环亚酰胺水解酶在大肠杆菌中的重组表达与纯化  

Expression and Purification of Cyclic Imide Hydrolase in E.coli

作　　者：史鹏[1] 张学尧[1] 王丽丽[1] 石亚伟[1] 

机构地区：[1]教育部化学生物学与分子工程重点实验室山西大学生物技术研究所,山西太原030006

出　　处：《山西大学学报（自然科学版）》2008年第2期252-257,共6页Journal of Shanxi University(Natural Science Edition)

基　　金：山西省自然科学基金(20031042);山西省高校科技开发项目(20051204)

摘　　要：以重组质粒pEI为模板，PCR扩增环亚酰胺酶基因序列，插入pMAL—s表达载体中，重组质粒转入E．ColiBL21（DE3）后，经IPTG诱导可溶性表达融合蛋白MBP—CIH，利用Amylose亲和柱一步纯化获得MBP—CIH，再经人鼻病毒3C蛋白酶切除亲和臂MBP，硫酸铵沉淀、Phenyl—Sepharose疏水层析获得电泳纯的CIH，纯化倍数为12．7．活力回收29％，比活为36．3U／mg．重组环亚酰胺水解酶的最适pH为8．0～9．0，最适温度50℃，在温度低于55℃时酶的性质相对稳定，Ni^2＋、Co^2＋能显著提高酶活力，Mn^2＋、Ca^2＋和Mg^2＋对酶活没明显有影响，Cu^2＋、Zn^2＋、Cd^2＋、Fe^2＋以及金属螯合剂8-羟基喹啉、EDTA则对酶有不同程度的抑制作用．初步说明该酶是一个金属依赖性酶。The gene of cyclic imide hydrolase was amplified by PCR with the recombinant plasmid pEI as the template and inserted into the vector pMAL-s. The recombinant pMAL-s-cih was transferred into E. coli BL21, and the target protein was expressed as soluble form by IPTG induction. A purification procedure for the CIH was performed by a three-step. Amylose affinity column, human rhinovirus 3C protease digestion and ammonium sulfate fractionation, followed by Phenyl-Sepharose hydrophobic interaction chromatography. With the above processes,the overall recovery of enzymatic activity was 29% and the specific activity for substrate DL-hydantoin achieved 36.4 U/mg with more that 95% purity as estimated by SDS-PAGE analysis. The optimal pH of the CIH is 8. 0-9. 0. The enzyme activity is stable at 55 ℃ ,and its optimal temperature is at 50 ℃. Divalent metal ions,including Ni^2＋ ,Co^2＋ obviously enhanced the enzymatic activity,and Mn^2＋, Ca^2＋ and Mg^2＋ had no obviously influence,while Cu^2＋, Zn^2＋ ,Cd^2＋ and Fe^2＋ to varies extents had the inhibitory effect. Metal cheators such as 8-HQSA and EDTA also were as the inhibitory with different levels.

关 键 词：环亚酰胺水解酶 亲和层析 人鼻病毒3C蛋白酶 疏水层析 

分 类 号：Q814[生物学—生物工程]