铜绿假单胞菌磷脂酶C的重组表达、复性及酶学性质

Expression,renaturation and characterization of Pseudomonas aeruginosa phospholipase C

为进一步研究磷脂酶C(phospholipase C)的性质,以一株实验室前期验证的产磷脂酶C铜绿假单胞菌(Pseudomonas aeruginosa)的染色体为模板,扩增得到磷脂酶C的编码基因PAplc。构建重组大肠杆菌表达质粒pET28a-PAplc,并转化大肠杆菌BL21(DE3)。SDS-PAGE分析发现,重组PAplc蛋白主要以包涵体形式出现,分子量8.5×10^(4)。以直接稀释法复性,蛋白回收率27.5%,纯度大于98%。以p-NPPC法测得重组PAplc比酶活为63.79 U/mg,最适反应温度为60℃,最适反应pH为7.5;温度低于50℃、pH为7.0~9.0时重组PAplc比较稳定。Zn^(2+)、Cu^(2+)和Co^(2+)对重组PAplc酶活有明显的抑制作用;低浓度Mg^(2+)、Ca^(2+)、Mn^(2+)对其酶活有促进作用。

To characterize phospholipase C,the phospholipase C gene PAplc was amplified by using a laboratory-proven chromosome of the phospholipase C(Pseudomonas aeruginosa).Recombinant expression plasmid pET28a-PAplc was constructed and transformed into E.coli BL21(DE3).SDS-PAGE analysis show that the recombinant PAplc protein occurred mainly in the form of inclusion bodies with a molecular weight of 8.5×10^(4).The refolding of the target protein was achieved by direct dilution method with the recovery of 27.5%and the purity more than 98%.The recombinant PAplc activity was 63.79 U/mg by p-NPPC method.The optimum reaction temperature was 60℃,and the optimum pH was 7.5.The enzyme was stable below 50℃,and pH between 7.0 and 9.0.Metal ions like Zn^(2+),Cu^(2+)and Co^(2+)inhibited the recombinant PAplc activity,whereas ions of Mg^(2+),Ca^(2+)and Mn^(2+)at low concentrations promoted the activities of the enzyme.