Method for producing gamma-glutamylvalylglycine or a salt thereof

Patent metadata

Field	Value
Publication number	US-9677106-B2
Application number	US-201615235311-A
Country	US
Kind code	B2
Filing date	Aug 12, 2016
Priority date	Oct 7, 2011
Publication date	Jun 13, 2017
Grant date	Jun 13, 2017

Abstract

A method for producing γ-Glu-Val-Gly comprising the step of reacting Val-Gly with a γ-glutamyl group donor in the presence of a γ-glutamyltransferase, a microorganism containing the enzyme, or a processed product thereof to generate γ-Glu-Val-Gly, wherein the γ-glutamyltransferase consists of a large subunit and a small subunit, and the small subunit has a specific mutation.

First claim

What is claimed is:

1. A method for producing γ-Glu-Val-Gly, comprising:
reacting Val-Gly with a γ-glutamyl group donor in the presence of a γ-glutamyltransferase such that γ-Glu-Val-Gly is generated,
wherein the γ-glutamyltransferase comprises a large subunit and a small subunit,
the large subunit has an amino acid sequence having no less than 90% sequence identity to the amino acid sequence of the positions 26 to 390 of the amino acid sequence of SEQ ID NO: 2,
the small subunit has an amino acid sequence having no less than 90% sequence identity to the amino acid sequence of the positions 391 to 580 of the amino acid sequence of SEQ ID NO: 2, and
the tyrosine at the position corresponding to position 444 of SEQ ID NO: 2 is modified by a substitution.

2. The method according to claim 1 , wherein the tyrosine at the position corresponding to position 444 of SEQ ID NO: 2 is substituted with aspartic acid, glutamic acid, asparagine, or alanine.

3. The method according to claim 1 , wherein the small subunit has an amino acid substitution corresponding to an amino acid substitution in SEQ ID NO: 2 selected from the group consisting of Y444D, Y444E, Y444A, (T413A+Y444E), (T413H+Y444E), (T413N+Y444E), (Q430N+Y444E), (Q430N+Y444D), (Q430N+Y444N), (P441A+Y444E), (V443A+Y444E), (V443E+Y444E), (V443G+Y444E), (V443L+Y444E), (V443N+Y444E), (V443Q+Y444E), (Y444E+L446A), (Y444E+A453S), (Y444E+D472T), (Y444E+G484A), (Y444E+G484S), (Y444E+S498C), (Y444E+Q542H), (Y444E+D561N), (T413N+Y444E+V443A), (T413N+Y444E+A453S), (T413N+Y444E+S498C), (T413N+Y444E+Q542H), (G484S+Y444E+V443A), (G484S+Y444E+Q542H), (Q430N+Y444E+T413N), (T413H+Y444E+G484S), (T413N+Y444E+G484S), (T413N+Y444E+G484S+V443A), (T413N+Y444E+G484S+A453S), (T413N+Y444E+G484S+Q542H), (T413N+Y444E+G484S+S572K), (T413N+Y444E+G484S+Q430N), and (T413N+Y444E+G484E+S498C), in SEQ ID NO: 2.

4. The method according to claim 1 , wherein the small subunit has the amino acid sequence of SEQ ID NO: 13 except that the tyrosine at the position 54 of SEQ ID NO: 13 corresponding to the position 444 of SEQ ID NO: 2 is modified by a substitution.

5. The method according to claim 1 , wherein the small subunit has the amino acid sequence of
the positions 391 to 580 of SEQ ID NO: 2 except that the tyrosine at the position 444 of SEQ ID NO: 2 is substituted with aspartic acid, glutamic acid, asparagine, or alanine,
the positions 391 to 580 of SEQ ID NO: 3 except that the tyrosine at the position 444 of SEQ ID NO: 3 corresponding to the position 444 of SEQ ID NO: 2 is substituted with aspartic acid, glutamic acid, asparagine, or alanine,
the positions 392 to 581 of SEQ ID NO: 4 except that the tyrosine at the position 445 of SEQ ID NO: 4 corresponding to the position 444 of SEQ ID NO: 2 is substituted with aspartic acid, glutamic acid, asparagine, or alanine,
the positions 388 to 577 of SEQ ID NO: 5 except that the tyrosine at the position 441 of SEQ ID NO: 5 corresponding to the position 444 of SEQ ID NO: 2 is substituted with aspartic acid, glutamic acid, asparagine, or alanine,
the positions 391 to 580 of SEQ ID NO: 6 except that the tyrosine at the position 444 of SEQ ID NO: 6 corresponding to the position 444 of SEQ ID NO: 2 is substituted with aspartic acid, glutamic acid, asparagine, or alanine,
the positions 391 to 580 of SEQ ID NO: 7 except that the tyrosine at the position 444 of SEQ ID NO: 7 corresponding to the position 444 of SEQ ID NO: 2 is substituted with aspartic acid, glutamic acid, asparagine, or alanine,
the positions 391 to 580 of SEQ ID NO: 8 except that the tyrosine at the position 444 of SEQ ID NO: 8 corresponding to the position 444 of SEQ ID NO: 2 is substituted with aspartic acid, glutamic acid, asparagine, or alanine,
the positions 400 to 589 of SEQ ID NO: 9 except that the tyrosine at the position 453 of SEQ ID NO: 9 corresponding to the position 444 of SEQ ID NO: 2 is substituted with aspartic acid, glutamic acid, asparagine, or alanine,
the positions 391 to 580 of SEQ ID NO: 10 except that the tyrosine at the position 444 of SEQ ID NO: 10 corresponding to the position 444 of SEQ ID NO: 2 is substituted with aspartic acid, glutamic acid, asparagine, or alanine,
the positions 392 to 581 of SEQ ID NO: 11 except that the tyrosine at the position 445 of SEQ ID NO: 11 corresponding to the position 444 of SEQ ID NO: 2 is substituted with aspartic acid, glutamic acid, asparagine, or alanine, or
the positions 392 to 581 of SEQ ID NO: 12 except that the tyrosine at the position 445 of SEQ ID NO: 12 corresponding to the position 444 of SEQ ID NO: 2 is substituted with aspartic acid, glutamic acid, asparagine, or alanine.

