氧化葡糖杆菌sndh-sdh基因簇的克隆表达

目的:从氧化葡糖杆菌H763中克隆sndh-sdh基因簇,在大肠杆菌和氧化葡糖杆菌621H中分别表达山梨酮脱氢酶-山梨糖脱氢酶(SNDH-SDH),并检测其活性。方法与结果:以氧化葡糖杆菌H763基因组DNA为模板,PCR扩增包括启动子、结构基因及终止序列在内的sndh-sdh基因簇,回收3533 bp的扩增产物,连入pMD18T载体,转化至大肠杆菌DH5α中表达;以山梨糖或木糖为底物,DCIP法检测菌体裂解液,DCIP检测液颜色由蓝绿色变为黄色,表明大肠杆菌表达产物具有脱氢酶活性。构建pBBR1MCS2-sndh-sdh载体,通过接合转移导入氧化葡糖杆菌621H,重组葡糖杆菌在以山梨醇或山梨糖为底物的培养基中培养,采用薄层层析检测法检测其培养上清中的代谢产物,层析板上显示了2-酮基-L-古龙酸斑点。结论:重组大肠杆菌DH5α和氧化葡糖杆菌621H中均表达了有脱氢酶活性的SNDH-SDH。

Cloning, Expression and Activity Analysis of sndh-sdh Gene Cluster of Gluconobacter oxydans

Objective: To clone the sndh-sdh gene cluster from Gluconobacter oxydans H763 and investigate the expression and biological activity of sorbosone dehydrogenase-sorbose dehydrogenase(SNDH-SDH) in E.coli DH5α and G.oxydans 621H.Methods & Results: The sndh-sdh gene cluster,including promoter,structural gene and ter minator sequence,was directly amplified using PCR method from G.oxydans H763 genome DNA.Then the 3533 bp amplification product was retrieved and ligated into the vector pMD18T and transformed into E.coli DH5α.Its expression and biological activity were examined by DCIP assay when sorbose or xylose was used as substrate.Af ter the cell lysis was added to the DCIP detection solution,its color changed from bluish green to yellow,which suggested that the expression product had dehydrogenase activity.The vector pBBR1MCS2-sndh-sdh was construct ed and conjugated into G.oxydans 621H.After the recombinant strain was cultivated in the medium contained sor bitol or sorbose as substrate,the metabolic products in the supernatant were assayed by thin-layer chromatogra phy,and chromatoplate showed the 2-keto-L-gulonic acid(2-KLG) spots.Conclusion: The above results indicated that the SNDH-SDH has expressed in E.coli DH5α and G.oxydans 621H.