β-脱卤酶的基因克隆表达及其酶学性质

Cloning, expression and characterization of β-dehalogenase

摘要:

根据GenBank中的序列设计引物, 克隆芽孢杆菌中的β-脱卤酶基因(命名为bhd)。以pET30a(+)为载体、Escherichia coli BL21(DE3)-CondonPlus为宿主菌, 实现了bhd的高效表达。使用HisTrapTMFF亲和层析柱纯化重组β-脱卤酶, 分子量约为23.1 kD。酶学性质研究表明, 纯化的重组β-脱卤酶水解3-氯丙酸制备3-羟基丙酸的最适反应体系为30 °C, 100 mmol/L, pH 7.0的磷酸钠缓冲液。在最适反应条件下, 重组β-脱卤酶的比活为16.2 U/mg, Km和Vmax分别为3.26 μmol/L和17.86 mmol/(min·g protein)。在最适反应条件下, 以10 mmol/L 3-氯丙酸为底物, 反应36 h的转化率在93%以上。

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.