# US 11,299,722 B2 · Modified lipase and use thereof > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 11,299,722 B2 - Document ID: US11299722B2 - Status: Granted - Kind: B2 - Application number: 16/639,871 - Filed: 2018-08-17 - Published: 2020-11-19 - Granted: 2022-04-12 - Earliest claimed: 2017-09-01 - Assignees: Amano Enzyme Inc. - Applicants: Amano Enzyme Inc. - Inventors: Kazunori Yoshida; Tetsuya Takahashi; Kazuhiko Ishikawa - Web page: https://patentgrep.com/patent/US11299722B2 ## CPC - C12N 9/20: Triglyceride splitting, e.g. by means of lipase - C12Y 301/01003: Triacylglycerol lipase (3.1.1.3) - C02F 3/342: {characterised by the enzymes used} - C02F 3/343: {for digestion of grease, fat, oil} - C11B 3/003: {by enzymes or microorganisms, living or dead} - C11C 1/045: {using enzymes or microorganisms, living or dead} - C12P 7/64: Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats - C02F 2101/32: Hydrocarbons, e.g. oil - C12R 2001/72: Candida - C12R 2001/84: Pichia - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - C12Y: ENZYMES - C02F: TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE (separation in general B01D; special arrangements on waterborne vessels of installations for treating water, waste water or sewage, e.g. for producing fresh water, B63J; adding materials to water to prevent corrosion C23F; treating radioactively-contaminated liquids G21F9/04; regeneration of reactants for recirculation into processes, see the relevant places for the processes) - C11B: PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES (drying-oils C09F) - 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 - 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)} - C12R: INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS ## Abstract The present invention has an object of providing a modified lipase having excellent stability and reactivity at a high temperature. Provided is a modified lipase with improved reactivity and/or stability at a high temperature, the modified lipase having an amino acid sequence including one or more amino acid substitutions which are selected from the group consisting of T130C-S153C, A249P, F259Y, S282P, S283Y and S300P in the amino acid sequence represented by SEQ ID NO: 1, or an amino acid sequence having 90% or more sequence identity therewith. ## Claims ### Claim 1 A modified lipase with improved reactivity and/or stability at a high temperature, as compared to a lipase consisting of the amino acid sequence of SEQ ID NO: 1, the modified lipase having the amino acid sequence of SEQ ID NO: 1 except as having one or more amino acid substitutions selected from the group consisting of T130C-S153C, A249P, F259Y, 5282P, 5283Y, and S300P, or alternatively the modified lipase having an amino acid sequence having 92% or more sequence identity with the amino acid sequence of SEQ ID NO: 1 with one or more amino acid substitutions selected from the group consisting of T130C-S153C, A249P, F259Y, S282P, S283Y, and S300P, with the proviso that the amino acid sequence of the modified lipase is different from the amino acid sequence of SEQ ID NO: 1 at a position or positions other than the position(s) at which the amino acid substitution(s) is(are) made; wherein the high temperature is a temperature above 40 Celsius degree. ### Claim 2 (depends on 1) The modified lipase according to claim 1, wherein the amino acid substitution contained in the amino acid sequence of the modified lipase is T130C-S153C or S283Y, and the modified lipase has improved reactivity at a high temperature. ### Claim 3 (depends on 1) The modified lipase according to claim 1, wherein the amino acid substitution contained in the amino acid sequence of the modified lipase is A249P, S283Y, or S300P, and the modified lipase has improved stability at a high temperature. ### Claim 4 A lipase with improved reactivity and/or stability at a high temperature, as compared to a lipase consisting of the amino acid sequence of SEQ ID NO: 1, comprising the amino acid sequence of any of SEQ ID NOs: 2 to 7, or an amino acid sequence having 92% or more sequence identity with the amino acid sequence of any of SEQ ID Nos: 2 to 7, with the proviso that the amino acid sequence is different at a position or positions other than those corresponding to cysteine at positions 130 and 153 in the amino acid sequence of SEQ ID NO: 2, proline at position 249 in the amino acid sequence of SEQ ID NO: 3, tyrosine at position 259 in the amino acid sequence of SEQ ID NO: 4, proline at position 282 in the amino acid sequence of SEQ ID NO: 5, tyrosine at position 283 in the amino acid sequence of SEQ ID NO: 6, and proline at position 300 in the amino acid sequence of SEQ ID NO: 7, when the respective amino acid sequences are used as a reference sequence to determine the sequence identity; wherein the high temperature is a temperature above 40 Celsius degree. ### Claim 5 (depends on 1) A gene encoding the modified lipase according to claim 1. ### Claim 6 (depends on 5) The gene according to claim 5, comprising a base sequence of any of SEQ ID NOs: 8 to 19. ### Claim 7 (depends on 5) A recombinant DNA containing the gene according to claim 5. ### Claim 8 (depends on 7) A microorganism carrying the recombinant DNA according to claim 7. ### Claim 9 (depends on 8) The microorganism according to claim 8, wherein the host is Escherichia coli, Candida cylindracea, Aspergillus oryzae, Bacillus subtilis, or Pichia pastoris. ### Claim 10 (depends on 1) An enzyme preparation comprising the modified lipase according to claim 1. ### Claim 11 (depends on 1) A method for degradation of fats and oils comprising allowing the modified lipase according to claim 1 or an enzyme preparation comprising the modified lipase according to claim 1 to act on the fats and oils to carry out an enzyme reaction. ### Claim 12 (depends on 11) The method for degradation according to claim 11, wherein the enzyme reaction is carried out at 30° C. to 70° C. ### Claim 13 A modified lipase with improved reactivity and/or stability at a high temperature, as compared to a lipase consisting of the amino acid sequence of SEQ ID NO: 1, the modified lipase having the amino acid sequence of SEQ ID NO: 1 except as having the amino acid substitutions is T130C-S153C, or alternatively the modified lipase having an amino acid sequence having 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 1 with one or more amino acid substitutions of T130C-S153C, with the proviso that the amino acid sequence of the modified lipase is different from the amino acid sequence of SEQ ID NO: 1 at a position or positions other than the position(s) at which the amino acid substitution is made; wherein the high temperature is a temperature above 40 Celsius degree.