Nitrilase from Pseudomonas fluorescens EBC191: cloning and heterologous expression of the gene and biochemical characterization of the recombinant enzyme

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Abstract
The gene encoding an enantioselective arylacetonitrilase was identified on a 3·8 kb DNA fragment from the genomic DNA ofPseudomonas fluorescensEBC191. The gene was isolated, sequenced and cloned into thel-rhamnose-inducible expression vector pJOE2775. The nitrilase was produced in large quantities and purified as a histidine-tagged enzyme from crude extracts ofl-rhamnose-induced cells ofEscherichia coliJM109. The purified nitrilase was significantly stabilized during storage by the addition of 1 M ammonium sulfate. The temperature optimum (50 °C), pH optimum (pH 6·5), and specific activity of the recombinant nitrilase were similar to those of the native enzyme fromP. fluorescensEBC191. The enzyme hydrolysed various phenylacetonitriles with different substituents in the 2-position and also heterocyclic and bicyclic arylacetonitriles to the corresponding carboxylic acids. The conversion of most arylacetonitriles was accompanied by the formation of different amounts of amides as by-products. The relative amounts of amides formed from different nitriles increased with an increasing negative inductive effect of the substituent in the 2-position. The acids and amides that were formed from chiral nitriles demonstrated in most cases opposite enantiomeric excesses. Thus mandelonitrile was converted by the nitrilase preferentially toR-mandelic acid andS-mandelic acid amide. The nitrilase gene is physically linked in the genome ofP. fluorescenswith genes encoding the degradative pathway for mandelic acid. This might suggest a natural function of the nitrilase in the degradation of mandelonitrile or similar naturally occurring hydroxynitriles.