木薯醇腈酶在枯草芽孢杆菌中的表达及活性检测  

Expression and characterization of hydroxynitrile lyase from Manihot esculenta in Bacillus subtilis

作　　者：张冬冬[1] 郭晓军[1] 刘慧娟[1] 雷白时[1] 牛振东[2] 朱宝成[1] 

机构地区：[1]河北农业大学生命科学学院,河北保定071001 [2]中国科学院微生物研究所,北京100091

出　　处：《河北农业大学学报》2009年第3期59-62,66,共5页Journal of Hebei Agricultural University

基　　金：河北省自然科学基金项目(398152)

摘　　要：醇腈酶(α-hydroxynitrile lyase,HNL)能够催化羰基化合物和HCN立体选择性的加工形成手性醇腈化合物。以含有木薯醇腈酶基因的重组质粒pET28a-HNL为模板,应用PCR扩增得到HNL基因,将其连接到pMD18-T载体中进行测序分析,然后通过Nde I和Xba I2个酶切位点将其连接到枯草芽孢杆菌表达载体pMA5Z2中,获得了含有HNL基因的重组质粒pMA5Z2-HNL,转化蛋白质三缺陷的枯草芽孢杆菌DB1342。SDS-PAGE显示HNL在枯草芽孢杆菌DB1342中获得表达,产物分泌到细胞外,每毫升发酵液中获得了2.108 U的醇腈酶。Hydroxynitrile lyase （HNL） can catalyze carbonyls and HCN which stereoselectively manufacture and form chiral cyanohydrin components. HNL gene was amplified by PCR using plasmid pET28a- HNL which contain the gene of hydroxynitrile lyase from Manihot esculenta as template and then ligated into pMD18 - T vector in order to execute sequence analysis. Then the amplified DNA fragment was ligated into the Bacillus subtilis expression vector pMA5Z2 using Nde Ⅰ and Xba Ⅰ. The recombinated plasmid pMA5Z2 - HNL was obtained and then transformed into triple- protease deficient Bacillus subtilis strain DB1342 competent cells. The result of SDS- PAGE showed that HNL was expressed in Bacillus subtilis strain DB1342 and the product secreted into the medium. Bioactive hydroxynitrile lyase was obtained with the enzyme activity about 2. 108 U/ mL determined by analysis of enzyme activity.

关 键 词：木薯醇腈酶 枯草芽孢杆菌 分泌表达 活性 

分 类 号：Q786[生物学—分子生物学]