带His-tag的解脂耶氏酵母脂肪酶Lip2在毕赤酵母中的表达及纯化

将去自身信号肽并且N-端带6×His标签的YlLip2基因克隆至表达载体pPIC9K中,电转化GS115获得高效表达脂肪酶His₆-YlLip2的基因工程菌。筛选到的阳性克隆子摇瓶发酵脂肪酶活力最高为400U/ml。对重组毕赤酵母在10 L发酵罐中表达His₆-YlLip2的分批补料发酵工艺进行了初步优化,探讨了培养基、pH、温度对生物量和重组蛋白表达量的影响。结果表明:采用FM22培养基,诱导温度为25℃,pH 5.0,甲醇诱导114 h后His₆-YlLip2的最高酶活力达到3160U/ml。SDS-PAGE分析表明,蛋白的分子量大约为38kDa。重组的His₆-YlLip2经镍柱一步纯化后的纯度达到95.43%,比酶活达到4250U/mg。

High-level Expression and Purification of Yarrowia lipolytica Lipase Lip2 with Six Hisditine Tags in Pichia pastoris

The mature Y. lipolytica lipase Lip2 gene without the signal sequence and with a 6×His-tag in N-terminal,was ligated into vector pPIC9K. The recombinant plasmid pPIC9K-Lip2H was linearized by rectriction enzyme Sal I,and then transformed into Pichia pastoris GS115 by electroporation. A clone exhibiting a maximum lipase activity of 400U/ml in shaking flask was chosen for 10 L bioreactor cultivation. The fed-batch fermentation process was preliminarily optimized.With respect to the effects on biomass and the expression level of His₆-YlLip2,the optimal basal salt medium was FM22,and the optimal parameters for induction pH and temperature were 5.0,25℃,respectively. After 114h methanol induction,the lipase activity of His₆-YlLip2 reached the highest value of 3160U/ml. SDS-PAGE analysis showed that the molecular weight of the aimed protein was about 38 kDa. Using a Ni-NTA affinity chromatography,the purity of recombinant His₆-YlLip2 reached 95.43% by one-step purification with the specific activity of 4250U/mg.