Abstract
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.
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