Methods for making L-glufosinate
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.
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; and
aminating the PPO to L-glufosinate by a transaminase (TA) enzyme, using an amine group from one or more amine donors,
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 amine donor is glutamate or L-glutamate.
3. The method of claim 1 , wherein the D-glufosinate is originally present in a racemic mixture of D- and L- glufosinate or salts thereof.
4. 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,
wherein the mutation at position 54 is selected from the group consisting of N54C, N54L, N54T, and N54V; the mutation at position 56 is T56M; and the mutation at position 58 is selected from the group consisting of F58A, F58G, F58H, F58K, F58N, F58Q, F58R, F58S, and F58T.
5. The method of claim 4 , wherein the mutations comprise N54V, F58Q, and M213S.
6. The method of claim 1 , wherein the TA enzyme is an enzyme encoded by SEQ ID NO: 1.
7. The method of claim 1 , wherein the TA enzyme is a GabT transaminase having the amino acid sequence set forth in SEQ ID NO: 3.
8. The method of claim 1 , wherein the reacting step and the aminating step are performed in a single container.
9. The method of claim 8 , wherein all reagents are substantially added at the start of the reaction.
10. The method of claim 8 , wherein the reagents for the reacting step and the reagents for the aminating step are added to the single container at different times.
11. The method of claim 1 , wherein the reacting step and the aminating step are performed in separate containers.
12. 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; and
aminating the PPO to L-glufosinate by a transaminase (TA) enzyme, using an amine group from one or more amine donors, wherein said 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,
wherein the mutation at position 54 is selected from the group consisting of N54C, N54L, N54T, and N54V; the mutation at position 56 is T56M; and the mutation at position 58 is selected from the group consisting of F58A, F58G, F58H, F58N, F58Q, F58R, F58S,and F58T;
wherein at least 70% of the D-glufosinate is converted to L-glufosinate; and
wherein the method is performed ex vivo.
13. The method of claim 12 , wherein the TA enzyme is a GabT transaminase having the amino acid sequence set forth in SEQ ID NO: 3.
14. The method of claim 12 , wherein the amine donor is glutamate.
15. The method of claim 13 , wherein the one or more mutations comprise N54V, F58Q, and M213S.
16. The method of claim 12 , wherein the reacting step and the aminating step are performed in a single container.
17. The method of claim 16 , wherein all reagents are substantially added at the start of the reaction.
18. The method of claim 16 , wherein the reagents for the reacting step and the reagents for the aminating step are added to the single container at different times.
19. The method of claim 12 , wherein the reacting step and the aminating step are performed in separate containers.
20. The method of claim 12 , wherein the DAAO and TA enzymes are immobilized to a support.
21. The method of claim 1 , wherein the DAAO and TA enzymes are immobilized to a support.