Purification,Gene Cloning,and Characterization of a Novel Halohydrin Dehalogenase from Agromyces mediolanus ZJB120203

A novel halohydrin dehalogenase (HHDH), catalyzing the transformation of 1,3-dichloro-2-propanol (1,3-DCP) to epichlorohydrin (ECH), was purified from Agromyces mediolanus ZJB120203. The molecular mass of the enzyme was estimated to be 28 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A 735-bp nucleotide fragment was obtained based on the N-terminal and internal amino acid sequences of the purified HHDH. The gene codes a protein sequence with 244 amino acid residues, and the protein sequence shows high similarity to Hhe A_AD2 (HHDH from Arthrobacter sp. AD2), defined as Hhe A_Am, which is the seventh reported HHDH. Expression of Hhe A_Am was carried out in Escherichia coli and purification was performed by nickel-affinity chromatography. The recombinant HheA_Am possessed an optimal pH of 8.5 and an optimal temperature of 50 °C and manifested a K _m of 4.58 mM and a V _max of 3.84 μmol/min^/mg. The activity of Hhe A_Am was not significantly affected by metal ions such as Zn²⁺, Ca²⁺, Cu²⁺, and EDTA, but was strongly inhibited by Hg²⁺ and Ag⁺. In particular, the Hhe A_Am exhibits an enantioselectivity for the conversion of prochiral 1,3-DCP to (S)-ECH. The applications of the Hhe A_Am as a catalyst for asymmetric synthesis are promising.