Molecular cloning and characterization of a cis-epoxysuccinate hydrolase from Bordetella sp. BK-52.
A cis-epoxysuccinate hydrolase (CESH) from Bordetella sp. BK-52 was purified 51.4-fold with a yield of 27.1% using ammonium sulphate precipitation, ionic exchange, hydrophobic interaction, molecular sieve chromatograph and an additional anion exchange chromatography. The CESH was stable in a broad range of temperature (up to 50 degrees C) and pH (4.0-10.0) with optima of 40 degrees C and pH6.5, respectively. It could be partially inhibited by EDTA-Na2, Ag+, SDS and DTT, while slightly enhanced by Ba2+ and Ca2+. The enzyme exhibited high stereospecificity in D(-)-tartaric acid (enantiomeric excess value higher than 99 %) with Km and Vmax value of 18.67 mM and 94.34 micronM/min/mg for disodium cis-epoxysuccinate, respectively. The Bordetella sp. BK-52 CESH gene, which contained 885 nucleotides (open reading frame) encoding 294 amino acids with a molecular mass of about 32 kDa, was successfully overexpressed in Escherichia coli using a T7/lac promoter vector and the enzyme activity increased 42-times compared to original strain. It may be an industrial biocatalyst for the preparation of D(-)-tartaric acid.
Pan
Haifeng
H
College of Life Science, Zhejiang University, Hangzhou 310058, China.
Bao
Wenna
W
Xie
Zhipeng
Z
Zhang
Jianguo
J
Li
Yongquan
Y
D000595:Amino Acid Sequence; D001483:Base Sequence; D001884:Bordetella / Q000201:enzymology* / Q000235:genetics; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D006867:Hydrolases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D013237:Stereoisomerism; D013644:Tartrates / Q000378:metabolism*
D004269:DNA, Bacterial; D013644:Tartrates; D006867:Hydrolases; C029768:tartaric acid