毕赤酵母展示表达南极假丝酵母脂肪酶B  

Display of Candida antarctica LipaseB on Cells Surface of Pichia pastoris

作　　者：苏国栋[1] 张少平[2] 尹钰[1] 林影[3] 

机构地区：[1]聊城大学生命科学学院生物工程实验室,聊城252004 [2]肇庆学院生命科学学院,肇庆526061 [3]华南理工大学生物科学与工程学院,广州510006

出　　处：《生物技术通报》2012年第8期107-112,共6页Biotechnology Bulletin

基　　金：国家自然科学基金项目(20976062);聊城大学博士科研究启动基金项目(31805)

摘　　要：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。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 fuorescence 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.

关 键 词：表面展示 脂肪酶 絮凝素 热稳定性 

分 类 号：Q78[生物学—分子生物学]