重组卤醇脱卤酶的表达优化及纯化  

Optimizing expression and purification of recombinant halohydrin dehalogenase from A. radiobacter AD1

作　　者：李阳[1] 汤丽霞[1] 郑楷[1] 王雄[1] 江荣香[1] 

机构地区：[1]电子科技大学生命科学与技术学院,四川成都610054

出　　处：《化工学报》2010年第12期3213-3219,共7页CIESC Journal

基　　金：国家自然科学基金项目(20872014) ;教育部出国留学人员启动资金~~

摘　　要：卤醇脱卤酶可以催化合成具有光学纯的环氧化物及β-取代醇等一类高价值手性药物中间体。卤醇脱卤酶的高效重组表达及纯化可以更好地促进该酶的应用。通过PCR扩增,将编码卤醇脱卤酶的基因克隆至pBAD表达载体中,经测序鉴定后,将其转入大肠杆菌TOP10中,对其重组表达条件进行优化,以提高蛋白表达量。同时,对该酶在已构建好的T7表达体系中的表达条件也进行了优化。经酶活测定及SDS-PAGE分析,选择最优条件进行500ml摇瓶的表达,在两种表达体系中,可溶性目的蛋白的含量均大大提高,占总蛋白含量35%左右。采用Q-Sepharose阴离子交换层析技术一步纯化,分别获得纯度为90%的卤醇脱卤酶34mg和41mg。同时,pBADHheC表达体系的构建为卤醇脱卤酶的生物催化特性改造奠定了基础。Optically pure epoxides and β-substituted alcohols,constituting one of the most important classes of building blocks for the synthesis of chiral drugs,could be obtained via reactions catalyzed by halohydrin dehloagenases.Researches aimed at the improvement of over-expression and purification of recombinant halohydrin dehalogenases will greatly facilitate its application.The encoding gene was successfully cloned into pBAD expression vector.Under the developed optimized expression conditions,halohydrin dehalogenase could be expressed as a soluble format in E.coli using the two expression systems.The soluble targeted protein was about 35% of the total soluble proteins as judged by activity measurements and SDS-PAGE analysis.From 0.5 L cultures of each,34 mg and 41 mg of 90% pure halohydrin dehalogenase were obtained by using one-step Q-Sepharose anion exchange chromatography,respectively.Meanwhile,the constructed pBADHheC plasmid would set a solid base for improving the biocatalytic properties of the enzyme.

关 键 词：卤醇脱卤酶 高效表达 T7表达体系 pBAD载体 纯化 

分 类 号：Q814.1[生物学—生物工程]