卡门柏青霉-PG3脂肪酶基因的克隆、表达及活性分析

以卡门柏青霉-PG3为出发菌株，提取其总RNA，应用反转录-聚合酶链式反应(RT-PCR)扩增出860bp左右的片段，核苷酸序列分析表明其与甘油单-双酰酯脂肪酶(MDGL)基因（mdlA）一致。将此基因克隆到原核表达载体pET-28a中，转化大肠杆菌BL21(DE3)，经诱导后，重组的脂肪酶（rMDGL）在宿主菌中得到表达，表达量可达菌体总蛋白量的45.2%。重组蛋白包涵体溶解、复性后用Ni²⁺组氨酸结合树脂螯合层析柱纯化，聚丙烯酰胺凝胶电泳(SDS-PAGE)显示为单一区带，其相对分子质量为41 000。以橄榄油为底物时没有检测到酶活性，以单硬脂酸甘油脂为底物时酶活性达到225nmol/s。该基因在原核系统中表达仍具有酶活性，说明MDGL的糖基化对其生物学活性并不是必不可少的。

Expression of gene encoding lipase from Penicillium camembetlii-PG3 in E. coli and its specific activtity

860bp cDNA fragment was amplified by RT-PCR method using total RNA extracted from Penicillium camembetlii-PG3. Analysis of nucleotide sequence of the fragment suggested that it was the same as the gene(mdlA) encoding mono-and diacylglycerol lipase(MDGL). The target gene was then inserted into the expression vector pET-28a and expressed in E.coli BL21 with IPTG induction. The recombinant protein(rMDGL) was highly expressed as an inclusion body and accumulated up to 45.2% of bacterial total protein. SDS-PAGE analysis showed that the relative molecular mass of rMDGL was about 41000. No activity of the purified rMDGL was detected towards olive oil and 225nmol/s was reached when using monostearoylglycerol as substrate. Analysis of biology activity of rMDGL suggested that glycolsylation might be not essential for MDGL.