Identification and Expression in E. coli of Novel Nitrile Hydratases from the Metagenome

A PCR‐based screening procedure using degenerate consensus primers was employed to identify nitrile hydratase (NHase) encoding genes in libraries of cloned environmental DNA. Screening of about 3 Gbp of metagenomic DNA led to the identification of twelve novel and highly diverse NHase α‐subunits, ten β‐subunits, and three NHase activator proteins. The disruption of the natural arrangement of the genes encoding the NHase subunits and a reduced temperature proved to be beneficial for the soluble expression in E. coli . Of nine NHases investigated, six were produced in an active conformation. The co‐expression of corresponding activator proteins resulted in the activation of a previously inactive enzyme and a marked increase in activity of another enzyme. Thus, the sequence‐homology based screening approach led to the identification and finally the production of novel and diverse NHase enzymes.