Method for preparing L-glufosinate ammonium by biological enzymatic de-racemization, glufosinate ammonium dehydrogenase mutant and use thereof

Patent metadata

Field	Value
Publication number	US-12522852-B2
Application number	US-202017910787-A
Country	US
Kind code	B2
Filing date	Dec 26, 2020
Priority date	Mar 18, 2020
Publication date	Jan 13, 2026
Grant date	Jan 13, 2026

Abstract

The present invention discloses a method for preparing L-glufosinate ammonium by biological enzymatic de-racemization, a glufosinate ammonium dehydrogenase mutant and a use thereof. The method for preparing L-glufosinate ammonium by biological enzymatic de-racemization includes catalyzing D,L-glufosinate ammonium as a raw material by a multi-enzyme catalysis system to obtain L-glufosinate ammonium. The enzyme catalysis system includes D-amino acid oxidase for catalyzing D-glufosinate ammonium in the D,L-glufosinate ammonium to 2-carbonyl-4-[hydroxy(methyl)phosphonyl]butanoic acid, and a glufosinate ammonium dehydrogenase mutant for catalytically reducing 2-carbonyl-4-[hydroxy(methyl)phosphonyl]butanoic acid to L-glufosinate ammonium. The glufosinate ammonium dehydrogenase mutant is obtained by mutation of glufosinate-ammonium dehydrogenase in wild fungi Thiopseudomonas denitrificans at a mutation site of V377S. The glufosinate ammonium dehydrogenase mutant in the present invention has better catalytic efficiency. When racemic D, L-glufosinate ammonium is used as a substrate for a catalytic reaction, the conversion rate is much higher than the conversion rate of a wild-type enzyme, and the yield of 2-carbonyl-4-[hydroxy(methyl)phosphonyl]butanoic acid (PPO for short) is also greatly improved.

First claim

The invention claimed is:

1 . A glufosinate ammonium dehydrogenase mutant that comprises SEQ ID NO: 4.

2 . A gene for coding the glufosinate ammonium dehydrogenase mutant according to claim 1 , wherein the gene has a nucleotide sequence comprising SEQ ID NO: 3.

3 . A recombinant bacterium comprising the gene according to claim 2 .

4 . A method for preparing L-glufosinate ammonium by biological enzymatic de-racemization, comprising catalyzing D,L-glufosinate ammonium as a raw material by a multi-enzyme catalysis system to obtain L-glufosinate ammonium, wherein the enzyme catalysis system comprises D-amino acid oxidase for catalyzing D-glufosinate ammonium in the D,L-glufosinate ammonium to 2-carbonyl-4-[hydroxy(methyl)phosphonyl]butanoic acid, and a glufosinate ammonium dehydrogenase mutant for catalytically reducing 2-carbonyl-4-[hydroxy (methyl)phosphonyl] butanoic acid to L-glufosinate ammonium, and
the glufosinate ammonium dehydrogenase mutant comprises SEQ ID NO: 4.

5 . The method according to claim 4 , wherein an amino acid sequence of the D-amino acid oxidase comprises SEQ ID NO: 9.

6 . The method according to claim 4 , wherein the enzyme catalysis system further comprises catalase for removing a byproduct including hydrogen peroxide.

7 . The method according to claim 4 , wherein the enzyme catalysis system further comprises a coenzyme cycling system, and the coenzyme cycling system is at least one of the following:
(1) a formate dehydrogenase coenzyme cycling system comprising a formate dehydrogenase, formate and a coenzyme; (2) a glucose dehydrogenase coenzyme cycling system comprising a glucose dehydrogenase, glucose and a coenzyme; and (3) an alcohol dehydrogenase coenzyme cycling system comprising an alcohol dehydrogenase, isopropanol and a coenzyme.

8 . The method according to claim 7 , wherein an amino acid sequence of the formate dehydrogenase comprises SEQ ID NO: 13; an amino acid sequence of the glucose dehydrogenase comprises SEQ ID NO: 11; or and an amino acid sequence of the alcohol dehydrogenase comprises SEQ ID NO: 15.

Assignees

• Univ Zhejiang Technology

Inventors

• Xue Yaping
• Cheng Feng
• Cao Chenghao
• Zheng Yuguo
• Zou Shuping
• Xu Jianmiao

Classifications

• C12Y111/01006

  Catalase (1.11.1.6) · CPC title

• C12Y104/03003

  D-Amino-acid oxidase (1.4.3.3) · CPC title

• C12Y102/01002

  Formate dehydrogenase (1.2.1.2) · CPC title

• C12Y101/9901

  Glucose dehydrogenase (acceptor) (1.1.99.10) · CPC title

• C12Y101/01001

  Alcohol dehydrogenase (1.1.1.1) · CPC title