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

A polymerase chain reaction (PCR) protocol was developed for the specific d etection 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 t he PCR, thus affording a simple and rapid method for screening NHase genes. The primer pairs, NHCo1/NHCo2 and NHFe1/NHFe2 yield PCR products correspon ding to a partial coding sequence of cobalt and iron NHase genes, respectiv ely. Using the PCR method, both types of iron- and cobalt-NHase-encoding ge nes were detected in DNA from pure cultures and soil samples. Furthermore c onsensus primers allowed rapid cloning and expression of novel NHases in Es cherichia coli. (C) 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.