Whole cell catalyst
View Patent ↗A whole cell catalyst is described comprising a hydantoinase, a racemase and a carbamoylase. Thus this catalyst is able to degrade hydantoins directly into the amino acids. Additionally, a process for the production of this catalysts and for the production of amino acids is claimed.
1. A microorganism which
(A) is transformed with DNAs encoding (i) a hydantoinase, (ii) a hydantoin racemase, and (iii) a D- or L-specific carbamoylase, and
(B) converts 5-monosubstituted hydantoins to L- or D-amino acids,
wherein the DNAs encoding the hydantoinase, the hydantoin racemase, and the D- or L-specific carbamoylase are overexpressed in the microorganism according to the turnover rates of the respective enzymes, to reduce the accumulation of intermediates in the conversion of the 5-monosubstituted hydantoins to L- or D- amino acids.
2. The microorganism of claim 1 , which is a bacterium.
3. The microorganism of claim 1 , which is an Escherichia coli.
4. The microorganism of claim 1 , wherein the DNAs encoding the hydantoinase, the hydantoin racemase, and the D- or L-specific carbamoylase are overexpressed in a rhamnose-inducible E. coli promoter cassette.
5. The microorganism of claim 1 , wherein the DNA encoding the hydantoinase comprises SEQ ID NO: 8.
6. The microorganism of claim 1 , wherein the DNA encoding the hydantoin racemase comprises SEQ ID NO: 10.
7. The microorganism of claim 1 , wherein the DNA encoding the L-specific carbamoylase comprises SEQ ID NO: 6.
8. The microorganism of claim 1 , wherein the DNA encoding the hydantoinase encodes an amino acid sequence comprising SEQ ID NO: 9.
9. The microorganism of claim 1 , wherein the DNA encoding the hydantoin racemase encodes an amino acid sequence comprising SEQ ID NO: 11.
10. The microorganism of claim 1 , wherein the DNA encoding the L-specific carbamoylase encodes an amino acid sequence comprising SEQ ID NO: 7.
11. The microorganism of claim 1 , which is transformed with at least one plasmid containing the DNAs encoding the hydantoinase, the hydantoin racemase, and the D- or L-specific carbamoylase.
12. A process for producing the microorganism of claim 1 , comprising, transforming a microorganism with one or more plasmids containing the DNAs encoding the hydantoinase, the hydantoin racemase, and the D- or L-specific carbamoylase.
13. The process of claim 12 , wherein the one or more plasmids are selected from the group consisting of pBW31, pBW32, pBW34, pBW35, pBW53.
14. The process of claim 12 , wherein the one or more plasmids are selected from the group consisting of pBW31 and pBW32; pBW34 and pBW35; pBW34 and pBW53; and pBW32 and pBW34.
15. The process of claim 12 , wherein the one or more plasmids are derived from pSC101, pACYC184, or pBR322.
16. The process of claim 12 , wherein the microorganism is an E. coli having a chromosomally inserted hydantoinase gene.
17. The process of claim 12 , in which a rhamnose inducible E. coli promoter cassette is used.
18. A process for the production of enantiomerically enriched amino acids, comprising culturing the microorganism of claim 1 in a culture medium.
19. The process of claim 18 , which is performed in an enzyme-membrane reactor.
20. A plasmid selected from the group consisting of pBW31 (DSM 15809), pBW32 (DSM 15810), pBW34 (DSM 15811), pBW35 (DSM 15812), pBW53 (DSM 15813) and pAW229.
21. A primer selected from the group consisting of S2480 (SEQ ID NO: 1), S2248 (SEQ ID NO: 2), S2249 (SEQ ID NO: 3), S2517 (SEQ ID NO: 4), and S2518 (SEQ ID NO: 5).
22. A microorganism comprising one or more plasmids of claim 20 .
23. The microorganism of claim 1 , wherein the microorganism is transformed with a plasmid comprising the amplification product obtained by PCR amplification, using a primer pair, and wherein at least one primer of said pair is selected from the group consisting of S2480, S2248, S2249, S2517 and S2518.
24. The microorganism of claim 23 , wherein the DNAs encoding the hydantoinase, the hydantoin racemase, and the D- or L-specific carbamoylase are obtained from a microorganism of the genus Arthrobacter aurescens.
25. The microorganism of claim 1 , wherein the DNAs encoding the hydantoinase, the hydantoin racemase, and the D- or L-specific carbamoylase are obtained from a microorganism of the genus Arthrobacter aurescens.
26. The microorganism of claim 1 , wherein the DNAs encoding the hydantoin racemase, and the D- or L-specific carbamoylase are obtained from a microorganism of the genus Arthrobacter aurescens.
27. The microorganism of claim 26 , wherein the DNA encoding the hydantoinase is obtain from a microbial source.