# US 5,346,823 A · Subtilisin modifications to enhance oxidative stability

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- Number: US 5,346,823 A
- Document ID: US5346823A
- Status: Granted
- Kind: A
- Application number: 08/036,592
- Filed: 1993-03-24
- Granted: 1994-09-13
- Earliest claimed: 1984-05-29
- Assignees: Genencor, Inc.
- Inventors: David A. Estell; James A. Wells; Richard R. Bott
- Web page: https://patentgrep.com/patent/US5346823A

## CPC

- C12N 1/205: Bacterial isolates
- C11D 3/386: Preparations containing enzymes {, e.g. protease or amylase}
- C12N 9/54: bacteria being Bacillus
- C12N 15/102: {Mutagenizing nucleic acids}
- C12N 15/75: for Bacillus
- C12R 2001/07: Bacillus
- 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
- C12R: INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS

## Abstract

There are provided methods for making a mutant Bacillus subtilisin having altered oxidative stability, the methods comprising obtaining DNA fragment consisting of a region coding for a Bacillus subtilisin, and introducing a mutation into said DNA fragment such that the mutation is introduced in a region encoding a methionine, tryptophan, cysteine or lysine, sensitive to oxidation, such that upon expression of the mutant subtilisin one or more codon regions encoding for methionine, tryptophan, cysteine or lysine is replaced with an amino acid other than methionine, tryptophan, cysteine or lysine, preferably alanine or serine.

## Claims

### Claim 1

A method for making a mutant Bacillus subtilisin having altered oxidative stability, the method comprising:
a) obtaining a DNA fragment consisting essentially of a region encoding a Bacillus subtilisin;
b) substituting codons encoding either serine of alanine into said DNA fragment within a codon region encoding a methionine, tryptophan, cysteine or lysine provided that said methionine, tryptophan, cysteine or lysine is oxidized in the presence of an oxidizing agent;
c) transforming a Bacillus host cell with said mutated DNA;
d) expressing said mutant subtilisin;
e) recovering said mutant subtilisin; and
f) screening said mutant subtilisin for improved oxidative stability by assaying residual enzyme activity of the mutant in the presence of an oxidizing agent.

### Claim 2

An isolated DNA segment encoding a mutant Bacillus subtilisin having altered oxidative stability wherein a codon encoding either serine of alanine is substituted for one or more codons selected from the group consisting of codons specifying methionine, tryptophan, cysteine or lysine, wherein said substituted codons are present in a DNA segment encoding a native or a modified subtilisin, and wherein said methionine, tryptophan, cysteine or lysine to be substituted is oxidized in the presence of an oxidizing agent.

### Claim 3 (depends on 2)

An expression vector comprising the DNA segment of claim 2, wherein said DNA segment is operably linked to one or more regulatory DNA sequence elements suitable for the expression of a mutant Bacillus subtilisin.

### Claim 4 (depends on 3)

A host cell transformed with the expression vector of claim 3.