title: Gene cloning, expression, and characterization of a nitrilase from Alcaligenes faecalis ZJUTB10.
abstract: Nitrilases are important industrial enzymes that convert nitriles directly into the corresponding carboxylic acids. In the current work, the fragment with a length of 1068 bp that encodes the A. faecalis ZJUTB10 nitrilase was obtained. Moreover, a catalytic triad was proposed and verified by site-directed mutagenesis, and the detailed mechanism of this nitrilase was clarified. The substrate specificity study demonstrated that the A. faecalis ZJUTB10 nitrilase belongs to the family of arylacetonitrilases. The optimum pH and temperature for the purified nitrilase was 7-8 and 40 °C, respectively. Mg(2+) stimulated hydrolytic activity, whereas Cu(2+), Co(2+), Ni(2+), Ag(+), and Hg(2+) showed a strong inhibitory effect. The K(m) and v(max) for mandelonitrile were 4.74 mM and 15.85 μmol min(-1) mg(-1) protein, respectively. After 30 min reaction using the nitrilase, mandelonitrile at the concentration of 20 mM was completely hydrolyzed and the enantiomeric excess against (R)-(-)-mandelic acid was >99%. Characteristics investigation indicates that this nitrilase is promising in catalysis applications.
authors.affiliation: Institute of Bioengineering, Zhejiang University of Technology, Hangzhou 310014, People's Republic of China.
authors.forename: Zhi-Qiang
authors.initials: ZQ
authors.lastname: Liu
authors.forename: Li-Zhu
authors.initials: LZ
authors.lastname: Dong
authors.forename: Feng
authors.initials: F
authors.lastname: Cheng
authors.forename: Ya-Ping
authors.initials: YP
authors.lastname: Xue
authors.forename: Yuan-Shan
authors.initials: YS
authors.lastname: Wang
authors.forename: Jie-Nv
authors.initials: JN
authors.lastname: Ding
authors.forename: Yu-Guo
authors.initials: YG
authors.lastname: Zheng
authors.forename: Yin-Chu
authors.initials: YC
authors.lastname: Shen
mesh_terms: D042481:Alcaligenes faecalis / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics*; D000595:Amino Acid Sequence; D000619:Aminohydrolases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D002384:Catalysis; D003001:Cloning, Molecular; D004795:Enzyme Stability; D007700:Kinetics; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D000619:Aminohydrolases; C021867:nitrilase