US2016010072A1PendingUtilityA1

Proteases comprising one or more combinable mutations

60
Assignee: DANISCO US INCPriority: Nov 11, 2008Filed: Jun 29, 2015Published: Jan 14, 2016
Est. expiryNov 11, 2028(~2.3 yrs left)· nominal 20-yr term from priority
Inventors:Wolfgang AehleLuis G. Cascao-PereiraDavid A. EstellFrits GoedegebuurJames T. Kellis, Jr.Ayrookaran J. PouloseBrian F. Schmidt
C12Y 304/21062C12N 9/54C11D 3/38663C11D 3/38609C11D 3/386C12N 15/52C11D 3/38618C12N 15/09C11D 3/38636C11D 3/38654C11D 3/38681C11D 3/38627C11D 3/38645
60
PatentIndex Score
0
Cited by
0
References
0
Claims

Abstract

The present invention provides engineered protease variants. In particular, the protease variants comprise combinable mutations at selected surface positions that affect the charge and/or hydrophobicity of the enzyme to enhance at least one desired property of the resulting variant enzyme in a chosen application. Compositions comprising the protease variants, and methods for using the same are also provided.

Claims

exact text as granted — not AI-modified
1 - 8 . (canceled) 
     
     
         9 . An isolated nucleic acid encoding an isolated subtilisin protease variant of a parent  Bacillus  subtilisin, wherein said subtilisin variant is a mature form having proteolytic activity and comprising a substitution at two or more positions selected from positions 24, 45, 101, 109, 118, 213 and 217, wherein said positions are numbered by correspondence with the amino acid sequence of  B. amyloliquefaciens  subtilisin BPN′ set forth as SEQ ID NO:1, and wherein said parent has the sequence of SEQ ID NO:4 or SEQ ID NO:7. 
     
     
         10 . An expression vector comprising the nucleic acid of  claim 9 . 
     
     
         11 . A host cell comprising the expression vector set forth  claim 10 . 
     
     
         12 - 19 . (canceled) 
     
     
         20 . The nucleic acid of  claim 9 , wherein said isolated subtilisin variant further comprises a relative protein expression level performance index (TCA PI) and/or a stain removal activity performance index (BMI PI) that is greater than or equal to 0.5. 
     
     
         21 . The nucleic acid of  claim 20 , wherein said parent  Bacillus  subtilisin of said isolated subtilisin variant is GG36 and wherein said substitution at two or more positions is selected from: S24Q, S24E, S24L, S24R, R45Q, R45E, R45L, S101Q, S101E, S101L, S101R, Q109E, Q109L, Q109 R, G118Q, G118E, G118L, G118R, T213Q, T213L, T213R, T213E, L217Q, and L217E, wherein the positions correspond to the positions of BPN′ subtilisin of SEQ ID NO:1. 
     
     
         22 . The nucleic acid of  claim 20 , wherein said parent  Bacillus  subtilisin of said isolated subtilisin variant is FNA and wherein said substitution at two or more positions is selected from: S24Q, S24E, S24L, S24R, A45Q, A45E, A45L, A45R, S101Q, S101E, S101L, S101R, N109Q, N109E, N109L, N109R, K213Q, K213E, K213L, K213R, L217Q, L217E, wherein the positions correspond to the positions of BPN′ subtilisin of SEQ ID NO:1. 
     
     
         23 . The nucleic acid of  claim 20 , wherein said parent  Bacillus  subtilisin of said isolated subtilisin variant is GG36 and wherein said substitution at two or more positions is selected from S24Q-R45Q-S101Q-G118Q-T213Q, R45Q-S101Q-G118Q-T213Q, S101Q-G118Q-T213Q, G118Q-T213Q, S24E-R45Q-S101Q-G118Q-T213Q, S24E-R45E-S101Q-G118Q-T213Q, S24E-R45E-S101E-G118Q-T213Q, S24E-R45E-S101E-Q109E-G118Q-T213Q, S24E-R45E-S101E-Q109E-G118E-T213Q, S24E-R45E-S101E-Q109E-G118E-T213E, S24L-R45Q-S101Q-G118Q-T213Q, S24L-R45L-S101Q-G118Q-T213Q, S24L-R45L-S101L-G118Q-T213Q, S24L-R45L-S101L-Q109L-G118Q-T213Q, S24L-R45L-S101L-Q109L-G118L-T213Q, S24L-R45L-S101L-Q109L-G118L-T213L, S24R-R45Q-S101Q-G118Q-T213Q, S24R-S101Q-G118Q-T213Q, S24R-S101R-G118Q-T213Q, S24R-S101R-Q109R-G118Q-T213Q, S24R-S101R-Q109R-G118R-T213Q, S24R-S101R-Q109R-G118R-T213R, S24E-S101R-Q109R-G118R-T213R, S24E-R45E-S101R-Q109R-G118R-T213R, S24E-R45E-S101E-Q109R-G118R-T213R, S24E-R45E-S101E-Q109E-G118R-T213R, S24E-R45E-S101E-Q109E-G118E-T213R, S24E-R45E-S101E-Q109E-G118E-T213E, S24Q-R45Q-S101Q-G118Q-T213Q-L217Q, and S24Q-R45Q-S101Q-G118Q-T213Q-L217E, wherein the positions correspond to the positions of BPN′ subtilisin of SEQ ID NO:1. 
     
