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 an L-amino acid dehydrogenase (LAAD) enzyme in the presence of an ammonia source,
wherein at least 70% of the D-glufosinate is converted to L-glufosinate; and
wherein the method is performed ex vivo.
2. The method according to claim 1 , wherein the D-glufosinate is originally present in a racemic mixture of D- and L-glufosinate or salts thereof.
3. The method according to claim 1 , wherein the DAAO enzyme has the amino acid sequence of SEQ ID NO: 2.
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.
5. The method of claim 4 , wherein the mutation at position 54 is selected from the group consisting of N54C, N54L, N54T, and N54V.
6. The method of claim 4 , wherein the mutation at position 56 is T56M.
7. The method of claim 4 , wherein the mutation at position 58 is selected from the group consisting of F58A, F58G, F58H, F58K, F58N, F58Q, F58R, F58S, and F58T.
8. The method of claim 4 , wherein the mutation at position 213 is M213S.
9. The method of claim 4 , wherein the mutant DAAO comprises mutations at positions 54 and 56, using SEQ ID NO: 2 as a reference sequence.
10. The method of claim 4 , wherein the mutant DAAO comprises mutations F58Q or F58H, using SEQ ID NO: 2 as a reference sequence.
11. The method of claim 4 , wherein the mutant DAAO comprises mutations N54V and F58Q, using SEQ ID NO: 2 as a reference sequence.
12. The method of claim 4 , wherein the mutant DAAO comprises mutations N54V, F58Q, and M213S, using SEQ ID NO: 2 as a reference sequence.
13. The method of claim 1 , wherein the reacting step and the aminating step are performed in a single container.
14. The method of claim 13 , wherein all reagents are substantially added at the start of the reaction.
15. The method of claim 13 , wherein the reagents for the reacting step and the reagents for the aminating step are added to the single container at different times.
16. The method of claim 1 , wherein the reacting step and the aminating step are performed in separate containers.
17. The method of claim 1 , wherein the aerating comprises introducing oxygen, oxygen enriched air, an oxygen enriched gas stream, or air.
18. The method of claim 1 , wherein the aerating is conducted intermittently or continuously.