# US 10,308,920 B2 · Hydrolases, nucleic acids encoding them and methods for making and using them > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,308,920 B2 - Document ID: US10308920B2 - Status: Granted - Kind: B2 - Application number: 14/877,335 - Filed: 2015-10-07 - Published: 2016-03-03 - Granted: 2019-06-04 - Earliest claimed: 2003-03-07 - Assignees: Dsm IP Assets B.V. - Applicants: Dsm IP Assets B.V. - Inventors: Uwe T. Bornscheuer; David Weiner; Tim Hitchman; Jonathan Lyon; Sirirung Wongsakul - Web page: https://patentgrep.com/patent/US10308920B2 ## CPC - C12N 9/20: Triglyceride splitting, e.g. by means of lipase - A23C 9/20: Dietetic milk products not covered by groups A23C9/12 - A23C9/18 - A23C 19/05: Treating milk before coagulation; Separating whey from curd - A23C 19/063: {Addition of, or treatment with, enzymes or cell-free extracts of microorganisms} - A23K 10/14: Pretreatment of feeding-stuffs with enzymes - A23K 20/189: Enzymes - A23L 29/06: {Enzymes} - A61K 8/0216: {Solid or semisolid forms} - A61K 8/375: {the alcohol moiety containing more than one hydroxy group} - A61K 8/606: {Nucleosides; Nucleotides; Nucleic acids} - A61K 8/66: Enzymes - A61K 38/465: {acting on ester bonds (3.1), e.g. lipases, ribonucleases} - A61P 39/02: Antidotes - A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00 - A61Q 5/06: Preparations for styling the hair, e.g. by temporary shaping or colouring - A61Q 11/00: Preparations for care of the teeth, of the oral cavity or of dentures; Dentifrices, e.g. toothpastes; Mouth rinses - A61Q 19/00: Preparations for care of the skin - C10M 129/70: of monocarboxylic acids - C10M 129/76: containing free hydroxy or carboxyl groups - C11C 3/08: with fatty acids - C11D 3/386: Preparations containing enzymes {, e.g. protease or amylase} - C11D 3/38627: {containing lipase} - C11D 3/38636: {containing enzymes other than protease, amylase, lipase, cellulase, oxidase or reductase} - C12C 1/047: Influencing the germination by chemical or physical means - C12C 5/004: {Enzymes} - C12C 11/003: {Fermentation of beerwort} - C12N 9/14: Hydrolases (3) - C12N 9/16: acting on ester bonds (3.1) - C12N 9/18: Carboxylic ester hydrolases {(3.1.1)} - C12P 7/6418: by hydrolysis of fatty acid esters - C12Q 1/34: involving hydrolase - D06M 16/003: {with enzymes or microorganisms} - G01N 33/573: for enzymes or isoenzymes - A23V 2002/00: Food compositions, function of food ingredients or processes for food or foodstuffs - A61K 2800/86: Products or compounds obtained by genetic engineering - C10M 2207/281: of (cyclo)aliphatic monocarboxylic acids - C10M 2207/289: containing free hydroxy groups - C10M 2207/40: Fatty vegetable or animal oils - C12Y 301/01003: Triacylglycerol lipase (3.1.1.3) - D06M 2200/12: Hydrophobic properties - D06M 2200/30: Flame or heat resistance, fire retardancy properties - D06M 2200/50: Modified hand or grip properties; Softening compositions - G01N 2500/02: Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor) - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - A23C: DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; PREPARATION THEREOF (preservation thereof A23B11/00) - A23K: FODDER - A23L: FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF (preservation thereof A23B) - A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D) - A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS - A61Q: SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS - C10M: LUBRICATING COMPOSITIONS (well drilling compositions C09K8/02); USE OF CHEMICAL SUBSTANCES EITHER ALONE OR AS LUBRICATING INGREDIENTS IN A LUBRICATING COMPOSITION (mould release, i.e. separating, agents for metals B22C3/00, for plastics or substances in a plastic state, in general B29C33/56, for glass C03B40/02; textile lubricating compositions D06M11/00, D06M13/00, D06M15/00; immersion oils for microscopy G02B21/33) - C11C: FATTY ACIDS FROM FATS, OILS OR WAXES; CANDLES; FATS, OILS OR FATTY ACIDS BY CHEMICAL MODIFICATION OF FATS, OILS, OR FATTY ACIDS OBTAINED THEREFROM - C11D: DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL - C12C: BEER; PREPARATION OF BEER BY FERMENTATION (ageing or ripening by storing C12H1/22; methods for reducing the alcohol content after fermentation C12H3/00; methods for increasing the alcohol content after fermentation C12H6/00; venting devices for casks, barrels or the like C12L9/00); PREPARATION OF MALT FOR MAKING BEER; PREPARATION OF HOPS FOR MAKING BEER - C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)} - C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES - D06M: TREATMENT, NOT PROVIDED FOR ELSEWHERE IN CLASS D06, OF FIBRES, THREADS, YARNS, FABRICS, FEATHERS OR FIBROUS GOODS MADE FROM SUCH MATERIALS - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) - A23V: INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION - C12Y: ENZYMES ## Abstract The invention provides hydrolases, polynucleotides encoding them, and methods of making and using these polynucleotides and polypeptides. In one aspect, the invention is directed to polypeptides, e.g., enzymes, having a hydrolase activity, e.g., an esterase, acylase, lipase, phospholipase (e.g., phospholipase A, B, C and D activity, patatin activity, lipid acyl hydrolase (LAH) activity) or protease activity, including thermostable and thermotolerant hydrolase activity, and polynucleotides encoding these enzymes, and making and using these polynucleotides and polypeptides. The hydrolase activities of the polypeptides and peptides of the invention include esterase activity, lipase activity (hydrolysis of lipids), acidolysis reactions (to replace an esterified fatty acid with a free fatty acid), transesterification reactions (exchange of fatty acids between triglycerides), ester synthesis, ester interchange reactions, phospholipase activity and protease activity (hydrolysis of peptide bonds). The polypeptides of the invention can be used in a variety of pharmaceutical, agricultural and industrial contexts, including the manufacture of cosmetics and nutraceuticals. In another aspect, the polypeptides of the invention are used to synthesize enantiomerically pure chiral products. ## Claims ### Claim 1 A variant polypeptide having a lipase activity, wherein the variant polypeptide is selected from: (i) an amino acid sequence having at least 95% sequence identity to SEQ ID NO:16 and containing at least one substitution modification relative to SEQ ID NO:16; or (ii) an amino acid sequence containing at least one substitution modification relative to SEQ ID NO:16, encoded by a nucleic acid having at least 95% sequence identity to SEQ ID NO:15. ### Claim 2 (depends on 1) A protein preparation comprising the variant polypeptide as set forth in claim 1, wherein the protein preparation comprises a liquid, a solid, or a gel. ### Claim 3 (depends on 1) A food supplement for an animal comprising the variant polypeptide as set forth in claim 1. ### Claim 4 (depends on 1) A method for hydrolyzing a triacylglycerol (TAG), a diacylglycerol (DAG) or a monoacylglycerol (MAG) comprising the following steps: (a) providing the variant polypeptide as set forth in claim 1; (b) providing a composition comprising a triacylglycerol (TAG), a diacylglycerol (DAG) or a monoacylglycerol (MAG); and (c) contacting the polypeptide of step (a) with the composition of step (b) under conditions wherein the polypeptide hydrolyzes the triacylglycerol (TAG), diacylglycerol (DAG), or monoacylglycerol (MAG). ### Claim 5 (depends on 1) A detergent composition comprising the variant polypeptide as set forth in claim 1. ### Claim 6 (depends on 5) The detergent composition of claim 5, wherein the variant polyeptide is a nonsurface-active hydrolase or a surface-active hydrolase. ### Claim 7 (depends on 5) The detergent composition of claim 5, wherein the variant polypeptide is formulated in a nonaqueous liquid composition, a cast solid, a granular form, a particulate form, a compressed tablet, a gel form, a paste, or a slurry form. ### Claim 8 (depends on 1) A method for refining a lubricant comprising the following steps: (a) providing the variant polypeptide as set forth in claim 1; (b) providing a lubricant; and (c) treating the lubricant with the polypeptide of (a) under conditions wherein the polypeptide can selective hydrolyze oils in the lubricant, thereby refining it. ### Claim 9 (depends on 1) A method of treating a fabric comprising the following steps: (a) providing the variant polypeptide as set forth in claim 1; (b) providing a fabric; and (c) treating the fabric with the polypeptide of (a) under condition wherein the polypeptide can selectively hydrolyze carboxylic esters thereby treating the fabric. ### Claim 10 (depends on 9) The method of claim 9, wherein treatment of the fabric comprises improvement of the hand and drape of the final fabric, dyeing, obtaining flame retardancy, obtaining water repellency, obtaining optical brightness, or obtaining resin finishing. ### Claim 11 (depends on 1) A method for removing or decreasing the amount of a food or oil stain comprising contacting the variant polypeptide of claim 1 with the food or oil stain under conditions wherein the variant polypeptide can hydrolyze food or oil stain. ### Claim 12 (depends on 1, 11) The method of claim 11, wherein the variant polypeptide of claim 1 has an enhanced stability to denaturation by surfactants and to heat deactivation. ### Claim 13 (depends on 1) A dietary composition comprising the variant polypeptide as set forth in claim 1. ### Claim 14 (depends on 1) A method of reducing fat content in milk or vegetable-based dietary compositions comprising the following steps: (a) providing the variant polypeptide as set forth in claim 1; (b) providing a composition comprising a milk or a vegetable oil, and (c) treating the composition of step (b) with the polypeptide of (a) under conditions wherein the polypeptide can hydrolyze the oil or fat in the composition of (b), thereby reducing its fat content. ### Claim 15 (depends on 1) A method of catalyzing an interesterification or transesterification reaction to produce new triglycerides comprising the following steps: (a) providing the variant polypeptide of claim 1, wherein the polypeptide can catalyze an interesterification or transterification reaction; (b) providing a mixture of triglycerides (TAG), a diacylglycerol (DAG), a monoacylglycerol (MAG) or free fatty acids; and (c) treating the composition of step (b) with the polypeptide of (a) under conditions wherein the polypeptide can catalyze exchange of free fatty acids with the acyl groups of triglycerides (TAG), the diacylglycerol (DAG), the monoacylglycerol (MAG), thereby producing new triglycerides enriched in the added fatty acids. ### Claim 16 (depends on 1) A method for ameliorating or preventing lipopolysaccharide (LPS)-mediated toxicity comprising administering to a patient a pharmaceutical composition comprising the variant polypeptide as set forth in claim 1. ### Claim 17 (depends on 1) A method for detoxifying an endotoxin comprising contacting the endotoxin with the variant polypeptide as set forth in claim 1.