| Nitrilase is an important member of hydrolysis enzyme, it can convert nitriles into carboxylic acid and ammonium. This property brings about high value and broad application prospect in organic synthesis and industrial production.Starting from amino acids homologous sequences of nitrilase, a nitilase gene was cloned by methods of degenerate PCR and TAIL-PCR from Rhodobacter sp. LHS-305which was stored in our laboratory. A recombinant expression plasmid of nitrilase gene was constructed, and finally got the recombinant nitrilase by inducible expression and affinity chromatography purification, then detailed properties of this nitrilase was studied.The nitrilase gene consists of969bp nucleotides encoding a peptide of322amino acid residues. Its nucleotide sequence had the highest similarity of82%to nitrilase reported before. By Ni-NTA affinity chromatography, recombinant nitrilase was purified, and displayed a single band in SDS-PAGE. Compared with the standard protein marker, molecular mass of the enzyme was calculated as40kDa.Then, the properties of this recombinant nitrilase was studied. The optimum temperature was40℃, optimum pH value was7.0. The recombinant nitrilase activity was inhibited by Cu2+, Zn2+, and Ag+, the presence of Ca2+, Mg2+, Ni+was found to have little effect on the nitrilase activity, DTT and EDTA has slight inhibitory effect to enzyme activity. The enzyme displayed broad substrate specificity, exhibited high hydrolysis activities for aliphatic nitriles and aromatic nitriles. Catalysis of aliphatic dinitriles was regioselective, and these aliphatic dinitriles were converted completely to the corresponding mononitrile-monoacids. The kinetic parameters of the recombinant enzyme were determined with Fmax and Km of77.5μmol·min-1·mg-1and73.1mM, respectively.These studies laid the foundation for further study of the catalytic mechanism of this enzyme. |
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