title: Expression and Characterization of a Novel Nitrilase from Hyperthermophilic Bacterium Thermotoga maritima MSB8.
abstract: The present study describes the gene cloning, overexpression and characterization of a novel nitrilase from hyperthermophilic bacterium Thermotoga maritima MSB8. The nitrilase gene consisted of 804 base pairs, encoding a protein of 268 amino acid residues with a molecular mass of 30.07 kDa after SDS-PAGE analysis. The optimal temperature and pH of the purified enzyme were 45°C and 7.5, respectively. The enzyme demonstrated good temperature tolerance, with 40% residual activity after 60 min of heat treatment at 75°C. The kinetic constants Vmax and Km of this nitrilase toward 3-cyanopyridine were 3.12 μmol/min/mg and 7.63 mM, respectively. Furthermore, this novel nitrilase exhibited a broad spectrum toward the hydrolysis of the aliphatic nitriles among the tested substrates, and particularly was specific to aliphatic dinitriles like succinonitrile, which was distinguished from most nitrilases ever reported. The catalytic efficiency kcat/Km was 0.44 /mM/s toward succinonitrile. This distinct characteristic might enable this nitrilase to be a potential candidate for industrial applications for biosynthesis of carboxylic acid.
authors.affiliation: Institute of Life Sciences, Jiangsu University, Zhenjiang 212000, P.R. China.
authors.forename: Zhi
authors.initials: Z
authors.lastname: Chen
authors.affiliation: Institute of Life Sciences, Jiangsu University, Zhenjiang 212000, P.R. China.|National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, P.R. China.
authors.forename: Huayou
authors.initials: H
authors.lastname: Chen
authors.affiliation: Institute of Life Sciences, Jiangsu University, Zhenjiang 212000, P.R. China.
authors.forename: Zhong
authors.initials: Z
authors.lastname: Ni
authors.affiliation: Institute of Life Sciences, Jiangsu University, Zhenjiang 212000, P.R. China.
authors.forename: Rui
authors.initials: R
authors.lastname: Tian
authors.affiliation: Institute of Life Sciences, Jiangsu University, Zhenjiang 212000, P.R. China.
authors.forename: Tianxi
authors.initials: T
authors.lastname: Zhang
authors.affiliation: Institute of Life Sciences, Jiangsu University, Zhenjiang 212000, P.R. China.
authors.forename: Jinru
authors.initials: J
authors.lastname: Jia
authors.affiliation: Institute of Life Sciences, Jiangsu University, Zhenjiang 212000, P.R. China.
authors.forename: Shengli
authors.initials: S
authors.lastname: Yang
mesh_terms: D000595:Amino Acid Sequence; D000619:Aminohydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009570:Nitriles / Q000378:metabolism; D011725:Pyridines / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013696:Temperature; D020124:Thermotoga maritima / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism
chemical_list: D009570:Nitriles; D011725:Pyridines; D000619:Aminohydrolases; C021867:nitrilase; C020729:3-cyanopyridine
keywords: Aliphatic dinitriles; Hyperthermophilic bacterium; Nitrilase; Temperature tolerance; Thermotoga maritima