Gene cloning, overexpression, and characterization of the nitrilase from Rhodococcus rhodochrous tg1-A6 in E. coli.
A DNA fragment containing the entire coding sequence of nitrilase gene was amplified from Rhodococcus rhodochrous tg1-A6 with high nitrilase activity using PCR and sequenced. The open reading frame of the nitrilase gene contains 1,101 base pairs, which encodes a putative polypeptide of 366 amino acid residues. The nitrilase gene was cloned into an expression vector pET-28a and expressed in an Escherichia coli strain BL21(DE3). The enzymatic activity of nitrilase, which converts various nitriles to the corresponding carboxylic acids, was detected to reach 24.5 U/ml at 9 h in the recombinant bacteria.
Luo
Hui
H
Department of Biological Science and Technology, University of Science and Technology Beijing, People's Republic of China. luohui99@yahoo.com.cn
Fan
Lu
L
Chang
Yanhong
Y
Ma
Jinwei
J
Yu
Huimin
H
Shen
Zhongyao
Z
D000619:Aminohydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D012240:Rhodococcus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity
D001426:Bacterial Proteins; D000619:Aminohydrolases; C021867:nitrilase