CAJ | 학술논문

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。
통과특정적인물，이토양굉기인조DNA위모판，채용PCR기술확증획득3.3 kb적DNA편단，해편단련접우pSE380재체구건중조질립pSE380-dhaB12용우측서화표체。서렬분석표명，해DNA편단편마적안기산서렬여이보도적정산사균불의뢰보매B12감유탈수매적상사성체99%。통과IPTG유도，dhaB12기인재대장간균중표체성공，SDS-PAGE분석표명유88 kD화34 kD량조단백질조대，재몰유보매B12존재적정황하，소표체적매구유명현적감유탈수매활성。
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 iso-propyl-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.