Omega-transaminase mutants with activity improvements toward ketones and methods for producing optically pure amines

Patent metadata

Field	Value
Publication number	US-2016298092-A1
Application number	US-201615094625-A
Country	US
Kind code	A1
Filing date	Apr 8, 2016
Priority date	Apr 8, 2015
Publication date	Oct 13, 2016
Grant date	—

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.

First claim

What is claimed is:

1 . An omega-transaminase mutant comprising a point mutation of a wild-type omega-transaminase represented by an amino acid sequence of SEQ ID NO: 1, the point mutation is rendered by replacing tryptophan at position 58 with the otheramino acid.

2 . The omega-transaminase mutant according to claim 1 , wherein the other amino acid is an sterically less demanding amino acid having a lower molecular weight than that of an inherent amino acid of the wild-type omega-transaminase.

3 . The omega-transaminase mutant according to claim 2 , wherein the sterically less demanding amino acid is a neutral amino acid or an anionic amino acid.

4 . The omega-transaminase mutant according to claim 3 , wherein the neural amino acid is a hydrophobic amino acid or a polar uncharged amino acid.

5 . The omega-transaminase mutant according to claim 4 , wherein the hydrophobic amino acid comprises any one hydrophobic amino acid selected from the group consisting of alanine, valine, leucine, isoleucine, proline, glycine, phenylalanine and methionine.

6 . The omega-transaminase mutant according to claim 4 , wherein the polar uncharged amino acid comprises any one amino acid selected from the group consisting of serine, threonine, cysteine, glutamine, asparagine and tyrosine.

7 . The omega-transaminase mutant according to claim 4 , wherein the anionic amino acid comprises any one amino acid selected from the group consisting of aspartic acid and glutamic acid.

8 . The omega-transaminase mutant according to claim 3 , wherein the omega-transaminase mutant comprises any one selected from the group consisting of amino acid sequences of SEQ ID NO: 2 to 17.

9 . The omega-transaminase mutant according to claim 1 , further comprising point mutations in addition to the W58 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 the other amino acid.

10 . The omega-transaminase mutant according to claim 9 , wherein the other amino acid is a neutral amino acid or an anionic amino acids.

11 . The omega-transaminase mutant according to claim 9 , wherein the point mutation is a point mutation is rendered by replacing leucine at position 57 with the other amino acid.

12 . The omega-transaminase mutant according to claim 11 , wherein the point mutation further comprises at least one point mutation selected from the group consisting of:
a point mutation is rendered by mutation of valine at position 154; and a point mutation is rendered by mutation of isoleucine at position 261.

13 . The omega-transaminase mutant according to claim 9 , wherein the omega-transaminase mutant comprises at least one selected from the group consisting of amino acid sequences of SEQ ID NOS: 18 to 30.

14 . A method of producing an omega-transaminase mutant comprising:
recombining genes encoding the amino acid sequence of the omega-transaminase mutant according to claim 1 with an expression vector; transforming host cells with the recombinant expression vector; and expressing the omega-transaminase mutant, followed by purification.

15 . A method of producing an optically active amine comprising:
adding the omega-transaminase mutant according to claim 1 to a substrate solution comprising an amino donor and a ketone; and subjecting the mixture to reaction.

16 . A method of producing an optically active amine using kinetic resolution comprising:
adding the omega-transaminase mutant according to claim 1 to a substrate solution comprising a racemic amine; and subjecting the racemic amine to selective deamination to obtain an optically active amine.

Assignees

• Industry-Academic Cooperation Foundation Yonsei Univ

Inventors

• Shin Jong-Shik
• Park Eul-Soo
• Han Sang-Woo

Classifications

• C12N9/1096 Primary

  transferring nitrogenous groups (2.6) · CPC title

• C12N15/63

  Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression · CPC title

• C12P13/001

  Amines; Imines · CPC title

• C12Y206/01018

  Beta-alanine-pyruvate transaminase (2.6.1.18) · CPC title

• C12Y206/01

  Transaminases (2.6.1) · CPC title