甲基对硫磷水解酶纯化与保存

The Purification and Preservation of Methyl Parathion Hydrolase

对带有重组表达质粒E.coli菌种进行了扩大培养,在IPTG的诱导下大量表达,获得了C-末端含有6个寡聚组氨酸的甲基对硫磷水解酶。经Ni-NTA亲和层析获得了具有较高生物活性的甲基对硫磷水解酶。本文对该酶纯化的梯度洗脱条件和酶动力学进行了考察,并通过环境对酶活性的影响检测,确定了将酶制备成冻干粉,更利于保存。

Through the cultivation of E. coli strains with the restructuring of expression plasmid under the induced by isopropyl-β- D -galatose, the methyl parathion hydrolase was expressed in E. coli BL21 （DE3） as a fusion protein tagged with （His） 6 at C - terminus. The fusion protein was purified to homogeneity by Ni - affinity chromatography under undenaturing condition, the protein could degrade methyl parathion effectively. The gradient elution aconditions were de- termined to reduce the hybrid protein and improve the protein puriry, and the biological activity and enzyme kinetic of the methyl parathion hydrolase was detected. The effects of storage environmental factors on the activity of methyl parathion hydrolase were analyzed. The enzyme prepared by the freeze - dried powder was more conductive to the preservation.