【摘要】 通过特定的引物,以土壤宏基因组DNA为模板,采用PCR技术扩增获得3.3 kb的DNA片段,该片段连接于pSE380载体构建重组质粒pSE380-dhaB12用于测序和表达。序列分析表明,该DNA片段编码的氨基酸序列与已报导的丁酸梭菌不依赖辅酶B12甘油脱水酶的相似性达99%。通过IPTG诱导,dhaB12基因在大肠杆菌中表达成功,SDS-PAGE分析表明有88 kD和34 kD两条蛋白质条带,在没有辅酶B12存在的情况下,所表达的酶具有明显的甘油脱水酶活性。更多还原

【Abstract】 With a given primer pair,a DNA segment of 3.3 kb was amplified by polymerase chain reaction(PCR) using a metagenomic DNA of a soil sample as template,and then cloned in vector pSE380,forming recombinant plasmids pSE380-dhaB12 for sequencing and expression.Sequence analysis indicated that the amino acid sequences encoded by this segment was 99% identical to that reported for coenzyme B12-independent glycerol dehydratase of Clostridium butyricum.The dhaB12 genes were successfully expressed in Escherichia coli by induction of isopropyl-beta-D-thiogalatopyranoside(IPTG),and synthesis of two proteins at 88 kD and 34 kD were determined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE).The expressed enzyme showed significant glycerol dehydratase activity in the absence of coenzyme B12.更多还原