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.
出处
《河北农业大学学报》
CAS
CSCD
北大核心
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)
关键词
木薯醇腈酶
枯草芽孢杆菌
分泌表达
活性
hydroxynitrile lyase from Manihot esculenta
Bacillus subtilis
secreted expression
activity
