酮古龙酸菌山梨酮脱氢酶的原核表达及活性鉴定

目的:克隆酮古龙酸菌Y25的山梨酮脱氢酶基因sndh2,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sndh2基因,连接到pET22b表达载体后转入大肠杆菌BL21(DE3)中,经IPTG诱导表达;对菌体裂解液进行SDS-PAGE分析;以D-木糖为底物,采用非变性聚丙烯酰胺凝胶电泳后活性染色及DCIP检测法鉴定表达产物的脱氢酶活性。结果:扩增得到1290 bp的山梨酮脱氢酶基因;构建了表达质粒pET22b-sndh2,SDS-PAGE结果显示获得相对分子质量为43.1×103的可溶性表达产物;非变性聚丙烯酰胺凝胶电泳胶上出现的蓝黑色条带及DCIP检测液颜色的变化说明表达产物在以D-木糖为底物时表现出脱氢酶活性。结论:在大肠杆菌中表达的山梨酮脱氢酶具有生物活性。

Prokaryotic Expression and Enzyme Activity Identification of L-Sor bone Dehydrogenase of Ketogulonigenium vulgare

Objective: To clone a L-sorbone dehydrogenase(SNDH) gene sndh2 from Ketogulonigenium vulgare Y25 and to investigate its expression in Escherichia coli and biological activity.Methods: The sndh2 gene was am plified by using PCR method from K.vulgare Y25 genome,and was constructed into the expression vector pET22b.The plasmid was transformed into BL21(DE3),and induced by IPTG.Then the whole cell lysate was analyzed by SDS-PAGE.The biological activity of the expression product was examined by native-PAGE and DCIP assay when D-xylose was used as a substrate.Results: The length of sndh2 gene was 1290 bp as expected.The expres sion plasmid pET22b-sndh2 was correctly constructed.SDS-PAGE analysis revealed the expression protein existed in supernatant with a molecular weight of 43.1 kD.The black-blue band on native-PAGE gel after active stain and the color change of DCIP detection solution as well demonstrated that the expression product had dehydroge nase activity when D-xylose was used as a substrate.Conclusion: SNDH2 expressed in E.coli BL21(DE3) has bioactivity.