US5316935A
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Subtilisin variants suitable for hydrolysis and synthesis in organic media
Assignee: California Institute of Technology
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Filing: 1992-04-06
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Granted: 1994-05-31
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Expires: 2012-04-06
Abstract
Abstract In accordance with the present invention, there are provided novel, modified subtilisin enzyme(s) having improved catalytic activity and/or stability in organic media.
Inventors
- 👤 Frances H. Arnold
- 👤 Keqin Chen
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-02 20:23
CPC Classifications
C; C12; C12N; C12N9/00; C12N9/14; C12N9/48; C12N9/50; C12N9/52; C12N9/54
Claims Analysis
Total Claims: 26
Claim 1
Independent Claim
84 words
A substantially pure modified subtilisin enzyme having improved stability and/or catalytic activity in organic media, relative to the stability and/or catalytic activity of unmodified subtilisin enzymes in organic media, wherein said modified subtilisin enzyme is selected from among the group consisting of mature, Class I subtilisins and wherein the modification consists of an amino acid substitution at amino acid sequence position 181 of said Class I subtilisin enzyme which corresponds substantially to position 181 of the mature subtilisin BPN', said substitution consisting of N181S.
Claim 2
Dependent Claim
84 words
A substantially pure modified subtilisin enzyme having improved stability and/or catalytic activity in organic media, relative to the stability and/or catalytic activity of unmodified subtilisin enzymes in organic media, wherein said modified subtilisin enzyme is selected from among the group consisting of mature, Class I subtilisins and wherein the modification consists of an amino acid substitution at amino acid sequence position 181 of said Class I subtilisin enzyme which corresponds substantially to position 181 of the mature subtilisin BPN', said substitution consisting of N181S.
Claim 3
Dependent Claim
43 words
A modified subtilisin enzyme according to claim 1 wherein said Class I subtilisin enzyme is selected from BASBPN, BSS168, BSSDY, BLSCAR, BAPB92, BYSYAB, BLS147, BSEPR, BSISP1, TVTHER, DNEBPR, XCEXPR, BSBPF, EFCYLA, SEEPIP, SPSCPA, LLSK11, SMEXSP, AVPRCA, MMPPC3, HSIPC2, HSFURI, DMFUR1, KLKEX1, or SCKEX2.
Claim 4
Dependent Claim
28 words
A modified subtilisin enzyme according to claim 2, wherein said Class I subtilisin enzyme is selected from BASBPN, BSS168, BSSDY, BLSCAR, BAPB92, BYSYAB, BLS147, BSEPR, BSISP1, or TVTHER.
Claim 5
Dependent Claim
16 words
An isolated DNA segment consisting essentially of a region encoding the modified enzyme of claim 1.
Claim 6
Dependent Claim
9 words
An expression vector containing DNA according to claim 4.
Claim 7
Dependent Claim
8 words
Host cells containing DNA according to claim 4.
Claim 8
Dependent Claim
9 words
Host cells containing expression vector according to claim 5.
Claim 9
Dependent Claim
24 words
Process for the production of modified enzyme according to claim 1, said process comprising expressing DNA encoding said modified enzyme in a suitable host.
Claim 10
Dependent Claim
69 words
A modified subtilisin enzyme according to claim 1 wherein said modification consists of one or more additional amino acid substitutions at amino acid sequence positions of said Class I subtilisin enzyme corresponding substantially to those of the amino acid sequence of the mature subtilisin BPN', said substitutions selected from the group consisting of D60N, D97G, Q103R, G131D, S182G, S188P, D248A, D248L, D248N or T255A, and optionally E156G and/or N218S.
Claim 11
Dependent Claim
14 words
A modified subtilisin enzyme according to claim 9 wherein said substitution consists of D60N.
Claim 12
Dependent Claim
14 words
A modified subtilisin enzyme according to claim 9 wherein said substitution consists of D97G.
Claim 13
Dependent Claim
14 words
A modified subtilisin enzyme according to claim 9 wherein said substitution consists of Q103R.
Claim 14
Dependent Claim
14 words
A modified subtilisin enzyme according to claim 9 wherein said substitution consists of G131D.
Claim 15
Dependent Claim
14 words
A modified subtilisin enzyme according to claim 9 wherein said substitution consists of E156F.
Claim 16
Dependent Claim
14 words
A modified subtilisin enzyme according to claim 9 wherein said substitution consists of S182G.
Claim 17
Dependent Claim
14 words
A modified subtilisin enzyme according to claim 9 wherein said substitution consists of S188P.
Claim 18
Dependent Claim
14 words
A modified subtilisin enzyme according to claim 9 wherein said substitution consists of N218S.
Claim 19
Dependent Claim
14 words
A modified subtilisin enzyme according to claim 9 wherein said substitution consists of D248N.
Claim 20
Dependent Claim
14 words
A modified subtilisin enzyme according to claim 9 wherein said substitution consists of T255A.
Claim 21
Dependent Claim
17 words
A modified subtilisin enzyme according to claim 9 wherein said substitution consists of D60N, Q103R and N218S.
Claim 22
Dependent Claim
18 words
A modified subtilisin enzyme according to claim 9 wherein said substitution consists of D60N, D97G, Q103R and N218S.
Claim 23
Dependent Claim
17 words
A modified subtilisin enzyme according to claim 9 wherein said substitution consist of D60N, G131D and S188P.
Claim 24
Dependent Claim
18 words
A modified subtilisin enzyme according to claim 9 wherein said substitution consists of D97G, Q103R, E156G, and T255A.
Claim 25
Dependent Claim
22 words
A modified subtilisin enzyme according to claim 9 wherein said substitution consists of D60N, D97G, Q103R, G131D, E156G, S182G, S188P and N218S.
Claim 26
Dependent Claim
24 words
A modified subtilisin enzyme according to claim 9 wherein said substitution consists of D60N, D97G, Q103R, G131D, E156G, S182G, S188P, N218S, D248L and T255A.
References Cited (Backward Citations)
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