Rapid and specific identification of nitrile hydratase (NHase)-encoding genes in soil samples by polymerase chain...

A polymerase chain reaction (PCR) protocol was developed for the specific detection of genes coding nitrile hydratase (NHase). Primer design was based on the highly conserved sequences found in the coding region of the alpha-subunit gene corresponding to the metal-binding site. Purified genomic DNA from bacterial strains or directly from soil can serve as the target for the PCR, thus affording a simple and rapid method for screening NHase genes. The primer pairs, NHCo1/NHCo2 and NHFe1/NHFe2 yield PCR products corresponding to a partial coding sequence of cobalt and iron NHase genes, respectively. Using the PCR method, both types of iron- and cobalt-NHase-encoding genes were detected in DNA from pure cultures and soil samples. Furthermore consensus primers allowed rapid cloning and expression of novel NHases in Escherichia coli.