假单胞菌AD39菌株三嗪水解酶基因trzN的克隆、表达和酶鉴定  

Cloning,Expression and Enzyme Characterization of Triazine Hydrolase Gene trzN from Pseudomonas sp. AD39

作　　者：赵雅芳[1,2] 赵化冰[1] 孙素菊[2] 蔡宝立[3] 

机构地区：[1]中国人民武装警察部队后勤学院,天津市职业与环境危害生物标志物重点实验室,天津300162 [2]河北医科大学公共卫生学院,河北石家庄050017 [3]南开大学生命科学学院,天津300071

出　　处：《南开大学学报（自然科学版）》2012年第6期1-6,共6页Acta Scientiarum Naturalium Universitatis Nankaiensis

基　　金：国家自然科学基金(40971183)

摘　　要：来自Pseudomonassp.AD39菌株的三嗪水解酶TrzN,能催化除草剂阿特拉津的水解脱氯反应,将阿特拉津转化成羟基阿特拉津.AD39菌株的TrzN以组氨酸标签蛋白的形式在大肠杆菌中表达,重组蛋白用镍柱亲和层析法纯化.纯化的TrzN蛋白能以阿特拉津、阿特拉通、杀草净、扑草净、莠灭净和西玛津等6种三嗪类除草剂为催化底物,其中对莠灭净的催化活力最高.酶学实验表明,催化反应的最适pH为8.0,Co2+、Fe2+、Ca2+、Mg2+和Mn2+等金属离子对TrzN有激活作用,Zn2+、Cu2+和Ni2+等金属离子对TrzN的酶活力有抑制作用.酶动力学实验显示,TrzN蛋白的Km为41.47μmol.L-1,Vmax为2.48μmol.min-1mg-1.The TrzN from Pseudomonas sp.AD39 is able to transform the herbicide atrazine to hydroxyatrazine by hydrolytic dechlorination reaction.The TrzN protein from strain AD39 was cloned and expressed in Escherichia coli as a His-tagged protein.The recombinant protein was purified via nickel column chromatography.The purified TrzN protein was tested with six s-triazine ring compounds（atrazine,atratone,dipropetry,prometryne,ametryn and simazine）.All of them were substrates and the highest activity was exhibited with ametryn.The enzymology experiments indicated that the optimal pH for TrzN catalysis reaction was 8.0,Co^2＋,Fe^2＋,Ca^2＋,Mg^2＋ and Mn^2＋ could increase TrzN activity,however,Zn2＋,Cu2＋ and Ni2＋ could inhibit TrzN activity.The enzyme kinetic experiments showed that Km and Vmax of TrzN protein were 41.47 μmol·L-1and 2.48 μmol·min-1·mg-1,respectively.

关 键 词：假单胞菌AD39 trzN表达 三嗪水解酶 酶学性质 酶动力学 

分 类 号：Q78[生物学—分子生物学]