# US 2013/0158115 A1 · Perhydrolase Variant Providing Improved Specific Activity In the Presence of Surfactant > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2013/0158115 A1 - Document ID: US20130158115A1 - Status: Published application - Kind: A1 - Application number: 13/712,281 - Filed: 2012-12-12 - Published: 2013-06-20 - Earliest claimed: 2011-12-19 - Assignees: E. I. Du Pont de Nemours and Company - Inventors: Robert DICOSIMO; Hongxian HE; Neeraj PANDEY; Mark Scott PAYNE - Web page: https://patentgrep.com/patent/US20130158115A1 ## CPC - C12N 9/18: Carboxylic ester hydrolases {(3.1.1)} - B65D 25/00: Details of other kinds or types of rigid or semi-rigid containers - C12Y 301/01072: Acetylxylan esterase (3.1.1.72) - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - B65D: CONTAINERS FOR STORAGE OR TRANSPORT OF ARTICLES OR MATERIALS, e.g. BAGS, BARRELS, BOTTLES, BOXES, CANS, CARTONS, CRATES, DRUMS, JARS, TANKS, HOPPERS, FORWARDING CONTAINERS; ACCESSORIES, CLOSURES, OR FITTINGS THEREFOR; PACKAGING ELEMENTS; PACKAGES - C12Y: ENZYMES ## Abstract An acetyl xylan esterase variant having perhydrolytic activity is provided for producing peroxycarboxylic acids from carboxylic acid esters and a source of peroxygen. More specifically, a variant of the Thermotoga maritima C277T acetyl xylan esterase is provided having an improved specific activity when producing peroxycarboxylic acids in the presence of an anionic surfactant. The variant acetyl xylan esterase may be used to produce peroxycarboxylic acids suitable for use in a variety of applications such as cleaning, disinfecting, sanitizing, bleaching, wood pulp processing, and paper pulp processing applications. ## Claims ### Claim 1 An isolated nucleic acid molecule selected from the group consisting of: (a) a polynucleotide encoding a polypeptide having perhydrolytic activity, said polypeptide comprising the amino acid sequence of SEQ ID NO: 4; (b) a polynucleotide comprising the nucleic acid sequence of SEQ ID NO: 8; and (c) a polynucleotide fully complementary to the polynucleotide of (a) or (b). ### Claim 2 (depends on 1) A vector comprising the nucleic acid molecule of claim 1. ### Claim 3 (depends on 1) A recombinant DNA construct comprising the nucleic acid molecule of claim 1 operably-linked to a suitable regulatory sequence. ### Claim 4 (depends on 3) A recombinant host cell comprising the recombinant DNA construct of claim 3. ### Claim 5 (depends on 3) A method for expressing a polypeptide comprising transforming a host cell with the recombinant DNA construct of claim 3. ### Claim 6 An isolated polypeptide having perhydrolytic activity comprising the amino acid sequence of SEQ ID NO: 4. ### Claim 7 (depends on 6) The polypeptide of claim 6; wherein said polypeptide is characterized by a peracetic acid formation specific activity that is higher in the presence of a surfactant than the peracetic acid formation specific activity of the Thermotoga maritima C277T acetyl xylan esterase provided as amino acid sequence SEQ ID NO: 3. ### Claim 8 (depends on 6) A process for producing a peroxycarboxylic acid comprising: (a) providing a set of reaction components comprising: (1) at least one substrate selected from the group consisting of: (i) one or more esters having the structure [X]mR5 wherein X=an ester group of the formula R6—C(O)O; R6=C1 to C7 linear, branched or cyclic hydrocarbyl moiety, optionally substituted with hydroxyl groups or C1 to C4 alkoxy groups, wherein R6 optionally comprises one or more ether linkages for R6=C2 to C7; R5=a C1 to C6 linear, branched, or cyclic hydrocarbyl moiety or a five-membered cyclic heteroaromatic moiety or six-membered cyclic aromatic or heteroaromatic moiety optionally substituted with hydroxyl groups; wherein each carbon atom in R5 individually comprises no more than one hydroxyl