# US 5,352,594 A · Selection and method of making enzymes for perhydrolysis system and for altering substrate specificity, specific activity and catalytic efficiency

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- Number: US 5,352,594 A
- Document ID: US5352594A
- Status: Granted
- Kind: A
- Application number: 07/908,596
- Filed: 1992-06-30
- Granted: 1994-10-04
- Earliest claimed: 1984-05-29
- Assignees: Genecor, Inc.
- Inventors: Ayrookaran J. Poulouse
- Web page: https://patentgrep.com/patent/US5352594A

## CPC

- C12P 7/6418: by hydrolysis of fatty acid esters
- C11D 3/386: Preparations containing enzymes {, e.g. protease or amylase}
- C12N 1/205: Bacterial isolates
- C12N 9/14: Hydrolases (3)
- C12N 9/16: acting on ester bonds (3.1)
- C12N 9/20: Triglyceride splitting, e.g. by means of lipase
- C12N 9/50: Proteinases {, e.g. Endopeptidases (3.4.21-3.4.25)}
- C12N 9/54: bacteria being Bacillus
- C12N 9/63: {derived from plants}
- C12N 15/102: {Mutagenizing nucleic acids}
- C12N 15/75: for Bacillus
- C12N 9/52: derived from bacteria {or Archaea}
- C12N 9/58: derived from fungi
- C12R 2001/07: Bacillus
- Y10S 435/874: Pseudomonas
- Y10S 435/877: Pseudomonas putida
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}
- C11D: DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12R: INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- Y10S: TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS

## Abstract

The invention relates to methods of making and selecting esterase enzymes having an improved perhydrolysis to hydrolysis ratio, and varying K.sub.cat, K.sub.m, and K.sub.cat /K.sub.m and substrate specificity. Such enzymes are useful in peracid bleaching systems and other applications.

## Claims

### Claim 1

A method of altering the perhydrolysis/hydrolysis ratio of Pseudomonas mendocina lipase ATCC #53552, said lipase characterized in that it comprises a catalytic triad consisting of Ser126, His206 and Asp176, said method comprising:
a) replacing at least one amino acid in said lipase with a different amino acid, at a position within four (4) amino acid residue positions on the amino-terminal side or on the carboxyl-terminal side of Ser126, His206 or Asp176;
b) determining the perhydrolysis/hydrolysis ratio of said lipase of step a); and
c) selecting the lipase of step b) characterized in that the perhydrolysis/hydrolysis ratio of the lipase is increased by a factor of at least 2 when compared to native Pseudomonas mendocina lipase ATCC #53552.

### Claim 2 (depends on 1)

A method of claim 1 wherein the lipase has cutinase activity.

### Claim 3 (depends on 1)

A method of claim 1 wherein at least one amino acid is replaced in Pseudomonas mendocina lipase ATCC #53552 at a position selected from the group consisting of +127, +205, and +207.

### Claim 4

A method of altering the substrate specificity of Pseudomonas mendocina lipase ATCC #53552, said lipase characterized in that it comprises a catalytic triad consisting of Ser126, His206 and Asp176, said method comprising:
a) replacing at least one amino acid in said lipase with a different amino acid, at a position within four (4) amino acid residue positions on the amino-terminal side or on the carboxyl-terminal side of Ser126, His206 or Asp176;
b) determining the substrate specificity of said lipase of step a); and
c) selecting the lipase of step b) characterized in that the substrate specificity of the lipase is increased by a factor of at least 2 when compared to native Pseudomonas mendocina lipase ATCC #53552.

### Claim 5 (depends on 4)

A method of claim 4 wherein the lipase has cutinase activity.

### Claim 6 (depends on 4)

A method of claim 4 wherein at least one amino acid is replaced in Pseudomonas mendocina lipase ATCC #53552 at a position selected from the group consisting of +127, +175, +177, +203, +205 and +207.

### Claim 7

A method of altering the catalytic efficiency of Pseudomonas mendocina lipase ATCC #53552, said lipase characterized in that it comprises a catalytic triad consisting of Ser126, His206 and Asp176, said method comprising:
a) replacing at least one amino acid in said lipase with a different amino acid, at a position within four (4) amino acid residue positions on the amino-terminal side or on the carboxyl-terminal side of Ser126, His206 or Asp176;
b) determining the catalytic efficiency of said lipase of step a); and
c) selecting the lipase of step b) characterized in that the catalytic efficiency of the lipase is increased by at least 25% when compared to native Pseudomonas mendocina lipase ATCC #53552.

### Claim 8 (depends on 7)

A method of claim 7 wherein the lipase has cutinase activity.

### Claim 9 (depends on 7)

A method of claim 7 wherein at least one amino acid is replaced in Pseudomonas mendocina lipase ATCC #53552 at a position selected from the group consisting of +175, +177, +202, +203, +205 and +207.

### Claim 10 (depends on 3)

A method of claim 3 wherein at least two amino acids are replaced in Pseudomonas mendocina lipase ATCC #53552 at positions selected from the group consisting of +205/+207, +127/+207 and +127/+205.

### Claim 11 (depends on 6)

A method of claim 6 wherein at least two amino acids are replaced in Pseudomonas mendocina lipase ATCC #53552 at positions +202/+203.