IP Library › Granted Patent US 6,953,677
Granted Patent B2
US 6,953,677 · App. 10/045,063 · Granted Oct 11, 2005

Recombinant L-N-carbamoylase from Arthrobacter aurescens and method of producing L-amino acids therewith

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Quick Facts
Patent No.
US 6,953,677
App. No.
10/045,063
Granted
Oct 11, 2005
Kind
B2
Abstract

A novel rec-L-N-carbamoylase from Arthrobacter aurescens and its method of use for producing L-amino acids. The recombinantly produced L-carbamoylase is unexpectedly stable, so that an industrial method of producing L-amino acids can be established with it, in contrast to previously known L-carbamoylases.

Claims (44)

1. A method for production of a β-aryl-substituted L-amino acid or β-indole-substituted L-amino acid, comprising:

(a) fermenting an E. coli host cell that contains an isolated polynucleotide selected from the group consisting of:

(i) a nucleotide sequence as set forth in SEQ ID NO: 1; and

(ii) a nucleotide sequence encoding the polypeptide as set forth in SEQ ID NO: 2;

(b) expressing an Arthrobacter aurescens ' L-N-carbamoylase from step (a); and

(c) contacting the L-N carbamoylase of step (b) with N-carbamoyl or N-formyl amino acids to produce a β-aryl-substituted L-amino acid or β-indole-substituted L-amino-acid.

2. The method according to claim 1 , further comprising the step of immobilizing the L-N-carbamoylase onto carriers.

3. The method according to claim 2 , wherein the L-N-carbamoylase is covalently immobilized on EAH-sepharose.

4. The method according to claim 1 , wherein the induction of expression of L-N-carbamoylase is by rhamnose, IPTG, or lactose.

5. The method according to claim 1 , wherein N-formyl-D,L tryptophase, and N-carbamoyl-D,L-phenylalanine serve as substrates for the L-N-carbamoylase.

6. The method according to claim 1 , wherein the isolated polynucleotide is the hyuC gene of Arthrobacter aurescens.

7. A method for production of L-methionine comprising:

(a) fermenting an E. coli host cell that contains an isolated polynucleotide selected from the group consisting of:

(i) a nucleotide sequence as set forth in SEQ ID NO: 1; and

(ii) a nucleotide sequence encoding the polypeptide as set forth in SEQ ID NO: 2;

(b) expressing an Arthrobacter aurescens ' L-N-carbamoylase from step (a); and

(c) contacting the L-N carbamoylase of step (b) with N-carbamoyl-L-methionine to produce L-methionine.

8. The method according to claim 7 , further comprising the step of immobilizing the L-N-carbamoylase onto carriers.

9. The method according to claim 8 , wherein the L-N-carbamoylase is covalently immobilized on EAH-sepharose.

10. The method according to claim 7 , wherein the induction of expression of L-N-carbamoylase is by rhamnose, IPTG, or lactose.

11. The method according to claim 7 , wherein N-carbamoyl-L-methionine serve as substrates for the L-N-carbamoylase.

12. The method according to claim 7 , wherein the isolated polynucleotide is the hyuC gene of Arthrobacter aurescens.

13. A method for production of L-tryptophan, L-phenylalanine, or L-tyrosine, comprising:

(a) fermenting an E. coli host cell that contains an isolated polynucleotide selected from the group consisting of

(i) a nucleotide sequence as set forth in SEQ ED NO: 1; and

(ii) a nucleotide sequence encoding the polypeptide as set forth in SEQ ID NO: 2;

(b) expressing an Arthrobacter aurescens ' L-N-carbamoylase from step (a); and

(c) contacting the L-N carbamoylase of step (b) with N-carbamoyl or N-formyl amino acids to produce L-tryptophan, L-phenylalanine, or L-tyrosine.

14. The method according to claim 13 , further comprising the step of immobilizing the L-N-carbamoylase onto carriers.

15. The method according to claim 14 , wherein the L-N-carbamoylase is covalently immobilized on EAH-sepharose.

16. The method according to claim 13 , wherein the induction of expression of L-N-carbamoylase is by rhamnose, IPTG, or lactose.

17. The method according to claim 13 , wherein N-carbamoyl-L-methionine serve as substrates for the L-N-carbamoylase.

18. The method according to claim 13 , wherein the isolated polynucleotide is the hyuC gene of Arthrobacter aurescens.

19. A method for production of L-thienylalanine comprising:

(a) fermenting an E. coli host cell that contains an isolated polynucleotide selected from the group consisting of:

(i) a nucleotide sequence as set forth in SEQ ID NO: 1; and

(ii) a nucleotide sequence encoding the polypeptide as set forth in SEQ ID NO: 2;

(b) expressing an Arthrobacter aurescens ' L-N-carbamoylase from step (a); and

(c) contacting the L-N carbamoylase of step (b) with N-carbamoyl-L-thienylalanie to produce L-thienylalanine.

20. The method according to claim 19 , further comprising the step of immobilizing the L-N-carbamoylase onto carriers.

21. The method according to claim 20 , wherein the L-N-carbamoylase is covalently immobilized on EAH-sepharose.

22. The method according to claim 19 , wherein the induction of expression of L-N-carbamoylase is by rhamnose, IPTG, or lactose.

23. The method according to claim 19 , wherein N-carbamoyl-L-methionine serve as substrates for the L-N-carbamoylase.

24. The method according to claim 19 , wherein the isolated polynucleotide is the hyuC gene of Arthrobacter aurescens.