group or no more than one ester group or carboxylic acid group; wherein R5 optionally comprises one or more ether linkages; m=an integer ranging from 1 to the number of carbon atoms in R5; and wherein said esters have solubility in water of at least 5 ppm at 25° C.; (ii) one or more glycerides having the structure wherein R1=C1 to C21 straight chain or branched chain alkyl optionally substituted with an hydroxyl or a C1 to C4 alkoxy group and R3 and R4 are individually H or R1C(O); (iii) one or more esters of the formula: wherein R1 is a C1 to C7 straight chain or branched chain alkyl optionally substituted with an hydroxyl or a C1 to C4 alkoxy group and R2 is a C1 to C10 straight chain or branched chain alkyl, alkenyl, alkynyl, aryl, alkylaryl, alkylheteroaryl, heteroaryl, (CH2CH2O)n, or (CH2CH(CH3)—O)nH and n is 1 to 10; (iv) one or more acylated monosaccharides, acylated disaccharides, or acylated polysaccharides; and (v) any combination of (i) through (iv); (2) a source of peroxygen; and (3) an enzyme catalyst comprising the polypeptide of claim 6; (b) combining the set of reaction components under suitable reaction conditions whereby peroxycarboxylic acid is produced; and (c) optionally diluting the peroxycarboxylic acid produced in step (b). ### Claim 9 (depends on 8) The process of claim 8 further comprising the step of: d) contacting a hard surface or inanimate object with the peroxycarboxylic acid produced in step (b) or step (c); whereby said hard surface or said inanimate object is disinfected, bleached, destained or a combination thereof. ### Claim 10 (depends on 9) The process of claim 9 wherein the inanimate object is a medical instrument. ### Claim 11 (depends on 8) The process of claim 8 further comprising the step of: d) contacting an article of clothing or a textile with the peroxycarboxylic acid produced in step (b) or step (c); whereby the article of clothing or textile receives a benefit. ### Claim 12 (depends on 11) The process of claim 11 wherein the benefit is selected from the group consisting of disinfecting, sanitizing, bleaching, destaining, deodorizing, and combinations thereof. ### Claim 13 (depends on 8) The process of claim 8 further comprising the step of: d) contacting wood pulp or paper pulp with the peroxycarboxylic acid produced in step (b) or step (c); whereby the wood pulp or paper pulp is bleached. ### Claim 14 (depends on 8) The process of claim 8 wherein the substrate is selected from the group consisting of: monoacetin; diacetin; triacetin; monopropionin; dipropionin; tripropionin; monobutyrin; dibutyrin; tributyrin; glucose pentaacetate; β-D-galactose pentaacetate; sorbitol hexaacetate; sucrose octaacetate; xylose tetraacetate; acetylated xylan; acetylated xylan fragments; β-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-hexanediol, 2,5-hexanediol, 1,6-hexanediol, 4-acetoxybenzoic acid; and mixtures thereof. ### Claim 15 (depends on 14) The process of claim 14 wherein the substrate is triacetin. ### Claim 16 (depends on 8) The process of claim 8 wherein the peroxycarboxylic acid produced is peracetic acid, perpropionic acid, perbutyric acid, perlactic acid, perglycolic acid, permethoxyacetic acid, per-β-hydroxybutyric acid, or mixtures thereof. ### Claim 17 (depends on 8) The process of claim 8 wherein the enzyme catalyst is in the form of a microbial cell, a permeabilized microbial cell, a microbial cell extract, a partially purified enzyme, or a purified enzyme. ### Claim 18 (depends on 6) A composition comprising: (a) a set of reaction components comprising: (1) at least one substrate selected from the group consisting of: (i) one or more esters having the structure [X]mR5 wherein X=an ester group of the formula R6—C(O)O; R6=C1 to C7 linear, branched or cyclic hydrocarbyl moiety, optionally substituted with hydroxyl