利用α凝集素在毕赤酵母表面展示脂肪酶

Surface Display Lipase B of Candida Antarctica on Pichia pastoris by Using α-agglulinin

目的:将南极假丝酵母脂肪酶B(CALB)通过α凝集素3’末端功能区域展示在毕赤酵母表面。方法:采用PCR方法扩增得到CALB成熟肽编码基因,将其连接到α凝集素3’末端的上游再与穿梭载体pPIC9K连接,构建表面展示载体p KNS-CALB。检测其水解活力和相关酶学性质。结果:展示CALB的毕赤酵母在甲醇的诱导下,表现出水解活性,最高可达382 U/g干细胞。对展示CALB的酶学性质研究表明:其最适温度为45℃,最适pH为8.0,60℃水浴4 h后残留酶活力高于最大酶活力的50%,其水解对硝基苯酚丁酸酯的酶活力最高。结论:利用α凝集素成功将CALB展示于毕赤酵母表面,酶活力有较大提高。

Objective:Using 3'-terminal of α-agglulinin to display Lipase B of Candida antarctica(CALB) in P.pastoris.Method:The CALB and α-agglulinin encoded gene were amplified by PCR technique and recombinant into expression vector pPIC9K.The recombinant vector was named pKNS-CALB.The hydrolysis activity and its character of the displayed CALB were detected.Result:Using methanol to induce the CALB expression in P.pastoris.The hydrolysis activity of surface displayed lipase is 382U/g dry cell.The results have shown that the properties of displayed CALB is the optimum temperature of 45℃,optimum pH 8.0,50℃ incubation for 4h,the residual activity of the maximum activity of 50%.The displayed CALB has the highest activity for the p-nitrophenol.Conclusion:Construct of surface displaying CALB system by using α-agglulinin in P.pastoris successfully.However,the activity of displayed lipase has improved greatly.