Mutant glutamate-cysteine ligase and method for manufacturing gamma glutamyl-valyl-glycine

Patent metadata

Field	Value
Publication number	US-10113161-B2
Application number	US-201615223662-A
Country	US
Kind code	B2
Filing date	Jul 29, 2016
Priority date	Jan 31, 2014
Publication date	Oct 30, 2018
Grant date	Oct 30, 2018

Abstract

A mutant glutamate-cysteine ligase (GSHA) suitable for generating γ-Glu-Val, and a method for producing γ-Glu-Val-Gly using the same are provided. γ-Glu-Val is produced by using a mutant GSHA having a specific mutation with Glu and Val as raw materials, and γ-Glu-Val-Gly is further produced by using γ-Glu-Val and Gly as raw materials. γ-Glu-Val-Gly is produced by using a mutant GSHA having a specific mutation with Glu, Val, and Gly as raw materials.

First claim

The invention claimed is:

1. A method for producing γ-Glu-Val and/or a salt thereof, the method comprising:
contacting a mutant glutamate-cysteine ligase with Glu and Val to produce γ-Glu-Val and/or a salt thereof,
wherein the mutant glutamate-cysteine ligase comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 2 wherein the amino acid residue corresponding to at least one residue selected from the group consisting of L135, Q144, Y241, N243, and Y300 of SEQ ID NO: 2 is mutated to a different amino acid in the mutant glutamate-cysteine ligase, and
wherein the mutant has γ-glutamylvaline synthetase activity.

2. A method for producing γ-Glu-Val-Gly and/or a salt thereof, the method comprising:
contacting a mutant glutamate-cysteine ligase with Glu and Val to produce γ-Glu-Val;
contacting a glutathione synthetase with the produced γ-Glu-Val and Gly to produce γ-Glu-Val-Gly and/or a salt thereof,
wherein the mutant glutamate-cysteine ligase comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 2 wherein the amino acid residue corresponding to at least one residue selected from the group consisting of L135, Q144, Y241, N243, and Y300 of SEQ ID NO: 2 is mutated to a different amino acid in the mutant glutamate-cysteine ligase, and
wherein the mutant has γ-glutamylvaline synthetase activity.

3. A method for producing γ-Glu-Val-Gly and/or a salt thereof, the method comprising:
contacting a mutant glutamate-cysteine ligase and a glutathione synthetase with Glu, Val, and Gly to produce γ-Glu-Val-Gly and/or a salt thereof,
wherein the mutant glutamate-cysteine ligase comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 2 wherein the amino acid residue corresponding to at least one residue selected from the group consisting of L135, Q144, Y241, N243, and Y300 of SEQ ID NO: 2 is mutated to a different amino acid in the mutant glutamate-cysteine ligase, and
wherein the mutant has γ-glutamylvaline synthetase activity.

4. The method according to claim 1 , wherein the mutant glutamate-cysteine ligase is purified.

5. The method according to claim 1 , wherein the mutant glutamate-cysteine ligase is immobilized.

6. The method according to claim 1 , wherein the mutant glutamate-cysteine ligase is contained in:
a culture broth of a microorganism producing the mutant glutamate-cysteine ligase,
cultured cells of a microorganism producing the mutant glutamate-cysteine ligase, or
a processed product of the cultured cells, wherein the processed product comprises the mutant glutamate-cysteine ligase.

7. The method according to claim 2 , wherein the glutathione synthetase is contained in:
a culture broth of a microorganism producing the glutathione synthetase,
cultured cells of a microorganism producing the glutathione synthetase, or
a processed product of the cultured cells, wherein the processed product comprises the glutathione synthetase.

8. The method according to claim 2 , wherein the mutant glutamate-cysteine ligase and the glutathione synthetase are contained in:
a culture broth of a microorganism producing the mutant glutamate-cysteine ligase and the glutathione synthetase,
cultured cells of a microorganism producing the mutant glutamate-cysteine ligase and the glutathione synthetase, or
a processed product of the cultured cells, wherein the processed product comprises the mutant glutamate-cysteine ligase and the glutathione synthetase.

9. The method according to claim 6 , wherein a γ-glutamyltransferase gene is disrupted in the microorganism.

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

11. The method according to claim 1 , wherein the contacting of the mutant glutamate-cysteine ligase is carried out in the presence of ATP.

