甲基对硫磷水解酶的重组表达及其纯化和性质研究  

Recombinant Expression of Methyl Parathion Hydrolase Gene and Its Purification and Characterization

作　　者：傅国平[1] 崔中利[1] 徐玮[1] 吴旭平[1] 李顺鹏[1] 

机构地区：[1]南京农业大学农业部农业环境微生物工程重点开放实验室

出　　处：《微生物学报》2004年第3期356-360,共5页Acta Microbiologica Sinica

基　　金：国家自然科学基金 ( 3 0 3 0 0 0 0 5 ) ;江苏省自然科学基金 (BK2 0 0 10 71) ;国家"863计划"( 2 0 0 1AA2 460 71;2 0 0 1AA2 14 12 1) ;农业部跨越计划项目 ;国家科技攻关 ( 2 0 0 2BA5 16A0 1)~~

摘　　要：用PCR方法获得甲基对硫磷水解酶编码基因 ,构建了重组表达质粒pET2 9a_mpd ,将其转化至EscherichiacoliBL2 1 (DE3)中 ,经IPTG诱导表达 ,得到C 末端含有 6个寡聚组氨酸的甲基对硫磷水解酶 ,用Ni NTA亲和层析纯化得到具有活性的甲基对硫磷水解酶。测定了环境因素对酶活性的影响及酶动力学参数。甲基对硫磷水解酶水解甲基对硫磷时 ,最适pH8 6～ 8 8,最佳反应温度 1 5℃ ;Mn2 + 、Zn2 + 、Cu2 + 可使酶活性增加 1 5 %～ 2 0 % ,Ca2 + 、Mg2 + 微弱地促进酶的作用 ,Ni2 + 对酶活性几乎无影响 ;1mmol LEDTA·Na2 + 几乎不影响酶的活性 ,而 1 0mmol LEDTA·Na2 + 对甲基对硫磷水解酶有较强的抑制作用。甲基对硫磷水解酶水解乙基对硫磷时 ,最适pH8 6。 2 5℃时 ,该酶对甲基对硫磷的米氏常数Km 为 (6 8 6± 5 1 ) μmol L ,kcat为 (4 5± 6 )S- 1 ;对乙基对硫磷的米氏常数Km 为 (5 9 5± 6 0 )μmol L ,kcat为 (8± 1 )S- 1 。kcat KmThe encoding region of mpd gene for methyl parathion hydrolase was subcloned by PCR. The recombinant plasmid pET29a-mpd was constructed and 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. A convenient and reliable method to assay the activity of methyl parathion hydrolase was established. The effects of environmental factors on the activity of methyl parathion hydrolase and its enzymatic kinetics were analyzed. The optimum pH value for enzymatic hydrolysis of methyl parathion was 8 6～8 8, the optimum temperature was 15℃. Mn 2+, Zn 2+, Cu 2+ increased the activity of methyl parathion hydrolase by 15%～20%. Ca 2+, Mg 2+ and Ni 2+ decreased enzyme activity slightly. 1mmol/L EDTA·Na 2+ almost had no effect on the enzyme activity, but 10mmol/L EDTA·Na 2+ inhibited enzyme activity strongly. The K m and kcat against methyl parathion at 25℃ was (68 6 ± 5 1)μmol/L and (45 ± 6)S -1 respectively. The optimum pH value for hydrolysis of parathion by this hydrolase was also 8 6～8 8, the K m and kcat against parathion at 25℃ was (59 5 ± 6 0)μmol/L and (8 ± 1) S -1 respectively. The values of kcat/K m of methyl parathion hydrolase to two substrates showed that the enzyme hydrolyzes methyl parathion more efficiently.

关 键 词：甲基对硫磷水解酶 基因表达 Ni-NTA亲和层析 酶活性 

分 类 号：Q55[生物学—生物化学]