Gene Cloning, Sequence Analyais and Gene Expression of A New Halohydrin Dehalogenase

Abstract:

Halohydrin dehalogenase catalyzes the synthesis of optically pure epoxides,β-substituted alcohol and other similar high-value chiral drug intermediates. Our laboratory screened an encoding gene Y from contaminated soil in Wuxi new district industrial park using metagenomic technology. The gene Y contains 765 bp nucleotides and encodes 254 amino acids. According to the Blast analysis,the amino acids sequence shares 81% homology with HheC from Agrobacterium tumefaciens and also has Ser132-Thr145-Arg149 catalytic triad which is the same with the known Halohydrin dehalogenases. Thus,we speculated it belongs to HheC. According to Escherichia coli BL21 codon preference,the optimized gene was synthesized and named as SyhheC. The constructed expression plasmid pET28a-SyhheC was transformed into E. coli BL21. The expressed enzyme with an apparent molecular weight of 34 000 by SDS-PAGE analysis was purified to homogeneity using Ni-NTA affinity chromatography and characterized using the substrate of 2-bromo-1-(4-nitrophenyl)ethanol. The activity of purified reSyHheC was 5.2 U/mg. This study provides a new strategy to excavate new enzymes with excellent characters from the soil.