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 sulfate precipitation, ionic exchange, hydrophobic interaction, molecular sieve chromatography and an additional anion-exchange chromatography. The CESH was stable in a broad range of temperature (up to 50°C) and pH (4.0-10.0) with optima of 40°C and pH 6.5, respectively. It could be partially inhibited by EDTA-Na 2 , Ag + , SDS, and DTT, and slightly enhanced by Ba 2+ and Ca 2+ . The enzyme exhibited high stereospecificity in D(-)-tartaric acid (enantiomeric excess value higher than 99%) with K m and V max values of 18.67 mM and 94.34 μM/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 was increased 42-times compared with the original strain. It may be an industrial biocatalyst for the preparation of D(-)-tartaric acid.