The gene encoding an enantioselective arylacetonitrilase was identified on a 3?8 kb DNA fragment from the genomic DNA of Pseudomonas fluorescens EBC191. The gene was isolated, sequenced and cloned into the L-rhamnose-inducible expression vector pJOE2775. The nitrilase was produced in large quantities and purified as a histidine-tagged enzyme from crude extracts of L-rhamnose-induced cells of Escherichia coli JM109. The purified nitrilase was significantly stabilized during storage by the addition of 1 M ammonium sulfate. The temperature optimum (50 6C), pH optimum (pH 6?5), and specific activity of the recombinant nitrilase were similar to those of the native enzyme from P. fluorescens EBC191. 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 to R-mandelic acid and S-mandelic acid amide. The nitrilase gene is physically linked in the genome of P. fluorescens with 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.

Sphingomonas xenophaga BN6 degrades various (substituted) naphthalenesulfonates to the corresponding (substituted) salicylates. A gene cluster was identified on the plasmid pBN6 which coded for several enzymes participating in the degradative pathway for naphthalenesulfonates. A DNA fragment of 16 915 bp was sequenced which contained 17 ORFs. The genes encoding the 1,2-dihydroxynaphthalene dioxygenase, 2-hydroxychromene-2-carboxylate isomerase, and 29-hydroxybenzalpyruvate aldolase of the naphthalenesulfonate pathway were identified on the DNA fragment and the encoded proteins heterologously expressed in Escherichia coli. Also, the genes encoding the ferredoxin and ferredoxin reductase of a multi-component, ring-hydroxylating naphthalenesulfonate dioxygenase were identified by insertional inactivation. The identified genes generally demonstrated the highest degree of homology to enzymes encoded by the phenanthrene-degrading organism Sphingomonas sp. P2, or the megaplasmid pNL1 of the naphthalene-and biphenyl-degrading strain Sphingomonas aromaticivorans F199. The genes of S. xenophaga BN6 participating in the degradation of naphthalenesulfonates also shared the same organization in three different transcriptional units as the genes involved in the degradation of naphthalene, biphenyl, and phenanthrene previously found in Sphingomonas sp. P2 and S. aromaticivorans F199. The genes were flanked in S. xenophaga BN6 by ORFs which specify proteins that show the highest homologies to proteins of mobile genetic elements.