有机磷农药降解菌YC-YH1中mpd和ophc2的克隆及酶学性质分析  

Cloning and Characterization of mpd and ophc2 from an Organophosphorus Pesticide-degrading Bacteria YC-YH1

作　　者：贾阳[1] 史延华[1] 任磊[1] 乔铖 王俊欢[1] 闫艳春[1] 

机构地区：[1]中国农业科学院研究生院,北京100081

出　　处：《生物技术通报》2015年第11期228-235,共8页Biotechnology Bulletin

基　　金：国家自然科学基金项目(31170119);中国农业科学院基础研究基金项目(0042011006;0042012003;0042014011)

摘　　要：从一株有机磷农药降解菌施氏假单胞菌Pseudomonas stutzeri YC-YH1中克隆到两个有机磷水解酶基因mpd和ophc2。将两个基因分别连接到表达载体p ET-32a并克隆到大肠杆菌BL21(DE3)中进行表达;利用Ni亲和层析柱纯化甲基对硫磷水解酶(MPH)和有机磷水解酶(OPHC2),并进行了酶学性质分析。MPH最高活力的反应温度为40℃,在p H8-12范围内,酶表现出较高的活力。OPHC2酶的最适反应温度为30℃,在p H 8-12的范围内酶活力较高。两种酶按1?1比例混合组成的混合酶在30-40℃范围内酶活力很高,达95%以上,混合酶在p H范围为8-12内仍具有较高的活力。多种金属离子会对酶的活性产生一定影响,酶对SDS和EDTA都有很高的敏感度,混合酶能够降低对金属离子的敏感性。We cloned two organophosphorus hydrolase genes mpd and ophc2 from an organophosphorus pesticide-degrading bacteria Pseudomonas stutzeri YC-YH1. The two genes were inserted into the expression vector p ET-32 a and transformed into E. coli BL21（DE3）respectively. We used Ni-affinity column to purify enzymes and then study the nature of the two enzymes. It was suggested that the optimal temperature of MPH is 40℃, and the MPH displays high activity in the p H range of 8 to 12. The optimal temperature of OPHC2 is 30℃. In the p H ranging of 8 to12, OPHC2 has higher activity. The mixture of MPH and OPHC2 in the proportion of 1？1 showsed higher activity in the range of 30℃ to 40℃, the enzyme showsed more than 95% activity in the range of p H8-12. A variety of metalions affect the activity of the enzymes and the enzymes are sensitive to SDS and EDTA, but the compound could reduce the sensitivity to the metalions.

关 键 词：有机磷水解酶基因 MPH OPHC2 纯化 酶学性质 

分 类 号：X592[环境科学与工程—环境工程] X172