Purification and characterization of nitrilase responsible for the enantioselective hydrolysis from Acinetobacter sp. AK 226.

A nitrilase was purified, apparently to homogeneity, from a cell extract of Acinetobacter sp. AK 226, which converts racemic 2-(4'-isobutylphenyl)propionitrile (Ibu-CN) to S-(+)-2-(4'-isobutyl-phenyl)propionic acid (S-(+)-ibuprofen). The molecular weight of the native enzyme was estimated as 580,000 upon gel filtration. The nitrilase hydrolyzed many kinds of nitrile compounds such as aliphatic, aromatic, and heterocyclic mononitriles or dinitriles. The amino-terminal amino acids were sequenced and found to be partly homologous to a nitrilase from Klebsiella pneumoniae subsp. ozanae. The purified enzyme had a pH optimum of 8.0 and a temperature optimum of 50 degrees C. The enzyme was not affected by chelating reagents, carbonyl reagents, reductants, most metal ions, or thiol reagents except silver ion, p-chloromercuribenzoate, and phenylmercuribenzoate. The reaction with racemic Ibu-CN resulted in the preferential production of S-(+)-ibuprofen, demonstrating that the nitrilase is highly enantioselective to S-(-)-Ibu-CN. In fact, the enzyme showed a 180-fold higher activity for racemic Ibu-CN than that for R-(+)-Ibu-CN.