     
         24 . The nucleic acid of  claim 20 , wherein said parent  Bacillus  subtilisin of said isolated subtilisin variant is GG36 and said isolated subtilisin variant comprises the substitution T213Q, wherein said position corresponds to the amino acid sequence of  B. amyloliquefaciens  subtilisin BPN′ set forth as SEQ ID NO:1. 
     
     
         25 . The nucleic acid of  claim 20 , wherein said parent  Bacillus  subtilisin of said isolated subtilisin variant is FNA and wherein said substitution at two or more positions is selected from S24Q-A45Q-S101Q-N109Q-N118Q-K213Q, A45Q-S101Q-N109Q-N118Q-K213Q, S101Q-N109Q-N118Q-K213Q, N109Q-N118Q-K213Q, N118Q-K213Q, S24E-A45Q-S101Q-N109Q-N118Q-K213Q, S24E-A45E-S101Q-N109Q-N118Q-K213Q, S24E-A45E-S101E-N109Q-N118Q-K213Q, S24E-A45E-S101E-N109E-N118Q-K213Q, S24E-A45E-S101E-N109E-N118E-K213Q, S24E-A45E-S101E-N109E-N118E-K213E, S24L-A45Q-S101Q-N109Q-N118Q-K213Q, S24L-A45L-S101Q-N109Q-N118Q-K213Q, S24L-A45L-S101L-N109Q-N118Q-K213Q, S24L-A45L-S101L-N109L-N118Q-K213Q, S24L-A45L-S101L-N109L-N118L-K213Q, S24L-A45L-S101L-N109L-N118L-K213L, S24R-A45Q-S101Q-N109Q-N118Q-K213Q, S24R-A45R-S101Q-N109Q-N118Q-K213Q, S24R-A45R-S101R-N109Q-N118Q-K213Q, S24R-A45R-S101R-N109R-N118Q-K213Q, S24R-A45R-S101R-N109R-N118R-K213Q, S24R-A45R-S101R-N109R-N118R-K213R, S24E-A45R-S101R-N109R-N118R-K213R, S24E-A45E-S101R-N109R-N118R-K213R, S24E-A45E-S101E-N109R-N118R-K213R, S24E-A45E-S101E-N109E-N118R-K213R, S24E-A45E-S101E-N109E-N118E-K213R, S24E-A45E-S101E-N109E-N118E-K213E, S24Q-A45Q-S101Q-N109Q-N118Q-K213Q-L217Q, and S24Q-A45Q-S101Q-N109Q-N118Q-K213Q-L217E, wherein the positions correspond to the positions of BPN′ subtilisin of SEQ ID NO:1. 
     
     
         26 . The nucleic acid of  claim 20 , wherein said parent  Bacillus  subtilisin of said isolated subtilisin variant is FNA and said isolated subtilisin variant comprises the substitution K213Q, wherein said position is numbered by correspondence with the amino acid sequence of  B. amyloliquefaciens  subtilisin BPN′ set forth as SEQ ID NO:1. 
     
     
         27 . The nucleic acid of  claim 20 , wherein said isolated subtilisin variant comprises the substitution K213Q, wherein said position is numbered by correspondence with the amino acid sequence of  B. amyloliquefaciens  subtilisin BPN′ set forth as SEQ ID NO:1. 
     
     
         28 . The nucleic acid of  claim 9 , wherein said parent  Bacillus  subtilisin of said isolated subtilisin variant is GG36 and wherein said substitution at two or more positions is selected from: S24Q, S24E, S24L, S24R, R45Q, R45E, R45L, S101Q, S101E, S101L, S101R, Q109E, Q109L, Q109 R, G118Q, G118E, G118L, G118R, T213Q, T213L, T213R, T213E, L217Q, and L217E, wherein the positions correspond to the positions of BPN′ subtilisin of SEQ ID NO:1. 
     
     
         29 . The nucleic acid of  claim 9 , wherein said parent  Bacillus  subtilisin of said isolated subtilisin variant is FNA and wherein said substitution at two or more positions is selected from: S24Q, S24E, S24L, S24R, A45Q, A45E, A45L, A45R, S101Q, S101E, S101L, S101R, N109Q, N109E, N109L, N109R, K213Q, K213E, K213L, K213R, L217Q, L217E, wherein the positions correspond to the positions of BPN′ subtilisin of SEQ ID NO:1. 
     
