Methods for making L-glufosinate

Patent metadata

Field	Value
Publication number	US-12509709-B2
Application number	US-202519186223-A
Country	US
Kind code	B2
Filing date	Apr 22, 2025
Priority date	Mar 2, 2016
Publication date	Dec 30, 2025
Grant date	Dec 30, 2025

Abstract

Methods for the production of L-glufosinate (also known as phosphinothricin or (S)-2-amino-4-(hydroxy(methyl)phosphonoyl)butanoic acid) are provided. The methods comprise a two-step process. The first step involves the oxidative deamination of D-glufosinate to PPO (2-oxo-4-(hydroxy(methyl)phosphinoyl)butyric acid). The second step involves the specific amination of PPO to L-glufosinate, using an amine group from one or more amine donors. By combining these two reactions, the proportion of L-glufosinate in a mixture of L-glufosinate and D-glufosinate can be substantially increased.

First claim

What is claimed is:

1 . A method for making L-glufosinate, comprising:
reacting D-glufosinate with a D-amino acid oxidase (DAAO) enzyme to form PPO (2-oxo-4-(hydroxy(methyl)phosphinoyl)butyric acid) while aerating, wherein the DAAO enzyme has an activity of at least about 3 μmol/min*mg, wherein the reaction is conducted in the presence of a catalase; aminating the PPO to L-glufosinate by an L-amino acid dehydrogenase (LAAD) enzyme in the presence of an ammonia source and a redox cofactor in reduced form; and recycling the redox cofactor through reduction; wherein at least 70% of the D-glufosinate is converted to L-glufosinate; and
wherein the method is performed ex vivo.

2 . The method of claim 1 , wherein the DAAO enzyme has an activity of at least 3 μmol/min*mg.

3 . The method of claim 1 , wherein the DAAO enzyme has an activity of at least 4 μmol/min*mg.

4 . The method of claim 1 , wherein the D-glufosinate is originally present in a racemic mixture of D- and L-glufosinate or salts thereof.

5 . The method of claim 1 , wherein the DAAO enzyme has the amino acid sequence of SEQ ID NO: 2.

6 . The method of claim 1 , wherein the DAAO enzyme is a mutant DAAO comprising one or more mutations at positions 54, 56, 58, 213, and 238, using SEQ ID NO: 2 as a reference sequence.

7 . The method of claim 6 , wherein the mutation at position 54 is selected from the group consisting of N54C, N54L, N54T, and N54V.

8 . The method of claim 6 , wherein the mutation at position 56 is T56M.

9 . The method of claim 6 , wherein the mutation at position 58 is selected from the group consisting of F58A, F58G, F58H, F58K, F58N, F58Q, F58R, F58S, and F58T.

10 . The method of claim 6 , wherein the mutation at position 213 is M213S.

11 . The method of claim 6 , wherein the mutant DAAO comprises mutations at positions 54 and 56, using SEQ ID NO: 2 as a reference sequence.

12 . The method of claim 6 , wherein the mutant DAAO comprises mutations F58Q or F58H, using SEQ ID NO: 2 as a reference sequence.

13 . The method of claim 6 , wherein the mutant DAAO comprises mutations N54V and F58Q, using SEQ ID NO: 2 as a reference sequence.

14 . The method of claim 6 , wherein the mutant DAAO comprises mutations N54V, F58Q, and M213S, using SEQ ID NO: 2 as a reference sequence.

15 . The method of claim 1 , wherein the reacting step and the aminating step are performed in a single container.

16 . The method of claim 15 , wherein all reagents are substantially added at the start of the reaction.

17 . The method of claim 15 , wherein the reagents for the reacting step and the reagents for the aminating step are added to the single container at different times.

18 . The method of claim 1 , wherein the reacting step and the aminating step are performed in separate containers.

19 . The method of claim 1 , wherein the aerating comprises introducing oxygen, oxygen enriched air, an oxygen enriched gas stream, or air.

20 . The method of claim 1 , wherein the aerating is conducted intermittently or continuously.

21 . The method of claim 6 , wherein the mutant DAAO comprises mutations N54V, T56N, F58H, and M213S, using SEQ ID NO: 2 as a reference sequence.

22 . The method of claim 1 , wherein at least 90% of the D-glufosinate is converted to L-glufosinate.

Assignees

• Basf Se

Inventors

• Green Brian Michael
• Gradley Michelle Lorraine

Classifications

• C12N9/10 Primary

  Transferases (2.) (ribonucleases C12N9/22) · CPC title

• C12N1/20

  Bacteria; Culture media therefor · CPC title

• C12Y206/01019

  4-Aminobutyrate—2-oxoglutarate transaminase (2.6.1.19) · CPC title

• C12Y104/03003

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

• A01N57/20

  containing acyclic or cycloaliphatic radicals · CPC title