EP0130756B1
Expired / Lapsed
EP
⏱️ 0.0 years remaining
Procaryotic carbonyl hydrolases, methods, dna, vectors and transformed hosts for producing them, and detergent compositions containing them
Assignee: Danisco US Inc
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Filing: 1984-06-22
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Granted: 1991-02-06
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Expires: 2004-06-22
Abstract
Abstract DNA sequences are disclosed encoding B. amyloliquefaciens and B. subtilis subtilisins and B. subtilis neutral protease. Using DNA derived from these sequences as hybridisation probes, genes for these and other closely related enzymes can be cloned from procaryotic sources, identified by their carbonyl hydrolytic acitivity and expressed in a suitable recombinant host. Moreover, the DNA can be mutated to produce enzymes with altered characteristics which can be screened for to identify desired changes, and thereafter the modified enzymes obtained by expression of the muted genes in suitable recombinant hosts.
Inventors
- 👤 Richard Ray Bott
- 👤 Eugenio Ferrari
- 👤 James Allen Wells
- 👤 David Aaron Estell
- 👤 Dennis James Henner
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-10-27 09:56
CPC Classifications
C; C11; C11D; C11D3/00; C11D3/16; C11D3/38; C11D3/386; C12; C12N; C12N1/00; C12N1/20; C12N1/205; C12N15/00; C12N15/09; C12N15/10; C12N15/102; C12N15/63; C12N15/74; C12N15/75; C12N9/00; C12N9/14; C12N9/48; C12N9/50; C12N9/52; C12N9/54; C12R; C12R2001/00; C12R2001/01; C12R2001/07
Claims Analysis
Total Claims: 24
Claim 1
Independent Claim
61 words
A process which comprises effecting a mutation in a Bacillus subtilisin enzyme or its pre- or preproenzyme in one or more of the positions corresponding to Tyr-1, Asn+155, Tyr+104, Met+222, Gly+166, Gly+169, Glu+156, Ser+33, Phe+189, Tyr+217 and Ala+152 in B. amyloliquefaciens subtilisin or its pre- or preproenzyme, and testing for a desired activity change in the enzyme resulting from said mutation.
Claim 2
Dependent Claim
61 words
A process which comprises effecting a mutation in a Bacillus subtilisin enzyme or its pre- or preproenzyme in one or more of the positions corresponding to Tyr-1, Asn+155, Tyr+104, Met+222, Gly+166, Gly+169, Glu+156, Ser+33, Phe+189, Tyr+217 and Ala+152 in B. amyloliquefaciens subtilisin or its pre- or preproenzyme, and testing for a desired activity change in the enzyme resulting from said mutation.
Claim 3
Dependent Claim
26 words
A process which comprises the manufacture of a mutant subtilisin enzyme having a desired activity change following its identification according to the procedure of claim 1.
Claim 4
Dependent Claim
35 words
A process of claim 1 or claim 2 wherein the mutation comprises the substitution of an amino acid at a position corresponding to +169 of B. amyloliquefaciens subtilisin, producing an enzyme of altered specific activity.
Claim 5
Dependent Claim
34 words
A process of claim 1 or claim 2 wherein the mutation comprises the substitution of an amino acid at a position corresponding to +156 of B. amyloliguefaciens, producing an enzyme of altered pH activity.
Claim 6
Dependent Claim
38 words
A process of claim 1 or claim 2 wherein the mutation comprises the substitution of an amino acid at a position corresponding to +222 of B. amyloliguefaciens, producing an enzyme of enhanced oxidation stability and/or altered pH activity.
Claim 7
Dependent Claim
21 words
A process of claim 5 wherein the mutation comprises the substitution of Ala or Ser for the residue at position +222.
Claim 8
Dependent Claim
38 words
A process of claim 1 or claim 2 wherein the mutation comprises the substitution of an amino acid at a position corresponding to +166 of B. amylaliquefaciens producing an enzyme of enhanced specific activity and/or altered substrate specificity.
Claim 9
Dependent Claim
17 words
The use of a mutant enzyme of any of claims 1 to 7 in a detergent composition.