12. The method according to claim 1 , wherein the amino acid residue is mutated in the mutant glutamate-cysteine ligase such that the mutation corresponds to at least one mutation in the amino acid sequence of SEQ ID NO: 2 selected from the group consisting of:
L135I, L135F, L135M, L135V, L135G, L135A, L135W, L135K, L135H, L135R, L135C, L135N, L135S, L135T, Q144F, Q144A, Q144N, Q144S, Q144D, Q144T, Q144R, Q144H, Q144G, Q144K, Q144Y, Q144W, Q144C, Q144M, Q144P, Q144V, Q144L, Q144I, Y241A, N243I, N243W, N243K, N243R, N243H, Y300A, Y300H, Y300R, and Y300K.

13. The method according to claim 3 , wherein the mutant glutamate-cysteine ligase is contained in:
a culture broth of a microorganism producing the mutant glutamate-cysteine ligase,
cultured cells of a microorganism producing the mutant glutamate-cysteine ligase, or
a processed product of the cultured cells, wherein the processed product comprises the mutant glutamate-cysteine ligase.

14. The method according to claim 3 , wherein the mutant glutamate-cysteine ligase and the glutathione synthetase are contained in:
a culture broth of a microorganism producing the mutant glutamate-cysteine ligase and the glutathione synthetase,
cultured cells of a microorganism producing the mutant glutamate-cysteine ligase and the glutathione synthetase, or
a processed product of the cultured cells, wherein the processed product comprises the mutant glutamate-cysteine ligase and the glutathione synthetase.

15. The method according to claim 13 , wherein a γ-glutamyltransferase gene is disrupted in the microorganism.

16. The method according to claim 13 , wherein the microorganism is Escherichia coli.

17. The method according to claim 14 , wherein a γ-glutamyltransferase gene is disrupted in the microorganism.

18. The method according to claim 14 , wherein the microorganism is Escherichia coli.

19. The method according to claim 3 , wherein the amino acid residue is mutated in the mutant glutamate-cysteine ligase such that the mutation corresponds to at least one mutation in the amino acid sequence of SEQ ID NO: 2 selected from the group consisting of:
L135I, L135F, L135M, L135V, L135G, L135A, L135W, L135K, L135H, L135R, L135C, L135N, L135S, L135T, Q144F, Q144A, Q144N, Q144S, Q144D, Q144T, Q144R, Q144H, Q144G, Q144K, Q144Y, Q144W, Q144C, Q144M, Q144P, Q144V, Q144L, Q144I, Y241A, N243I, N243W, N243K, N243R, N243H, Y300A, Y300H, Y300R, and Y300K.

20. The method according to claim 3 , wherein the amino acid residue is mutated in the mutant glutamate-cysteine ligase such that the mutation corresponds to at least one mutation in the amino acid sequence of SEQ ID NO: 2 selected from the group consisting of:
L135I/Q144R, L135I/Q144D, L135I/Q144A, L135I/Q144L, L135I/N243W, L135I/N243F, L135F/Q144A, L135F/N243W, L135M/Q144R, L135M/Q144A, L135M/Q144L, L135M/N243W, L135M/N243F, L135M/Q144H, L135M/Q144N, L135M/N243Y, L135M/N243R, L135M/N243C, L135V/Q144R, L135V/Q144D, L135V/Q144A, L135V/Q144L, L135V/Q144V, L135V/Q144K, L135V/Q144C, L135V/Q144T, L135H/Q144R, L135G/Q144L, L135A/Q144L, L135V/N243W, L135V/N243F, L135V/N243P, Q144R/N243W, Q144R/N243F, Q144D/N243W, Q144D/N243F, Q144A/N243W, Q144A/N243F, Q144L/N243W, Q144L/N243F, L135M/Q144F, L135M/N243A, L135V/N243G, L135V/N243A, L135V/N243L, L135V/N243Y, L135V/N243K, L135V/N243R, L135V/N243H, L135V/N243D, L135V/N243E, L135V/N243C, L135V/N243Q, L135V/N243S, L135V/N243T, L135V/Q144I, L135V/Q144P, L135V/Q144W, L135V/Q144H, L135V/Q144E, L135V/Q144N, L135V/Q144S, L135K/Q144L, L135H/Q144L, L135D/Q144L, L135C/Q144L, L135Q/Q144L, L135N/Q144L, L135S/Q144L, and L135T/Q144L.

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Assignees

• Ajinomoto Kk

Inventors

• Sasahara Ayako
• Tabuchi Eri
• Suzuki Hideyuki
• Tagami Uno
• Kashiwagi Tatsuki
• Ito Takayuki
• Nozaki Hiroyuki
• Suzuki Shunichi

Classifications

• C12P21/02

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

• C12N9/93 Primary

  Ligases (6) · CPC title

• C12Y603/02002

  Glutamate-cysteine ligase (6.3.2.2) · 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