圆弧青霉BD26碱性脂肪酶基因的克隆及其在大肠杆菌中的表达

Cloning and Expression of Alkaline Lipase Gene from Penicillium cyclopium Mutant in Escherichia coli

采用反转录聚合酶链式反应(RT-PCR),以圆弧青霉BD26总RNA为模板,扩增出774 bp cDNA片段,将该基因片段克隆到表达载体pET-28a(+)上并转化Escherichia coil BL21(DE3),得到重组菌株BL21/ Lip PD。经IPTG诱导,菌体在固体琼脂显色平板上形成明显透明圈。SDS-PAGE电泳显示该脂肪酶分子质量约为27 ku。表达条件优化结果表明,当工程菌培养至OD_(600)为0.5时,加入IPTG至终浓度为1.0 mmol/L,在32℃诱导培养1 h,比酶活达29.30 U/mg。

The total RNA of Penicillium cyclopium BD26 was isolated by one step method with TRIzol reagent. The 774-bp Lip PD gene of lipase was amplified by RT-PCR. The target gene was inserted into the expression vector pET-28a, which was finally expressed in Escherichia coli BL21（DE3） by IPTG induction. The activity of the recombined lipase was analyzed according to the diameters of the halos on solid agar plate. SDS-PAGE analysis showed that the relative molecular mass of lipase was about 27 ku The activity of the recombinant lipase could be measured using the olive oil as the substrate. With the optimal induction temperature 32℃, strain density OD600 0.5, IPTG concentration 1.0 mmol/L and induction time 3h, the specific activity of the recombinant lipase was up to 29.30 U/mg.