CAJ | 학술논문

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。
목적：극륭동고룡산균Y25적산리순탈경매기인sldh，재대장간균중진행표체병검측표체산물적활성。방법：이동고룡산균Y25기인조DNA위모판，PCR확증sldh기인，련접도표체재체pTIG，전입대장간균BL21（DE3）， IPTG유도표체；취표체균체、균체렬해상청화침정진행SDS-PAGE분석；이산리순위저물，통과활성전영、체외전화급휴지세포전화진행sldh기인표체산물적활성검측。결과：확증득도1740 bp적산리순탈경매기인，구건료표체질립pTIG-sldh병재대장간균중획득표체，SDS-PAGE결과현시표체산물위가용성형식，상대분자질량약58×103；활성전영결과설명표체산물재이산리순위저물시표현출탈경매활성，이경체외전화화휴지세포전화후박층층석검측출전화산물산리당적존재。결론：재대장간균중실현료동고룡산균산리순탈경매적가용성표체，차표체적중조탈경매능장산리순탈경생성산리당。
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.vul-gare 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 expres-sion 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 con-structed. 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-sorbi-tol to L-sorbose by dehydrogenation.