# US 8,569,034 B2 · Thermolysin variants and detergent compositions therewith > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 8,569,034 B2 - Document ID: US8569034B2 - Status: Granted - Kind: B2 - Application number: 12/740,782 - Filed: 2008-10-28 - Published: 2012-01-12 - Granted: 2013-10-29 - Earliest claimed: 2007-11-01 - Assignees: Danisco US Inc. - Applicants: David A. Estell; Ronaldus W. J. Hommes; Amy D. Liu; Andrew Shaw - Inventors: David A. Estell; Ronaldus W. J. Hommes; Amy D. Liu; Andrew Shaw - Web page: https://patentgrep.com/patent/US8569034B2 ## CPC - C12N 9/54: bacteria being Bacillus - C11D 3/386: Preparations containing enzymes {, e.g. protease or amylase} - C11D 3/38618: {Protease or amylase in liquid compositions only} - C11D 3/38636: {containing enzymes other than protease, amylase, lipase, cellulase, oxidase or reductase} - C12Y 304/24027: Thermolysin (3.4.24.27) - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - C11D: DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL - C12Y: ENZYMES ## Abstract The present invention provides methods and compositions comprising at least one thermolysin-like neutral protease enzyme with improved storage stability and/or catalytic activity. In some embodiments, the thermolysin finds use in cleaning and other applications comprising detergent. In some particularly preferred embodiments, the present invention provides methods and compositions comprising thermolysin formulated and/or engineered to resist detergent-induced inactivation. ## Claims ### Claim 1 A composition comprising: (i) an isolated thermolysin variant, having at least 95% amino acid identity with SEQ ID NO: 3 and having a substitution at position Y151 (tyrosine), wherein the thermolysin variant has activity to cleave at the N-terminal of hydrophobic amino acids and (ii) the neutral metalloprotease inhibitor galardin. ### Claim 2 (depends on 1) The composition of claim 1, wherein the thermolysin variant has improved enzyme activity stability and/or wash performance as compared to SEQ ID NO: 3. ### Claim 3 (depends on 1) The composition of claim 1, wherein the isolated thermolysin variant further comprises one or more substitutions at positions equivalent to positions 6, 7, 49, 56, 58, 61, 63, 65, 75, 128, 156, 196, 273, 278, and 280 of the amino acid sequence set forth as SEQ ID NO: 3. ### Claim 4 (depends on 1) The composition of claim 1, wherein the isolated thermolysin variant further comprises one or more substitutions at positions equivalent to positions 4, 6, 7, 36, 49, 53, 56, 58, 61, 63, 65, 75, 85, 108, 128, 129, 156, 194, 195, 196, 261, 265, 273, 278, 280 and 297 of the amino acid sequence set forth as SEQ ID NO:3. ### Claim 5 (depends on 1) The composition of claim 1, wherein the isolated thermolysin variant further comprises one or more substitutions chosen from substitutions T006G, T006H, T006I, T006K, T006M, T006N, T006P, T006Q, T006R, T006V, T006W, T006Y, V007F, V007H, V007K, V007L, V007M, V007P, V007Q, V007R, V007T, V007Y, T049G, T049H, T049I, T049K, T049L, T049N, T049P, T049Q, T049W, A058I, A058P, A058R, F063I, F063L, F063P, S065K, S065Y, Y075G, Y075M, Y075T, Q128H, Q128I, Q128L, Q128M, Q128V, Q128Y, I156M, I156R, I156T, I156W, G196R, Q273I, Q273P, Q273Y, T278K, T278M, T278P, N280K, N280R, T006A, T006C, T049D, T049I, T049L, T049M, T049N, T049S, A056C, A056R, A056Y, A058S, S065C, S065E, S065I, S065T, S065V, S065Y, Q128C, Q128I, Q128M, Q128T, Q128V, Q128Y, I156E, I156H, I156K, I156M, I156R, I156T, I156W, G196D, G196H, Q273A, Q273N, Q273T, Q273W, Q273Y, T278C, T278H, T278M, T278N, T278S, T278Y, N280E, N280I, N280L, N280M, N280S, T006C, T049D, T049N, T049Q, T049S, A056C, A056E, A058C, A058E, Q061E, Q061M, S065C, S065D, S065E, S065P, S065V, S065W, S065Y, Q128C, Q128I, Q128M, Q128T, Q128V, Q128Y, and I156E. ### Claim 6 (depends on 1) The composition of claim 1, wherein the isolated thermolysin variant further comprises an improvement in enzyme activity stability and/or wash performance as compared to SEQ ID NO: 3. ### Claim 7 (depends on 6) The composition of claim 6, wherein the improvement comprises one or more of improved thermostability, improved wash performance under lower pH conditions, improved performance under higher pH conditions, and improved autolytic stability. ### Claim 8 (depends on 6) The composition of claim 6, wherein the isolated thermolysin variant further comprises at least one calcium ion and/or zinc ion. ### Claim 9 (depends on 6) The isolated thermolysin variant of claim 6, further comprising at least one stabilizer. ### Claim 10 (depends on 9) The isolated thermolysin variant of claim 9, wherein said stabilizer is chosen from borax, glycerol, zinc ions, calcium ions, and calcium formate. ### Claim 11 (depends on 6) The isolated thermolysin variant of claim 6, wherein said composition is a cleaning composition. ### Claim 12 (depends on 11) The isolated thermolysin variant of claim 11, wherein said cleaning composition is a detergent. ### Claim 13 (depends on 1) The composition of claim 1, wherein the composition further comprises at least one additional enzyme selected from the group consisting of proteases, amylases, lipases, mannanases, pectinases, cutinases, oxidoreductases, hemicellulases, and cellulases. ### Claim 14 (depends on 1) The composition of claim 1, wherein the composition comprises at least about 0.0001 weight percent of said thermolysin variant. ### Claim 15 (depends on 1) The composition of claim 1, wherein the composition comprises from about 0.001 to about 0.5 weight percent of said thermolysin variant. ### Claim 16 (depends on 1) The composition of claim 1, wherein the composition further comprising at least one adjunct ingredient. ### Claim 17 (depends on 1) The composition of claim 1, wherein the composition further comprises a sufficient amount of a pH modifier to provide the composition with a pH of from about 3 to about 5. ### Claim 18 (depends on 17) The composition of claim 17, further comprising at least one surfactant. ### Claim 19 (depends on 18) The composition of claim 18, wherein said surfactant is a sodium alkyl sulfate surfactant comprising an ethylene oxide moiety. ### Claim 20 (depends on 1) The composition of claim 1, wherein the composition is a liquid.