南极假丝酵母脂肪酶B基因在大肠杆菌中的表达和发酵优化

旨在利用大肠杆菌实现南极假丝酵母脂肪酶B(CALB)基因的高效可溶性表达,并降低生产成本。构建带有不同信号肽的CALB基因表达质粒,转化至不同大肠杆菌宿主中,在摇瓶中进行基础培养基、诱导条件、培养基组成成分和进程曲线的优化。结果显示,带有PelB信号肽的重组菌pET25b-CALB-1/Rosetta(DE3)在20℃下使用0.5%(W/V)乳糖在TY培养基中诱导表达效果最好,优化后的合成培养基成分为1.75%(W/V)山梨醇、2.25%(W/V)鱼蛋白胨、1%(W/V)安琪酵母提取物、0.75%(W/V)Na2HPO4。在摇瓶中诱导60 h后,CALB酶活力最高达到35.67 U/mL,相比于初始发酵酶活力提高了17.77倍,是目前大肠杆菌生产CALB的最高水平。成功地构建了大肠杆菌表达系统,经过系统优化后,CALB的高水平可溶性表达得以实现。

Expression and Fermentation Optimization of Candida antarctica Lipase B in Escherichia coli

In order to achieve efficient soluble expression of Candida antarctica lipase B(CALB)in Escherichia coli at lower cost,CALB gene expression plasmids with different signal peptides were constructed and transformed into different E.coli hosts,and then a series of fermentation experiments were employed in shaking flasks to optimize the culture medium types,inducing conditions,components of medium and time course of cultivation.The results showed that the recombinant strain pET25b-CALB-1/Rosetta(DE3)with PelB signal peptide was induced to have the best expression in TY medium at 20℃employing 0.5%(W/V)lactose.The optimal composition of fermentation medium was 1.75%(W/V)sorbitol,2.25%(W/V)fish peptone,1%(W/V)Angel yeast extract and 0.75%(W/V)Na2HPO4.The recombination system achieved a maximum CALB activity of 35.67 U/mL after induction for 60 h,which was 17.77 times higher than that before optimization,i.e.,the highest one of CALB produced by E.coli at present.In conclusion,the expression system in E.coli was successfully established,and the high-level soluble expression of CALB was obtained after system optimization.