Several novel nitrilases were selected from meta-genomic libraries using cinnamonitrile and a mixture of six different nitriles as substrates. The nitrilase gene nit1 was expressed in Escherichia coli and the resulting protein was further examined concerning its biochemical properties. Nit1 turned out to be an aliphatic nitrilase favoring dinitriles over mononitriles. Stereochemical analysis revealed that Nit1 converted the dinitrile 2-methylglutaronitrile regioselectively. Hydrolysis at the omega-nitrile group of a dinitrile, such as catalyzed by Nit1, leads to omega-cyanocarboxylic acids, which are important precursors for chemical and pharmaceutical products. Nit1 metabolized 2-methylglutaronitrile to the corresponding omega-cyanocarboxylic acid 4-cyanopentanoic acid can be used for the production of the fine chemical 1,5-dimethyl-2-piperidone.