Cloning, expression and characterization of β-dehalogenase

Abstract:

The β-dehalogenase gene from Bacillus sp. was amplified by PCR with primers designed according to the sequence of the β-dehalogenase gene (named bhd) in GenBank. Then the bhd was overexpressed in Escherichia coli BL21(DE3)-CondonPlus with plasmid pET-30a(+). The recombinant β-dehalogenase (rBhd) was purified with HisTrapTMFF affinity chromatography and the molecular mass of the protein was about 23.1 kD. Further study on the enzymatic characteristics showed that, the hydrolysis reaction of 3-chloroproionic acid to 3-hydroxypropionic acid catalyzed by the purified rBhd should be carried out in 100 mmol/L sodium phosphate buffer at 30 °C. Under the optimum conditions, the specific activity, Km and Vmax of the enzyme were 16.2 U/mg, 3.26 μmol/L and 17.86 mmol/(min·g protein), respectively. When 10 mmol/L of 3-chloropropanoic acid was used as substrate, the conversion ratio reached 93% after reaction for 36 h.