Nitrilase from Pseudomonas fluorescens EBC191: cloning and heterologous expression of the gene and biochemical characterization of the recombinant enzyme.
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 degrees C), 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.
Kiziak
Christoph
C
Institut für Mikrobiologie, Universität Stuttgart, Allmandring 31, D-70569 Stuttgart, Germany.|Institut für Industrielle Genetik, Universität Stuttgart, Allmandring 31, D-70569 Stuttgart, Germany.
Conradt
Doris
D
Institut für Industrielle Genetik, Universität Stuttgart, Allmandring 31, D-70569 Stuttgart, Germany.
Stolz
Andreas
A
Institut für Mikrobiologie, Universität Stuttgart, Allmandring 31, D-70569 Stuttgart, Germany.
Mattes
Ralf
R
Institut für Industrielle Genetik, Universität Stuttgart, Allmandring 31, D-70569 Stuttgart, Germany.
Klein
Joachim
J
Institut für Industrielle Genetik, Universität Stuttgart, Allmandring 31, D-70569 Stuttgart, Germany.
D000619:Aminohydrolases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D015964:Gene Expression Regulation, Bacterial; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D011551:Pseudomonas fluorescens / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity; D013696:Temperature
D011994:Recombinant Proteins; D000619:Aminohydrolases; C021867:nitrilase