一种新型有机磷降解酶的二异丙基氟磷酸酯水解特性研究

Characteristics of a novel organophosphorus pesticide-degrading enzyme on diisopropyl fluorophosphates

有机磷降解酶是降解有机磷化合物(包括神经毒剂)的一类重要酶.本实验室克隆了铜绿假单胞菌HS-D38的有机磷降解酶基因opd(organophosphate degradation),并经定点突变与原核表达获得了一种新型重组有机磷降解酶OPD.本文研究了该重组有机磷降解酶OPD的分离纯化,及其降解有机磷神经毒剂模拟剂二异丙基氟磷酸酯(diisopropyl fluorophosphate,DFP)的性质.研究结果表明,重组有机磷水解酶OPD经Ni-NTA亲和色谱纯化后,比活力达到了218U/mg.该酶的最适温度为35℃,最适pH为8.0;金属离子Zn2+、Cr2+、Co2+对酶活性有激活作用.重组有机磷水解酶OPD对DFP的Km为1.05mmol.L-1,Vm为0.218mmol.L-1.min-1.

Organophosphate degradation enzyme has been reported to play an important role in the decontamination of organophosphorus nerve agents.The opd（organophosphate degradation） genes from Pseudomonas aeruginosa HS-D38 has been cloned,mutated and expressed.In this paper,the recombinant enzyme OPD was purified to homogeneity by Ni-NTA affinity chromatography.Employing nerve agent-analog diisopropyl fluorophosphates（DFP） as a substrate,the specific activity of the purified protein reached at 218 U/mg.The OPD has optimum activity at 35℃ and pH 8.0.It is stimulated by 1.0 mM of Zn2＋,Cr2＋ and Co2＋.The Michaelis constant Km is 1.05 mmol·L-1,and Vmax is 0.218 mmol·L-1·min-1.