毕赤酵母展示表达南极假丝酵母脂肪酶B

将南极假丝脂肪酶B(CALB)基因N端和C端,分别与酿酒酵母絮凝蛋白(Flo1p)絮凝结构域序列的N端(FS)和C端(FL)融合,构建成脂肪酶毕赤酵母表面展示载体KFS和KFL,并转化毕赤酵母GS115后获得重组子KFS-CALB和KFL-CALB。免疫荧光检测证实脂肪酶已展示于毕赤酵母细胞表面。甲醇诱导120 h后展示酶活性分别达到286 U/g干细胞和182 U/g干细胞。酶的热稳定性较游离酶有较大提高,50℃孵育4 h后KFS-CALB菌株的残留酶活力仍保持初始酶活力70%以上;KFL-CALB在50℃孵育2 h后的酶活力也达到初始酶活力50%,远远高于游离态的CALB,其在50℃孵育0.5 h后仅残留18%的初始酶活力。

Display of Candida antarctica LipaseB on Cells Surface of Pichia pastoris

P.pastoris display vectors of lipase were constructed by fusing N-terminal and C-terminal of FLO-flocculation domain to N-terminal and C-terminal of Candida antarctica LipaseB,respectively.The recombinant vectors were named KFS and KFL,respectively,and then transformed into P.pastoris GS115.The recombinants were named KFS-CALB and KFL-CALB.The fluorescence microscopy of immunolabeled P.pastoris revealed the lipase was displayed on the cell surface.The hydrolysis activity of lipase displayed on P.pastoris cell surface reached 286 U/g dry cell and 182 U/g dry cell after 120 h of induction.The thermalstability of displayed lipase was improved comparing with free lipase which only 18% of initial activity after incubation at 50℃ for 0.5 h,the residual activity of KFS-CALB was above 70% of initial activity after incubation at 50℃ for 4 h,and the residual activity of KFL-CALB was above 50% of initial activity after incubation at 50℃for 2 h.