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);
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 to obtain a composition comprising L-glufosinate, PPO, and D-glufosinate; and
subjecting the composition to further purification methods selected from ion exchange, extraction, salt formation, crystallization, and filtration.
2. The method of claim 1 , wherein the one or more amine donors is selected from the group consisting of glutamate and 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. A D-amino acid oxidase (DAAO) enzyme comprising the amino acid sequence set forth in SEQ ID NO:2, wherein said enzyme comprises one or more mutations at positions 54, 56, 58, 213, and 238;
wherein said mutation at position 54 is selected from the group consisting of N54C, N54L, N54T, and N54V; said mutation at position 56 is T56M; said mutation at position 58 is selected from the group consisting of F58A, F58G, F58H, F58K, F58N, F 58 Q, F 58 R, F58S, and F58T; and said mutation at position 213 is M213S.
5. The DAAO enzyme of claim 4 , wherein said enzyme comprises mutations F58K and M213S.
6. The DAAO enzyme of claim 4 , wherein said enzyme comprises mutations N54V, F58Q, and M213S.
7. The DAAO enzyme of claim 4 , wherein said enzyme comprises mutations F58H and M213S.
8. The DAAO enzyme of claim 5 , wherein said enzyme comprises mutations at positions 54 and 56, wherein said mutation at position 54 is selected from the group consisting of N54C, N54L, N54T, and N54V; and, said mutation at position 56 is T56M.
9. The DAAO enzyme of claim 8 , wherein said enzyme comprises mutations N54T and T56M.
10. The DAAO enzyme of claim 4 , wherein said enzyme comprises mutations F58Q or F58H.
11. The DAAO enzyme of claim 4 , wherein said enzyme comprises mutations N54V or N54T and F58Q or F58H.
12. The DAAO enzyme of claim 4 , wherein said enzyme comprises mutations N54T, F58Q, and M213S.
13. The DAAO enzyme of claim 11 , wherein said enzyme comprises mutation F58Q.
14. The DAAO enzyme of claim 13 , wherein said enzyme comprises mutation N54V.
15. The DAAO enzyme of claim 4 , wherein said enzyme comprises mutations N54V, N54C or N54T and F58Q, F58H, F58, or F58R.
16. The DAAO enzyme of claim 15 , wherein said enzyme comprises mutation M213S.
17. The DAAO enzyme of claim 4 , wherein said enzyme comprises mutation M213S.
18. The DAAO enzyme of claim 4 , wherein said enzyme comprises mutation N54V.