Gamma glutamyl-valine synthase, and method for producing gamma glutamyl-valyl-glycine

Patent metadata

Field	Value
Publication number	US-10508295-B2
Application number	US-201615234119-A
Country	US
Kind code	B2
Filing date	Aug 11, 2016
Priority date	Mar 5, 2014
Publication date	Dec 17, 2019
Grant date	Dec 17, 2019

Abstract

γ-Glu-Val synthetase suitable for generating γ-Glu-Val, and a method for producing γ-Glu-Val-Gly using the same are provided. By using γ-Glu-Val synthetase showing a ratio of γ-glutamylvaline synthetase activity to γ-glutamylglycine synthetase activity of 2.0 or higher, such as γ-Glu-Val synthetase of Kocuria rosea (AJ3132), γ-Glu-Val-Gly is produced from Glu, Val, and Gly as raw materials.

First claim

The invention claimed is:

1. A method for producing γ-Glu-Val-Gly and/or a salt thereof, the method comprising:
contacting a protein and glutathione synthetase with Glu, Val, and Gly to produce γ-Glu-Val-Gly; and
collecting the produced γ-Glu-Val-Gly,
wherein the protein comprises an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO: 6, 10, or 16, and has γ-glutamylvaline synthetase activity, and
a ratio of γ-glutamylvaline synthetase activity of the protein to γ-glutamylglycine synthetase activity of the protein is 15 or higher.

2. The method according to claim 1 , wherein the protein is purified.

3. The method according to claim 1 , wherein the protein is immobilized.

4. The method according to claim 1 , wherein the contacting of the protein and glutathione synthetase is carried out in the presence of ATP.

5. The method according to claim 1 , wherein the ratio of γ-glutamylvaline synthetase activity of the protein to γ-glutamylglycine synthetase activity of the protein is 20 or higher.

6. The method according to claim 1 , wherein the protein is contained in a culture broth of a microorganism having the protein, cultured cells of the microorganism, or a processed product of the cultured cells.

7. The method according to claim 1 , wherein the protein and the glutathione synthetase are contained in a culture broth of a microorganism having the protein and the glutathione synthetase, cultured cells of the microorganism, or a processed product of the cultured cells.

8. The method according to claim 6 , wherein the microorganism has been modified so that γ-glutamyltransferase activity is reduced as compared to γ-glutamyltransferase activity in a corresponding wild type of the microorganism.

9. The method according to claim 6 , wherein the microorganism is Escherichia coli.

10. The method according to claim 7 , wherein the microorganism has been modified so that γ-glutamyltransferase activity is reduced as compared to γ-glutamyltransferase activity in a corresponding wild type of the microorganism.

11. The method according to claim 7 , wherein the microorganism is Escherichia coli.

12. The method according to claim 1 , wherein the protein has an amino acid sequence having at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 6, 10, or 16, and has γ-glutamylvaline synthetase activity.

13. The method according to claim 1 , wherein the protein has the amino acid sequence of SEQ ID NO: 6, 10, or 16.

14. The method according to claim 1 , wherein an amount of γ-Glu-Val-Gly produced in the contacting is greater than an amount of γ-Glu-Gly-Gly produced in the contacting.

15. The method according to claim 1 , wherein a total amount of γ-Glu-Val and γ-Glu-Val-Gly produced in the contacting is greater than a total amount of γ-Glu-Gly and γ-Glu-Gly-Gly produced in the contacting.

16. The method according to claim 1 , wherein the glutathione synthetase is a protein encoded by the gshB gene of Escherichia coli or the GSH2 gene of Saccharomyces cerevisiae.

17. The method according to claim 13 , wherein the glutathione synthetase is a protein encoded by the gshB gene of Escherichia coli or the GSH2 gene of Saccharomyces cerevisiae.

18. The method according to claim 1 , wherein the glutathione synthetase has the amino acid sequence of SEQ ID NO: 28.

19. The method according to claim 13 , wherein the glutathione synthetase has the amino acid sequence of SEQ ID NO: 28.

Assignees

• Ajinomoto Kk

Inventors

• Tsuji Chihiro
• Nozaki Hiroyuki

Classifications

• C12P21/02 Primary

  having a known sequence of two or more amino acids, e.g. glutathione · CPC title

• C12Y603/02002

  Glutamate-cysteine ligase (6.3.2.2) · CPC title

• C12N15/70

  Vectors or expression systems specially adapted for E. coli · CPC title

• C12N9/93

  Ligases (6) · CPC title

• C07K5/0215

  containing natural amino acids, forming a peptide bond via their side chain functional group, e.g. epsilon-Lys, gamma-Glu · CPC title