# US 10,308,916 B2 · Omega-transaminase mutants with activity improvements toward ketones and methods for producing optically pure amines

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- Number: US 10,308,916 B2
- Document ID: US10308916B2
- Status: Granted
- Kind: B2
- Application number: 15/094,625
- Filed: 2016-04-08
- Published: 2016-10-13
- Granted: 2019-06-04
- Earliest claimed: 2015-04-08
- Assignees: Industry-Academic Cooperation Foundation, Yonsei University
- Applicants: Industry-Academic Cooperation Foundation, Yonsei University
- Inventors: Jong-Shik Shin; Eul-Soo Park; Sang-woo Han
- Web page: https://patentgrep.com/patent/US10308916B2

## CPC

- C12N 9/1096: {transferring nitrogenous groups (2.6)}
- C12N 15/09: Recombinant DNA-technology
- C12P 13/001: {Amines; Imines}
- C12Y 206/01018: Beta-alanine-pyruvate transaminase (2.6.1.18)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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)}
- C12Y: ENZYMES

## Abstract

Provided is an omega-transaminase mutant with improved activity using a point mutation in an active site of an omega-transaminase. Specifically, provided is a method for improving activity and extending a substrate spectrum of omega-transaminase by introducing a point mutation into a wild-type omega-transaminase rendered by replacing tryptophan at position 58 with the other amino acid.

## Claims

### Claim 1

An isolated omega-transaminase mutant of a wild-type omega-transaminase having the amino acid sequence of SEQ ID NO: 1 for producing an optically active amine from a ketone substrate,
wherein the omega-transaminase mutant has a point mutation at position 58 rendered by replacing tryptophan with a first different amino acid selected from the group consisting of alanine, cysteine, histidine, isoleucine, leucine, methionine, asparagine, glutamine, serine, threonine, valine, and tyrosine; and
wherein the omega-transaminase mutant has up to ten additional point mutations.

### Claim 2 (depends on 1)

The isolated omega-transaminase mutant according to claim 1, wherein the isolated omega-transaminase mutant has an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOS: 2 to 6 and 11 to 15.

### Claim 3

An isolated omega-transaminase mutant of a wild-type omega-transaminase having the amino acid sequence of SEQ ID NO: 1 for producing an optically active amine from a ketone substrate,
wherein the omega-transaminase mutant has a point mutation at position 58 rendered by replacing tryptophan with a first different amino acid,
wherein the omega-transaminase mutant has a second point mutation rendered by replacing at least one amino acid selected from the group consisting of leucine at position 57, valine at position 154, and isoleucine at position 261,
wherein the isolated omega-transaminase mutant has an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOS: 20 and 27 to 30; and
wherein the omega-transaminase mutant has up to ten additional point mutations.

### Claim 4 (depends on 1)

The isolated omega-transaminase mutant according to claim 1, further comprising a second point mutation,
wherein a second point mutation is rendered by replacing at least one amino acid selected from the group consisting of tyrosine at position 20, methionine at position 54, leucine at position 57, phenylalanine at position 86, valine at position 154, alanine at position 230, valine at position 233, isoleucine at position 261, threonine at position 324 and isoleucine at position 380 with a second different amino acid.

### Claim 5 (depends on 4)

The isolated omega-transaminase mutant according to claim 4, wherein the isolated omega-transaminase mutant has an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOS: 20 and 27 to 30.

### Claim 6 (depends on 1)

The isolated omega-transaminase mutant according to claim 1, wherein the isolated omega-transaminase mutant has the amino acid sequence of SEQ ID NO:2.

### Claim 7 (depends on 1)

The isolated omega-transaminase mutant according to claim 1, wherein the isolated omega-transaminase mutant consists of 456 amino acids.

### Claim 8 (depends on 1)

The isolated omega-transaminase mutant according to claim 1, wherein the isolated omega-transaminase mutant has at least 97% homology with SEQ ID NO:1.