     
         30 . The nucleic acid of  claim 9 , wherein said parent  Bacillus  subtilisin of said isolated subtilisin variant is GG36 and wherein said substitution at two or more positions is selected from S24Q-R45Q-S101Q-G118Q-T213Q, R45Q-S101Q-G118Q-T213Q, S101Q-G118Q-T213Q, G118Q-T213Q, S24E-R45Q-S101Q-G118Q-T213Q, S24E-R45E-S101Q-G118Q-T213Q, S24E-R45E-S101E-G118Q-T213Q, S24E-R45E-S101E-Q109E-G118Q-T213Q, S24E-R45E-S101E-Q109E-G118E-T213Q, S24E-R45E-S101E-Q109E-G118E-T213E, S24L-R45Q-S101Q-G118Q-T213Q, S24L-R45L-S101Q-G118Q-T213Q, S24L-R45L-S101L-G118Q-T213Q, S24L-R45L-S101L-Q109L-G118Q-T213Q, S24L-R45L-S101L-Q109L-G118L-T213Q, S24L-R45L-S101L-Q109L-G118L-T213L, S24R-R45Q-S101Q-G118Q-T213Q, S24R-S101Q-G118Q-T213Q, S24R-S101R-G118Q-T213Q, S24R-S101R-Q109R-G118Q-T213Q, S24R-S101R-Q109R-G118R-T213Q, S24R-S101R-Q109R-G118R-T213R, S24E-S101R-Q109R-G118R-T213R, S24E-R45E-S101R-Q109R-G118R-T213R, S24E-R45E-S101E-Q109R-G118R-T213R, S24E-R45E-S101E-Q109E-G118R-T213R, S24E-R45E-S101E-Q109E-G118E-T213R, S24E-R45E-S101E-Q109E-G118E-T213E, S24Q-R45Q-S101Q-G118Q-T213Q-L217Q, and S24Q-R45Q-S101Q-G118Q-T213Q-L217E, wherein the positions correspond to the positions of BPN′ subtilisin of SEQ ID NO:1. 
     
     
         31 . The nucleic acid of  claim 9 , wherein said parent  Bacillus  subtilisin of said isolated subtilisin variant is GG36 and said isolated subtilisin variant comprises the substitution T213Q, wherein said position corresponds to the amino acid sequence of  B. amyloliquefaciens  subtilisin BPN′ set forth as SEQ ID NO:1. 
     
     
         32 . The nucleic acid of  claim 9 , wherein said parent  Bacillus  subtilisin of said isolated subtilisin variant is FNA and wherein said substitution at two or more positions is selected from S24Q-A45Q-S101Q-N109Q-N118Q-K213Q, A45Q-S101Q-N109Q-N118Q-K213Q, S101Q-N109Q-N118Q-K213Q, N109Q-N118Q-K213Q, N118Q-K213Q, S24E-A45Q-S101Q-N109Q-N118Q-K213Q, S24E-A45E-S101Q-N109Q-N118Q-K213Q, S24E-A45E-S101E-N109Q-N118Q-K213Q, S24E-A45E-S101E-N109E-N118Q-K213Q, S24E-A45E-S101E-N109E-N118E-K213Q, S24E-A45E-S101E-N109E-N118E-K213E, S24L-A45Q-S101Q-N109Q-N118Q-K213Q, S24L-A45L-S101Q-N109Q-N118Q-K213Q, S24L-A45L-S101L-N109Q-N118Q-K213Q, S24L-A45L-S101L-N109L-N118Q-K213Q, S24L-A45L-S101L-N109L-N118L-K213Q, S24L-A45L-S101L-N109L-N118L-K213L, S24R-A45Q-S101Q-N109Q-N118Q-K213Q, S24R-A45R-S101Q-N109Q-N118Q-K213Q, S24R-A45R-S101R-N109Q-N118Q-K213Q, S24R-A45R-S101R-N109R-N118Q-K213Q, S24R-A45R-S101R-N109R-N118R-K213Q, S24R-A45R-S101R-N109R-N118R-K213R, S24E-A45R-S101R-N109R-N118R-K213R, S24E-A45E-S101R-N109R-N118R-K213R, S24E-A45E-S101E-N109R-N118R-K213R, S24E-A45E-S101E-N109E-N118R-K213R, S24E-A45E-S101E-N109E-N118E-K213R, S24E-A45E-S101E-N109E-N118E-K213E, S24Q-A45Q-S101Q-N109Q-N118Q-K213Q-L217Q, and S24Q-A45Q-S101Q-N109Q-N118Q-K213Q-L217E, wherein the positions correspond to the positions of BPN′ subtilisin of SEQ ID NO:1. 
     
     
         33 . The nucleic acid of  claim 9 , wherein said parent  Bacillus  subtilisin of said isolated subtilisin variant is FNA and said isolated subtilisin variant comprises the substitution K213Q, wherein said position is numbered by correspondence with the amino acid sequence of  B. amyloliquefaciens  subtilisin BPN′ set forth as SEQ ID NO:1. 
     
     
         34 . The nucleic acid of  claim 9 , wherein said isolated subtilisin variant comprises the substitution K213Q, wherein said position is numbered by correspondence with the amino acid sequence of  B. amyloliquefaciens  subtilisin BPN′ set forth as SEQ ID NO:1.

Join the waitlist — get patent alerts

Track US2016010072A1 — get alerts on status changes and closely related new filings.

We store only your email — no account needed. See our privacy policy.