Rhodococcus nitrile hydratase
The invention relates to a Rhodococcus polynucleotide cluster which contains nucleotide sequences which encode polypeptides having the activity of a nitrile hydratase, of an auxiliary protein P 15 K which activates this enzyme and of a cobalt transporter, to transformed microorganisms in which the nucleotide sequences encoding these proteins are present in increased quantity, and to the use of the transformed microorganisms for preparing amides from nitriles.
1. A process for enzymatically preparing an amide from a nitrile comprising:
a) converting a nitrile group-containing compound using a with transformed microorganism transformed with a vector expressing a nitrile hydratase enzyme having:
i) a beta subunit with an amino acid sequence that is at least 95% identical to that of SEQ ID NO:2; and
ii) an alpha subunit at least 95% identical to the sequence SEQ ID NO:3; and
b) purifying said amide.
2. The process of claim 1 , wherein said beta subunit comprises the amino acid sequence of SEQ ID NO:2 and said alpha subunit comprises the amino acid sequence of SEQ ID NO:3.
3. The process of claim 1 , wherein said beta subunit consists of the amino acid sequence of SEQ ID NO:2 and said alpha subunit consists of the amino acid sequence of SEQ ID NO:3.
4. The process of claim 1 , wherein said nitrile hydratase converts α-aminonitriles with a specific activity of> 50 U/mg of dry biomass.
5. The process of claim 1 , wherein resting cells which comprise said nitrile hydratase are used.
6. The process of claim 1 , wherein a nitrile of the general formula
is converted into the corresponding amide and in which:
X is OH, H, an alkyl having from 1 to 4 carbon atoms or NH 2 ;
R is H; a branched or unbranched saturated alkyl radical having from 1 to 12 carbon atoms and optionally substituted with NH 2 ; a branched or unbranched alkenyl radical having from 1 to 12 carbon atoms; a cycloalkyl group having from 3 to 6 C atoms; an alkylthio group-substituted alkylene radical, wherein said alkyl is a C 1 to C 3 radical and said alkylene is a bivalent C 3 to C 8 radical;
R′ is H, or an alkyl having from 1 to 3 with carbon atoms, and which is optionally substituted by one or two C 1 to C 3 alkyl groups or Cl, Br or F, or a monovalent alkylnitrile radical having from 1 to 6 carbon atoms.
7. The process of claim 1 , wherein said nitrile is methionine nitrile.
8. The process of claim 6 , wherein said beta subunit comprises the amino acid sequence of SEQ ID NO:2 and said alpha subunit comprises the amino acid sequence of SEQ II) NO:3.
9. The process of claim 8 , wherein said nitrile is methionine nitrile.
10. The process of claim 6 , wherein said beta subunit consists of the amino acid sequence of SEQ ID NO:2 and said alpha subunit consists of the amino acid sequence of SEQ ID NO:3.
11. The process of claim 10 , wherein said nitrile is metbionine nitrile.
12. The process of claim 6 , wherein R and R 1 are H.
13. The process of claim 12 , wherein X is OH.
14. The process of claim 12 , wherein X is an alkyl having from 1 to 4 carbon atoms.
15. The process of claim 12 , wherein X is NH 2 .
16. The process of claim 6 , wherein R is a branched or unbranched saturated alkyl radical having from 1 to 12 carbon atoms and optionally substituted with NH 2 .
17. The process of claim 16 , wherein X is an alkyl having from I to 4 carbon atoms.
18. The process of claim 16 , wherein X is NH 2 .
19. The process of claim 6 , wherein R is a cycloalkyl group having from 3 to 6 C atoms.
20. The process of claim 19 , wherein X is an alkyl having from 1 to 4 carbon atoms.