novel nitrilase from Rhodobacter sphaeroides LHS-305: cloning, heterologous expression and biochemical characterization

• Published in: World journal of microbiology & biotechnology 2014-01, Vol.30 (1), p.245-252
• Main Authors: Wang, Hualei, Li, Guinan, Li, Mingyang, Wei, Dongzhi, Wang, Xuedong
• Format: Article
• Language: English
• Subjects: acids; Aliphatic compounds; Amino acids; Aminohydrolases - chemistry; Aminohydrolases - genetics; Aminohydrolases - metabolism; Analysis; Applied Microbiology; Bacteria; Biochemistry; Biomedical and Life Sciences; Biotechnology; By products; Chromatography; Chromatography, Gel; Cloning; Cloning, Molecular; DNA, Bacterial - chemistry; DNA, Bacterial - genetics; E coli; Electrophoresis, Polyacrylamide Gel; Environmental Engineering/Biotechnology; Enzyme Stability; Enzymes; Escherichia coli; Escherichia coli - genetics; Gene Expression; gene overexpression; Genes; Hydrogen-Ion Concentration; hydrolysis; Kinetics; Life Sciences; Microbiology; Molecular Sequence Data; Molecular Weight; nitrilase; Nitriles; Nitriles - metabolism; Open Reading Frames; Original Paper; Pharmaceutical industry; polyacrylamide gel electrophoresis; Polypeptides; Protein Subunits; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Rhodobacter sphaeroides; Rhodobacter sphaeroides - enzymology; Rhodobacter sphaeroides - genetics; Sequence Analysis, DNA; size exclusion chromatography; Studies; Substrate Specificity; Temperature
• ISSN: 0959-3993; 1573-0972
• DOI: 10.1007/s11274-013-1445-7

In this study, a novel nitrilase gene from Rhodobacter sphaeroides was cloned and overexpressed in Escherichia coli. The open reading frame of the nitrilase gene includes 969 base pairs, which encodes a putative polypeptide of 322 amino acid residues. The molecular weight of the purified native nitrilase was about 560 kDa determined by size exclusion chromatography. This nitrilase showed one single band on SDS-PAGE with a molecular weight of 40 kDa. This suggested that the native nitrilase consisted of 14 subunits with identical size. The optimal pH and temperature of the purified enzyme were 7.0 and 40 °C, respectively. The kinetic parameters V ₘₐₓ and K ₘ toward 3-cyanopyridine were 77.5 μmol min⁻¹ mg⁻¹ and 73.1 mmol/l, respectively. The enzyme can easily convert aliphatic nitrile and aromatic nitriles to their corresponding acids. Furthermore, this enzyme demonstrated regioselectivity in hydrolysis of aliphatic dinitriles. This specific characteristic makes this nitrilase have a great potential for commercial production of various cyanocarboxylic acids by hydrolyzing readily available dinitriles.