima 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 hydroiyze 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: 10.
  • 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: 10.
  • R 5 a C1 to C6 linear, branched, or cyclic hydrocarbyl moiety optionally substituted with hydroxyl 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 hydroxy! groups or C1 to C4 alkoxy groups, optionally comprising one or more ether linkages.
  • Re is C2 to C7 linear hydrocarbyl moiety, optionally substituted with hydroxyl groups, and/or optionally comprising one or more ether linkages.
  • 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).
  • suitable substrates may also include one or more esters of the formula: wherein 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.
  • 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
  • R 2 is a C1 to C10 straight chain or branched chain alkyl, alkenyl, alkynyl, aryl, alkylaryl, alkylheteroaryl, heteroaryl, (CH 2 CH 2
  • 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), p-D-ribofuranose-1,2,3,5-tetraacetate, tri-O- acetyl-D-galactal, tri-O-acetyl-D-glucal, and acetylated cellulose.
  • the acetylated saccharide is selected from the group consisting of tri-O-acetyl-D-galactal, tri-O-acetyl-D- glucal, and acetylated cellulose.
  • suitable substrates are selected from the group consisting of: monoacetin; diacetin; triacetin; monopropionin; dipropionin;
  • tripropionin monobutyrin; dibutyrin; tributyrin; glucose pentaacetate; xylose tetraacetate; acetylated xylan; acetylated xylan fragments; ⁇ -D-ribofuranose- 1 ,2,3,5-tetraacetate; tri-O-acetyl-D-galactal; tri-O-acetyl-D-glucal; monoesters or diesters of 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-hexanedi
  • 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 (1 ,2,3-tributyrylglycerol); acetylated saccharides; and mixtures thereof.
  • 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- -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.
  • 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 1.11.1.6
  • 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 production host is an E. coli production host comprising a disrupted catalase gene selected from the group consisting of katG and katE (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 er a/.).
  • 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.
  • Rhodococcus Streptomyces, Brevibacterium, Corynebacteria, Mycobacterium, Deinococcus, Escherichia, Erwinia, Pantoea, Pseudomonas, Sphingomonas, Methylomonas, Methylobacter, Methylococcus, Methylosinus,
  • 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.
  • Thermotoga maritima Acetyl Xylan Esterase C277S Variant The coding sequence of a Thermotoga maritima acetyl xylan esterase (GEN BANK ® accession # NP_227893.1) was synthesized using codons optimized for expression in E co// (DNA 2.0, Menlo Park, Calif ), and cloned into pUC19 between Psi1 and Xba ⁇ 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.
  • 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 Thermotoga 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 I D 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/C277S 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 manufacture 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/Q179 C277S.
  • the nucleic acid sequence encoding Q179L/C277S is provided as SEQ ID NO: 25.
  • the amino acid sequence of the Q1791JC277S variant is provided as SEQ ID NO: 26. EXAMPLE 11
  • KLP18/pSW202/N275D/C277S, KLP18/pSW202/C277S/F317S, KLP18/pSW202/S35T/C277S, and KLP18/pSW202/Q179L/C277S were each grown as described in Examples 3, 5, 7, 9 and 1 1 , respectively.
  • the resulting celi pastes were re-suspended ⁇ 20% w/v) in 50 mM 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/Q179L/C277S (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, coli KLP18/pSW202
  • T. maritime C277S variant perhydrolase 10 pg/nriL 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.
  • Thermotoga maritima (U/mg (U/mg specific vs. vs.

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Abstract

L'invention concerne un variant d'acétyl xylan estérase qui présente une activité perhydrolytique et qui est utilisé afin de produire des acides peroxycarboxyliques à partir d'esters d'acides carboxyliques et d'une source de peroxygène. De façon plus précise, 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 l'hydrolyse d'acide peracétique (rapport PAAF/PAAH). Le variant d'acétyl xylan estérase peut être utilisé afin de 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.
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WO2012087790A1 (fr) * 2010-12-21 2012-06-28 E.I. Dupont De Nemours And Company Variant de perhydrolase permettant l'augmentation d'une activité spécifique
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WO2012087788A1 (fr) * 2010-12-21 2012-06-28 E.I. Dupont De Nemours And Company Variant de perhydrolase permettant l'augmentation d'une activité spécifique
WO2012087789A1 (fr) * 2010-12-21 2012-06-28 E.I. Dupont De Nemours And Company Variant de perhydrolase permettant l'augmentation d'une activité spécifique
WO2012087792A1 (fr) * 2010-12-21 2012-06-28 E.I. Dupont De Nemours And Company Variant de perhydrolase permettant l'augmentation d'une activité spécifique
WO2013096045A1 (fr) * 2011-12-19 2013-06-27 E. I. Du Pont De Nemours And Company Variants de perhydrolase présentant une activité spécifique améliorée en présence de tensioactif

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