# US 10,767,142 B2 · Proteases with improved enzyme stability in detergents > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,767,142 B2 - Document ID: US10767142B2 - Status: Granted - Kind: B2 - Application number: 16/087,391 - Filed: 2017-03-07 - Published: 2020-05-07 - Granted: 2020-09-08 - Earliest claimed: 2016-03-23 - Assignees: Henkel AG & Co. KGaA - Applicants: Henkel AG & Co. KGaA - Inventors: Nina Mussmann; Timothy O'Connell; Daniela Herbst; Inken Prueser; Christian Degering; Sabine Griemert; Thorsten Eggert; Christian Leggewie - Web page: https://patentgrep.com/patent/US10767142B2 ## CPC - C11D 3/38681: {Chemically modified or immobilised enzymes} - C11D 3/386: Preparations containing enzymes {, e.g. protease or amylase} - C12N 9/54: bacteria being Bacillus - C12Y 304/21062: Subtilisin (3.4.21.62) - C11D: DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - C12Y: ENZYMES ## Abstract The present disclosure relates to proteases having an amino acid sequence with at least about 70% sequence identity to the amino acid sequence given in SEQ ID NO:2, over the entire length thereof, and comprising an amino acid substitution at at least one of the positions P9, Q62, D101, N130, G166, N187, 5216, N238 or Q271, based in each case on the numbering according to SEQ ID NO:2. The present disclosure also relates to the production and use thereof. Such proteases exhibit very good stability, in particular storage stability, while at the same time having a good cleaning performance. ## Claims ### Claim 1 A protease having an amino-acid sequence which has an at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:2 over the entire length thereof and which has an amino acid substitution at one or more of the positions P9, Q62, D101, N130, G166, N187, S216, N238 and Q271, each based on the numbering according to SEQ ID NO:2. ### Claim 2 (depends on 1) The protease according to claim 1, wherein the amino acid substitution is selected from the group consisting of P9T/S/H, Q62E, D101E, N130D, G166S, N187H, S216C, N238K Q271E, and combinations thereof, each based on the numbering according to SEQ ID NO:2. ### Claim 3 (depends on 1) The protease according to claim 1, wherein the protease has one of the following amino acid substitution variants, each based on the numbering according to SEQ ID NO:2: (i) P9T; (ii) Q271E; (iii) P9S and N238K; (iv) P9H; (v) N187H; (vi) S216C; (vii) Q62E and N130D; (viii) Q62E, D101E, N130D, G166S and Q271E; or (ix) P9T, N187H, S216C and Q271E. ### Claim 4 (depends on 1) A protease wherein (a) said protease can be obtained as a starting molecule from a protease according to claim 1 by employing one or more conservative amino acid substitutions, the protease having at least one of the amino acid substitutions P9T/S/H, Q62E, D101E, N130D, G166S, N187H, S216C, N238K or Q271E at the positions which correspond to the positions 9, 62, 101, 130, 166, 187, 216, 238 and 271 according to SEQ ID NO:2; and/or (b) said protease can be obtained as a starting molecule from a protease according to claim 1 by employing fragmentation, deletion, insertion or substitution mutagenesis, and has an amino acid sequence which matches that of the starting molecule over a length of at least about 50 interconnected amino acids, the protease having at least one of the amino acid substitutions P9T/S/H, Q62E, D101E, N130D, G166S, N187H, S216C, N238K or Q271E at the positions which correspond to positions 9, 62, 101, 130, 166, 187, 216, 238 and 271 according to SEQ ID NO:2. ### Claim 5 A method for preparing a protease, comprising substituting an amino acid at one or more of the positions which correspond to positions 9, 62, 101, 130, 166, 187, 216, 238 and 271 in SEQ ID NO:2 in a starting protease of which the sequence is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:2 over the entire length thereof. ### Claim 6 (depends on 5) The method according to claim 5, further comprising one or both of the following method steps: (a) introducing one or more conservative amino-acid substitutions, wherein the protease has at least one of the amino acid substitutions P9T/S/H, Q62E, D101E, N130D, G166S, N187H, S216C, N238K or Q271E at the positions which correspond to the positions 9, 62, 101, 130, 166, 187, 216, 238 and 271 according to SEQ ID NO:2; (b) altering the amino-acid sequence by employing fragmentation, deletion, insertion or substitution mutagenesis such that the protease has an amino acid sequence which matches that of the starting molecule over a length of at least about 50 interconnected amino acids, wherein the protease has at least one of the amino acid substitutions P9T/S/H, Q62E, D101E, N130D, G166S, N187H, S216C, N238K or Q271E at the positions which correspond to positions 9, 62, 101, 130, 166, 187, 216, 238 and 271 according to SEQ ID NO:2. ### Claim 7 (depends on 1) The A nucleic acid coding for the protease according to claim 1. ### Claim 8 (depends on 7) A vector comprising the nucleic acid according to claim 7. ### Claim 9 (depends on 8) A non-human host cell comprising the vector according to claim 8. ### Claim 10 (depends on 1) A cleaning agent comprising the protease according to claim 1 and a surfactant. ### Claim 11 (depends on 10) A method of cleaning textiles or a hard surface, comprising contacting the textiles or hard surface with a cleaning agent according to claim 10. ### Claim 12 (depends on 1) The protease according to claim 1, wherein the protease has the amino acid substitution P9T based on the numbering according to SEQ ID NO:2. ### Claim 13 (depends on 1) The protease according to claim 1, wherein the protease has the amino acid substitution Q271E based on the numbering according to SEQ ID NO:2. ### Claim 14 (depends on 1) The protease according to claim 1, wherein the protease has the amino acid substitutions P9S and N238K based on the numbering according to SEQ ID NO:2. ### Claim 15 (depends on 1) The protease according to claim 1, wherein the protease has the amino acid substitution P9H based on the numbering according to SEQ ID NO:2. ### Claim 16 (depends on 1) The protease according to claim 1, wherein the protease has the amino acid substitution N187H based on the numbering according to SEQ ID NO:2. ### Claim 17 (depends on 1) The protease according to claim 1, wherein the protease has the amino acid substitution S216C based on the numbering according to SEQ ID NO:2. ### Claim 18 (depends on 1) The protease according to claim 1, wherein the protease has the amino acid substitutions Q62E and N130D based on the numbering according to SEQ ID NO:2. ### Claim 19 (depends on 1) The protease according to claim 1, wherein the protease has the amino acid substitutions Q62E, D101E, N130D, G166S and Q271E based on the numbering according to SEQ ID NO:2. ### Claim 20 (depends on 1) The protease according to claim 1, wherein the protease has the amino acid substitutions 9T, N187H, S216C and Q271E based on the numbering according to SEQ ID NO:2.