groups or C1 to C4 alkoxy groups, wherein R6 optionally comprises one or more ether linkages for R6=C2 to C7; R5=a C1 to C6 linear, branched, or cyclic hydrocarbyl moiety or a five-membered cyclic heteroaromatic moiety or six-membered cyclic aromatic or heteroaromatic moiety optionally substituted with hydroxyl groups; wherein each carbon atom in R5 individually comprises no more than one hydroxyl group or no more than one ester group or carboxylic acid group; wherein R5 optionally comprises one or more ether linkages; m=an integer ranging from 1 to the number of carbon atoms in R5; and wherein said esters have solubility in water of at least 5 ppm at 25° C.; (ii) one or more glycerides having the structure wherein R1=C1 to C21 straight chain or branched chain alkyl optionally substituted with an hydroxyl or a C1 to C4 alkoxy group and R3 and R4 are individually H or R1C(O); (iii) one or more esters of the formula: wherein R1 is a C1 to C7 straight chain or branched chain alkyl optionally substituted with an hydroxyl or a C1 to C4 alkoxy group and R2 is a C1 to C10 straight chain or branched chain alkyl, alkenyl, alkynyl, aryl, alkylaryl, alkylheteroaryl, heteroaryl, (CH2CH2O)n, or (CH2CH(CH3)—O)nH and n is 1 to 10; (iv) one or more acylated monosaccharides, acylated disaccharides, or acylated polysaccharides; and (v) any combination of (i) through (iv); (2) a source of peroxygen; and (3) an enzyme catalyst comprising the polypeptide of claim 6; and (b) at least one peroxycarboxylic acid formed upon combining the set of reaction components of (a). ### Claim 19 (depends on 6) A peracid generation and delivery system comprising: (a) a first compartment comprising (1) an enzyme catalyst comprising the polypeptide of claim 6; (2) at least one substrate selected from the group consisting of: (i) one or more esters having the structure [X]mR5 wherein X=an ester group of the formula R6—C(O)O; R6=C1 to C7 linear, branched or cyclic hydrocarbyl moiety, optionally substituted with hydroxyl groups or C1 to C4 alkoxy groups, wherein R6 optionally comprises one or more ether linkages for R6=C2 to C7; R5=a C1 to C6 linear, branched, or cyclic hydrocarbyl moiety or a five-membered cyclic heteroaromatic moiety or six-membered cyclic aromatic or heteroaromatic moiety optionally substituted with hydroxyl groups; wherein each carbon atom in R5 individually comprises no more than one hydroxyl group or no more than one ester group or carboxylic acid group; wherein R5 optionally comprises one or more ether linkages; m=an integer ranging from 1 to the number of carbon atoms in R5; and wherein said esters have solubility in water of at least 5 ppm at 25° C.; (ii) one or more glycerides having the structure wherein R1=C1 to C21 straight chain or branched chain alkyl optionally substituted with an hydroxyl or a C1 to C4 alkoxy group and R3 and R4 are individually H or R1C(O); (iii) one or more esters of the formula: wherein R1 is a C1 to C7 straight chain or branched chain alkyl optionally substituted with an hydroxyl or a C1 to C4 alkoxy group and R2 is a C1 to C10 straight chain or branched chain alkyl, alkenyl, alkynyl, aryl, alkylaryl, alkylheteroaryl, heteroaryl, (CH2CH2O)n, or (CH2CH(CH3)—O)nH and n is 1 to 10; (iv) one or more acylated monosaccharides, acylated disaccharides, or acylated polysaccharides; and (v) any combination of (i) through (iv); and (3) an optional buffer; and (b) a second compartment comprising (1) source of peroxygen; (2) a peroxide stabilizer; and (3) an optional buffer. ### Claim 20 (depends on 19) The peracid generation and delivery system of claim 19 wherein the substrate comprises triacetin. ### Claim 21 (depends on 6) A laundry care product comprising the polypeptide of claim 6. ### Claim 22 (depends on 6) A personal care product comprising the polypeptide of claim 6.