酮古龙酸菌山梨醇脱氢酶的原核表达及活性检测  

Prokaryotic Expression and Enzyme Activity Analysis of a D-Sorbitol Dehydrogenase of Ketogulonigenium vulgare

作　　者：赵楠[1,2] 熊向华[1] 葛欣[1] 汪建华[1] 夏焕章[2] 张惟材[1] 

机构地区：[1]军事医学科学院生物工程研究所,北京100071 [2]沈阳药科大学,辽宁沈阳110016

出　　处：《生物技术通讯》2013年第6期810-813,共4页Letters in Biotechnology

基　　金：国家自然科学基金(31100024)

摘　　要：目的:克隆酮古龙酸菌Y25的山梨醇脱氢酶基因sldh,在大肠杆菌中进行表达并检测表达产物的活性。方法:以酮古龙酸菌Y25基因组DNA为模板,PCR扩增sldh基因,连接到表达载体pTIG,转入大肠杆菌BL21(DE3),IPTG诱导表达;取表达菌体、菌体裂解上清和沉淀进行SDS-PAGE分析;以山梨醇为底物,通过活性电泳、体外转化及休止细胞转化进行sldh基因表达产物的活性检测。结果:扩增得到1740 bp的山梨醇脱氢酶基因,构建了表达质粒pTIG-sldh并在大肠杆菌中获得表达,SDS-PAGE结果显示表达产物为可溶性形式,相对分子质量约58×103;活性电泳结果说明表达产物在以山梨醇为底物时表现出脱氢酶活性,而经体外转化和休止细胞转化后薄层层析检测出转化产物山梨糖的存在。结论:在大肠杆菌中实现了酮古龙酸菌山梨醇脱氢酶的可溶性表达,且表达的重组脱氢酶能将山梨醇脱氢生成山梨糖。Objective： To clone a D-sorbitol dehydrogenase（SLDH） gene sldh from Ketogulonigenium vulgare Y25 and investigate its expression and biological activity in E.coli. Methods： The sldh gene was amplified from K.vulgare Y25 genome, then constructed into the pTIG vector. After the expression bacteria strain E.coli BL21（DE3） transformed with the recombinant plasmid was induced by IPTG, the whole cell, the supernatant and pellet of the cellular lysate were examined by SDS-PAGE. Using D-sorbitol as a substrate, the biological activity of the expression product was analyzed by Native-PAGE, the bioconversion in vitro and resting cells assay. Results： The length of amplified fragment was 1740 bp as expected and the expression plasmid pTIG-sldh was successfully constructed. It was revealed that the molecular weight of soluble expression protein band was about 58 kD by SDS-PAGE analysis. The result of Native-PAGE demonstrated that the expression product possesses dehydrogenase activity. In the bioconversion in vitro and resting cell experiment, L-sorbose was detected through the TLC assay. Conclusion： SLDH from K.vulgare was successfully solubly expressed in E.coli BL21（DE3） and transform D-sorbitol to L-sorbose by dehydrogenation.

关 键 词：酮古龙酸菌 山梨醇脱氢酶 原核表达 生物活性 活性电泳 

分 类 号：Q78[生物学—分子生物学]