Rhodococcus nitrile hydratase

An isolated polynucleotide cluster comprising four gene sequences, wherein said gene sequences encode:

• a) a polypeptide having and amino acid sequence at least 90% identical to that of SEQ ID NO:2 wherein said polypeptide is a subunit of an enzyme with nitrile hydratase activity;
• b) a polypeptide having and amino acid sequence at least 90% identical to that of SEQ ID NO:3;
• wherein said polypeptide is a subunit of an enzyme with nitrile hydratase activity;
• c) a polypeptide having and amino acid sequence at least 90% identical to that of SEQ ID NO:4, wherein said polypeptide has cobalt transporter activity;
• d) a polypeptide having and amino acid sequence at least 90% identical to that of SEQ ID NO:4, wherein said polypeptide has the activity of the auxiliary protein P15K.

An isolated polynucleotide, wherein said polynucleotide hybridizes, under stringent conditions, with a polynucleotide selected from the group consisting of:

• a) the complement of positions 1 to 1327 from SEQ ID NO:1;
• b) the complement of positions 1324 to 1737 from SEQ ID NO:1;
• and
• c) the complement of positions 2076 to 3146 of SEQ ID NO:1;
• and wherein said stringent conditions comprise washing in 5×SSC at a temperature of from 50 to 68° C.

A process for preparing a nitrile hydratase;

• comprising:
• a) fermenting a microorganism in which one or more of the four gene sequences of claim 1 is overexpressed, said fermentation being carried out in the presence of 2+from 0.15 to 4 mM Co, and under conditions which lead to the formation of the nitrile hydratase,
• b) allowing said nitrile hydratase to accumulate in said microorganism, and
• c) isolating said nitrile hydratase from the microorganism of step b).

A process for enzymically preparing an amide from a nitrile comprising:

• a) converting a nitrile group-containing compound using a Rhodococcus microorganism comprising a nitrile hydratase enzyme having:
• i) a beta subunit with an amino acid sequence that is at least 90% identical to that of SEQ ID NO:2;
• and
• ii) an alpha subunit at least 90% identical to the sequence SEQ ID NO:3;
• and
• b) purifying said amide.