Method for the production of glufosinate-ammonium

Patent metadata

Field	Value
Publication number	US-12031160-B2
Application number	US-201917419048-A
Country	US
Kind code	B2
Filing date	Jun 28, 2019
Priority date	Dec 28, 2018
Publication date	Jul 9, 2024
Grant date	Jul 9, 2024

Abstract

The present invention provides a glufosinate-ammonium dehydrogenase mutant and application in synthesis of L-glufosinate-ammonium thereof, the method uses 2-carbonyl-4-[(hydroxy)(methyl)phosphinoyl]-butyric acid or its salts as a substrate and the glufosinate-ammonium dehydrogenase or cells containing the glufosinate-ammonium dehydrogenase as a biocatalyst to carry out reductive amination, thereby obtaining L-glufosinate-ammonium. The method has features of high conversion rate of raw materials, high yield, easy separation and purification of the product, and high chiral purity; compared with other catalytic processes, the method in the present invention has features of relatively simple process and a conversion rate of raw materials up to 100%.

First claim

The invention claimed is:

1. A method for the production of L-glufosinate ammonium comprising contacting a variant of the polypeptide of SEQ ID NO: 6 having glufosinate-ammonium dehydrogenase activity with 2-carbonyl-4-[(hydroxy)(methyl)phosphinoyl]-butyric acid, wherein said variant comprises all of SEQ ID NO: 6 except for (i) a substitution that corresponds to the substitution L107R in the polypeptide of SEQ ID NO: 6, and (ii) one or more substitutions that correspond to the substitutions in the polypeptide of SEQ ID NO: 6 selected from the group consisting of F188P, G239K, G239Y, G239C and F357G.

2. The method of claim 1 , wherein said variant is in a catalyst, wherein said catalyst is in the form of wet cells that comprise said variant or a crude enzyme solution that comprises said variant, wherein said method comprises:
(a) adding said catalyst, 2-carbonyl-4-[(hydroxy)(methyl)phosphinoyl]-butyric acid, glucose and an inorganic amino donor to a pH 7.5 buffer to form a reaction solution;
(b) maintaining the reaction solution at 35° C. and 600 rpm for the conversion of 2-carbonyl-4-[(hydroxy)(methyl)phosphinoyl]-butyric acid into L-glufosinate ammonium; and
(c) subjecting the reaction solution to separation and purification to obtain L-glufosinate ammonium;
wherein the wet cells are obtained by culturing a recombinant engineered strain comprising a gene encoding the variant and a gene encoding a glucose dehydrogenase, and wherein the crude enzyme solution is obtained by subjecting the wet cells to ultrasonic disintegration.

3. The method of claim 2 , wherein the reaction solution (a) comprises 20 g/L to 100 g/L of wet cells, (b) has an initial concentration of 2-carbonyl-4-[(hydroxy)(methyl)phosphinoyl]-butyric acid from 10 mM to 500 mM, (c) has a glucose concentration from 12 mM to 600 mM, and (d) has an inorganic amino donor concentration from 50 mM to 1.5 M.

4. The method of claim 2 , wherein the catalyst is prepared by:
(a) inoculating LB medium containing 50 μg/mL ampicillin with a recombinant engineered strain comprising a gene encoding the variant and a gene encoding a glucose dehydrogenase, and incubating the LB medium at 37° C. and 200 rpm for 12 hours to obtain an inoculum;
(b) inoculating fresh LB medium containing 50 μg/mL ampicillin with 1% by volume of the inoculum of (a), and incubating at 37° C. and 150 rpm to obtain a culture;
(c) adding IPTG to the culture of (b) when OD600 reaches 0.6-0.8 and culturing for 16 hours;
(d) centrifuging the culture of (c) at 4° C. and 8000 rpm for 20 minutes and discarding the supernatant;
(e) collecting the pellet obtained from step (d) and washing the pellet with a pH 7.5, 20 mM phosphate buffer to obtain wet cells; and
(f) resuspending the wet cells of (e) with a pH 7.5, 100 mM PBS buffer and subjecting the wet cells to ultrasonic disintegration in an ice-water mixture for 10 minutes to obtain a crude enzyme solution, wherein the conditions of the ultrasonic disintegration are 400 W and 1 second on and 5 seconds off.

5. The method of claim 2 , wherein the variant comprises all of SEQ ID NO: 6 except for substitutions that corresponds to substitutions L107R, F188P, G239K and F357G in the polypeptide of SEQ ID NO: 6.

Assignees

• Univ Zhejiang Technology

Inventors

• Xue Yaping
• Cheng Feng
• Li Qinghua
• Zheng Yuguo
• Xu Jianmiao

Classifications

• C12P13/04

  Alpha- or beta- amino acids {(other amino acids C12P13/005)} · CPC title

• C12Y104/01

  with NAD+ or NADP+ as acceptor (1.4.1) · CPC title

• C12N9/0016 Primary

  with NAD or NADP as acceptor (1.4.1) · CPC title