Claim 10
Dependent Claim
25 words
The use according to claim 8 wherein the detergent is a linear alkyl benzene sulfonate, alkyl ethoxylated sulfate, sulfated linear alcohol or ethoxylated linear alcohol.
Claim 11
Dependent Claim
81 words
A process which comprises: providing a DNA sequence encoding a Bacillus subtilisin enzyme in which there has been made a mutation in one or more of the positions corresponding to Tyr-1, Asn+155, Tyr+104, Met+222, Gly+166, Gly+169, Glu+156, Ser+33, Phe+189, Tyr+217 and Ala+152 in B. amyloliquefaciens subtilisin or its pre- or preproenzyme, transforming a host cell with the DNA so that the coding sequence will be expressed therein, and testing for a desired activity change in the enzyme resulting from said mutation.
Claim 12
Dependent Claim
81 words
A process which comprises: providing a DNA sequence encoding a Bacillus subtilisin enzyme in which there has been made a mutation in one or more of the positions corresponding to Tyr-1, Asn+155, Tyr+104, Met+222, Gly+166, Gly+169, Glu+156, Ser+33, Phe+189, Tyr+217 and Ala+152 in B. amyloliquefaciens subtilisin or its pre- or preproenzyme, transforming a host cell with the DNA so that the coding sequence will be expressed therein, and testing for a desired activity change in the enzyme resulting from said mutation.
Claim 13
Dependent Claim
41 words
A process which comprises, following the identification of a desired mutant enzyme by the process of claim 10, expressing, in a recombinant host cell operably transformed therewith, a DNA sequence encoding said enzyme to obtain a mutant subtilisin, pro- or preprosubtilisin.
Claim 14
Dependent Claim
45 words
A process of claim 10 or claim 11 wherein the mutation is expressed as the substitution of one or more methionine, tryptophan, cysteine or lysine residues by a substituent amino acid residue not one of methionine, tryptophan, cysteine or lysine, preferably by alanine or serine.
Claim 15
Dependent Claim
47 words
A process of any one of claims 10, 11 and 12 wherein the mutation is expressed as a mutant exhibiting a change in one or more of the oxidation stability, Km, Kcat, Kcat/Km ratio, substrate specificity, specific activity or pH optimum of the enzyme prior to mutation.
Claim 16
Dependent Claim
15 words
A process of claim 13 wherein the mutation changes the oxidation stability of the enzyme.
Claim 17
Dependent Claim
15 words
The method of claim 13 wherein a mutant exhibiting a different substrate specificity is recovered.
Claim 18
Dependent Claim
37 words
A process of any one of claims 10 to 15 wherein the mutation comprises the substitution of an amino acid at a position corresponding to +169 of B. amyloliquefaciens subtilisin, producing an enzyme of altered specific activity.
Claim 19
Dependent Claim
37 words
A process of any one of claims 10 to 16 wherein the mutation comprises the substitution of an amino acid at a position corresponding to +156 of B. amyloliquefaciens, producing an enzyme of altered pH stability range.
Claim 20
Dependent Claim
21 words
A process of any one of claims 10 to 17 which further comprises incorporating the mutant enzyme into a detergent composition.
Claim 21
Dependent Claim
24 words
The process of claim 18 wherein the detergent is a linear alkyl benzene sulfonate, alkyl ethoxylated sulfate, sulfated linear alcohol or ethoxylated linear alcohol.
Claim 22
Dependent Claim
22 words
A process of any one of claims 10 to 19 wherein a subtilisin is produced in the form of prosubtilisin or preprosubtilisin.
Claim 23
Dependent Claim
24 words
A process of any one of claims 10 to 20 wherein the recombinant host cell is a strain of Bacillus, preferably a Bacillus subtilis.
Claim 24
Dependent Claim
29 words
A process of any one of claims 10 to 21 wherein the host cell was transformed with a DNA sequence encoding the enzyme operably linked to its signal sequence.
References Cited (Backward Citations)
No citation records indexed.
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