Purification and characterization of a cis-epoxysuccinic acid hydrolase from Nocardia tartaricans CAS-52, and expression in Escherichia coli

A highly enantioselective cis-epoxysuccinic acid hydrolase from Nocardia tartaricans was purified to electrophoretic homogeneity. The enzyme was purified 184-fold with a yield of 18.8 %. The purified cis-epoxysuccinic acid hydrolase had a monomeric molecular weight of 28 kDa, and its optimum conditions were 37 °C and pH 7–9. With sodium cis-epoxysuccinate as the substrate, Michaelis–Menten enzyme kinetics analysis gave a Km value of 35.71 mM and a Vmax of 2.65 mM min^?1. The enzyme was activated by Ni²⁺ and Al³⁺, while strongly inhibited by Fe³⁺, Fe²⁺, Cu²⁺, and Ag⁺. The cis-epoxysuccinic acid hydrolase gene was cloned, and its open reading frame sequence predicted a protein composed of 253 amino acids. A pET11a expression plasmid carrying the gene under the control of the T7 promoter was introduced into Escherichia coli, and the cis-epoxysuccinic acid hydrolase gene was successfully expressed in the recombinant strains.