Cloning，Analysis and Expression of a Novel Epoxide Hydrolase Gene（Vreh3） from Vigna radiata

Abstract:

Epoxide hydrolase can be effectively used in the preparation of optically active epoxides and vicinal diols，which are versatile intermediates for the preparation of highly value-added bioactive compounds such as pharmaceuticals. In this study，a gene encoding a novel epoxide hydrolase（VrEH3） was successfully cloned from Vigna radiata by RT-PCR and T-hairpin structure-mediated PCR amplification（THSO-PCR） techniques. The cloned coding region DNA sequence of Vreh3 is 1 178 bp in length，harboring two introns with sizes of 142 bp and 79 bp and a 957 bp open reading frame that encodes a 318-aa VrEH3. The theoretical molecular weight and calculated pI of VrEH3 were 36.2×103 and 5.59，respectively. The catalytic active center was constituted by a strictly conserved catalytic triad of Asp101-Asp262-His297. Subsequently，the expression plasmid pET-28a（+）-Vreh3 was constructed by ligated Vreh3 with pET-28a（+） and transformed into Escherichia coli BL21（DE3），forming a recombination strain E. coli/Vreh3. Using the E. coli/Vreh3 as the catalysis，the（R）-1-phenyl-1，2-ethanediol was obtained with a yield of 79.4% and an enantiomeric excess value of 94.7% by enantioconvergent hydrolysis of（R，S）-styrene oxide.