6. The method according to claim 1 , wherein the γ-glutamyl group donor is L-glutamine or a salt thereof.

7. The method according to claim 1 , wherein the reacting of Val-Gly with the γ-glutamyl group donor occurs in the presence of a microorganism containing the γ-glutamyltransferase, and the microorganism containing the γ-glutamyltransferase is a bacterium belonging to the family Enterobacteriaceae.

8. The method according to claim 7 , wherein the microorganism containing the γ-glutamyltransferase is an Escherichia bacterium.

9. The method according to claim 8 , wherein the microorganism containing the γ-glutamyltransferase is Escherichia coli.

10. The method according to claim 7 , wherein the microorganism containing the γ-glutamyltransferase is deficient in peptidase D.

11. The method according to claim 1 , wherein the reacting of Val-Gly with the γ-glutamyl group donor occurs in the presence of a metal chelating agent.

12. The method according to claim 1 ,
wherein the reacting of Val-Gly with the γ-glutamyl group donor occurs in the presence of a microorganism containing the γ-glutamyltransferase or in the presence of a processed product of the microorganism, and
the processed product of the microorganism has the γ-glutamyltransferase and is selected from the group consisting of the disrupted microorganism, an extract of the microorganism, a partially purified product of the microorganism, the purified γ-glutamyltransferase, the microorganism immobilized in acylamide or carrageenan, and the γ-glutamyltransferase immobilized on a solid phase.

13. The method according to claim 1 , wherein the small subunit has the amino acid sequence of SEQ ID NO: 13 except that the tyrosine at the position 54 of SEQ ID NO: 13 corresponding to the position 444 of SEQ ID NO: 2 is substituted with aspartic acid, glutamic acid, asparagine, or alanine.

14. The method according to claim 1 , wherein the small subunit has the amino acid sequence of
the positions 391 to 580 of SEQ ID NO: 3 except that the tyrosine at the position 444 of SEQ ID NO: 3 corresponding to the position 444 of SEQ ID NO: 2 is modified by a substitution,
the positions 392 to 581 of SEQ ID NO: 4 except that the tyrosine at the position 445 of SEQ ID NO: 4 corresponding to the position 444 of SEQ ID NO: 2 is modified by a substitution,
the positions 388 to 577 of SEQ ID NO: 5 except that the tyrosine at the position 441 of SEQ ID NO: 5 corresponding to the position 444 of SEQ ID NO: 2 is modified by a substitution,
the positions 391 to 580 of SEQ ID NO: 6 except that the tyrosine at the position 444 of SEQ ID NO: 6 corresponding to the position 444 of SEQ ID NO: 2 is modified by a substitution,
the positions 391 to 580 of SEQ ID NO: 7 except that the tyrosine at the position 444 of SEQ ID NO: 7 corresponding to the position 444 of SEQ ID NO: 2 is modified by a substitution,
the positions 391 to 580 of SEQ ID NO: 8 except that the tyrosine at the position 444 of SEQ ID NO: 8 corresponding to the position 44

Assignees

• Ajinomoto Kk

Inventors

• Nozaki Hiroyuki
• Abe Isao
• Takakura Jun
• Takeshita Rie
• Suzuki Hideyuki
• Suzuki Shunichi

Classifications

• C07K5/0819

  with the first amino acid being acidic · CPC title

• C12N9/104

  Aminoacyltransferases (2.3.2) · CPC title

• C12N15/70

  Vectors or expression systems specially adapted for E. coli · 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

• C12P21/02 Primary

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