Cloning, sequencing, and expression of a novel epoxide hydrolase gene from Rhodococcus opacus in Escherichia coli and characterization of enzyme.
An epoxide hydrolase gene of about 0.8 kb was cloned from Rhodococcus opacus ML-0004, and the open reading frame (ORF) sequence predicted a protein of 253 amino acids with a molecular mass of about 28 kDa. An expression plasmid carrying the gene under the control of the tac promotor was introduced into Escherichia coli, and the epoxide hydrolase gene was successfully expressed in the recombinant strains. Some characteristics of purified recombinant epoxide hydrolase were also studied. Epoxide hydrolase showed a high stereospecificity for L: (+)-tartaric acid, but not for D: (+)-tartaric acid. The epoxide hydrolase activity could be assayed at the pH ranging from 3.5 to 10.0, and its maximum activity was obtained between pH 7.0 and 7.5. The enzyme was sensitive to heat, decreasing slowly between 30 degrees C and 40 degrees C, and significantly at 45 degrees C. The enzyme activity was activated by Ca(2+) and Fe(2+), while strongly inhibited by Ag(+) and Hg(+), and slightly inhibited by Cu(2+), Zn(2+), Ba(2+), Ni(+), EDTA-Na(2) and fumarate.
Liu
Zhiqiang
Z
Institute of Bioengineering, Zhejiang University of Technology, Hangzhou, 310014, People's Republic of China. microliu@gmail.com
Li
Yin
Y
Xu
Yingying
Y
Ping
Lifeng
L
Zheng
Yuguo
Y
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004851:Epoxide Hydrolases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D007700:Kinetics; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012240:Rhodococcus / Q000201:enzymology* / Q000235:genetics; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity
D011994:Recombinant Proteins; D004851:Epoxide Hydrolases