Processes using amino acid dehydrogenases and ketoreductase-based cofactor regenerating system

Patent metadata

Field	Value
Publication number	US-2016281119-A1
Application number	US-201615184242-A
Country	US
Kind code	A1
Filing date	Jun 16, 2016
Priority date	Feb 10, 2010
Publication date	Sep 29, 2016
Grant date	—

Abstract

The present disclosure relates to the use of an amino acid dehydrogenase in combination with a cofactor regenerating system comprising a ketoreductase. In particular embodiments, the process can be used to prepare L-tert-leucine using a leucine dehydrogenase.

First claim

What is claimed is:

1 . A process for converting a compound mixture of formula IId which comprises a substrate for an amino acid dehydrogenase to a composition of formula I and a chiral amino acid of formula IIa:

comprising contacting the compound mixture of formula IId with an enantioselective amino acid dehydrogenase in a reaction medium comprising NAD + /NADH or NADP + /NADPH and a cofactor recycling system comprising a ketoreductase and a lower alkyl ketone, under conditions where the compound mixture of formula IId is converted to the composition of formula I and a chiral amino acid of formula IIa, and the lower alkyl ketone is converted to a lower secondary alcohol.

2 . The process of claim 1 , wherein the compound mixture of IId is a racemic mixture of formula IIe:

3 . The process of claim 1 , wherein the amino acid dehydrogenase comprises an L-amino acid dehydrogenase and the chiral amino acid of formula IIa is IIc

wherein the chiral amino acid of formula IIc is present in enantiomeric excess.

4 . The process of claim 3 , wherein the L-amino acid dehydrogenase is from Bacillus, Clostridium, Corynebacterium, Geobacillus, Natronobacterium, Synechocystis, Thermoactinomyces, Thermos, Thermomicrobium , or Carderia.

5 . The process of claim 4 , wherein the L-amino acid dehydrogenase is selected from L-alanine dehydrogenase, L-aspartate dehydrogenase, L-erythro-3,5-diaminohexanoate dehydrogenase, L-leucine dehydrogenase, L-glutamate dehydrogenase, lysine dehydrogenase, L-phenylalanine dehydrogenase, L-serine dehydrogenase. L-valine dehydrogenase, L-2,4-diaminopentanoate dehydrogenase, L-glutamate synthase, L-diaminopimelate dehydrogenase, L-N-methylalanine dehydrogenase, L-lysine 6-dehydrogenase, and L-tryptophan dehydrogenase.

6 . The process of claim 1 , wherein the amino acid dehydrogenase comprises a D-amino acid dehydrogenase and the chiral amino acid of formula IIa is IIb

wherein the chiral amino acid of formula of formula IIa is present in enantiomeric excess.

7 . The process of claim 6 , wherein the D-amino acid dehydrogenase is from Halobacterium, Methanosarcina, Pseudomonas, Pyrobaculum, Salmonella, Corynebacterium , and Escherichia.

8 . The process of claim 7 , wherein the D-amino acid dehydrogenase is from Pseudomonas aeruginosa, Pseudomonas fluorescens, Pyrobaculum islandicum, Salmonella typhimurium, Corynebacterium glutamicum , and Escherichia coli.

9 . The process of claim 8 , wherein the D-amino acid dehydrogenase is selected from D-alanine dehydrogenase, D-threonine dehydrogenase, and D-proline dehydrogenase.

10 . The process of claim 1 which is carried out in a cell free system.

11 . The process of claim 1 , wherein the amino acid dehydrogenase is present as a crude extract.

12 . The process of claim 1 , wherein the amino acid dehydrogenase is substantially purified.

13 . The process of claim 1 , wherein the ketoreductase is a wild type ketoreductase or an engineered ketoreductase.

14 . The process of claim 13 , wherein the ketoreductase is from Lactobacillus, Candida, Novosphingobium , or Saccharomyces.

15 . The process of claim 14 , wherein the ketoreductase of Lactobacillus is from Lactobacillus kefir, Lactobacillus brevis , or Lactobacillus minor.

16 . The process of claim 15 , wherein the ketoreductase of Candida is from Candida magnoliae.

17 . The process of claim 15 , wherein the ketoreductase of Saccharomyces is from Saccharomyces cerevisiae.

18 . The process of claim 15 , wherein the ketoreductase of Novosphingobium is from Novosphingobium aromaticivorans.

19 . The process of claim 1 , wherein the ketoreductase is an engineered ketoreductase characterized by increased thermostability, increased solvent stability, and/or increased enzymatic activity relative to the wild type ketoreductase.

20 . The process of claim 1 , wherein the ketoreductase comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24.

21 . The process of claim 1 , wherein the amino acid sequence is the amino acid sequence of SEQ ID NO:18.

22 . The process of claim 1 , wherein the ketoreductase is present as a crude extract.

23 . The process of claim 1 , wherein the ketoreductase is substantially purified.

24 . The process of claim 1 , further comprising the step of removing the lower secondary alcohol formed from the lower alkyl ketone from the reaction medium.

25 . The process of claim 1 , wherein the lower alkyl ketone is acetone and the lower secondary alcohol is isopropanol.

26 . The process of claim 25 , wherein the isopropanol is removed from the reaction medium.

27 . The process of claim 1 , wherein the reaction medium is at a pH of about 8.5 to about 10.

28 . The process of claim 27 , wherein the reaction medium is at a pH of about 8.5 to about 9.0.

29 . The process of claim 1 , wherein the reaction medium is at a temperature of about 25° C. to about 45° C.

30 . The process of claim 29 , wherein the reaction medium is at a temperature of about 35° C. to about 40° C.

31 . The process of claim 1 , wherein the lower alkyl ketone is present in at least 1.5 fold stoichiometric excess of the substrate of formula IIc.

Assignees

• Codexis Inc

Inventors

• Cabirol Fabien Louis
• Collier Steven J
• Daussmann Thomas
• Modukuru Naga K

Classifications

• C12P41/007 Primary

  by reactions involving acyl derivatives of racemic amines · CPC title

• C12Y101/00

  Oxidoreductases acting on the CH-OH group of donors (1.1) · CPC title

• C12N9/0004

  Oxidoreductases (1.) · CPC title

• C12P13/06 Primary

  Alanine; Leucine; Isoleucine; Serine; Homoserine · CPC title

• C12N9/0016

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