外源脂肪酶在毕氏酵母表面展示及发酵过程分析

The Surface Display of Lipase on a Pastoris pichia and Analysis of Fermention Process

分别考察基于絮凝素和凝集素2种不同展示系统,展示有外源脂肪酶CALB(Candidan Antarctica lipase B)的重组展示酵母的展示酶活、上清酶活,发现毕赤酵母作为宿主在外源脂肪酶展示过程会分泌目的蛋白至上清中,引起"外泄",进一步通过重组展示酵母外源蛋白鉴定、外源蛋白去糖基化、发酵过程中蛋白变化趋势观察,表明基于絮凝素系统的非共价锚定是引起展示过程中蛋白外泄比例过高的主要原因,研究为实现脂肪酶在毕赤酵母细胞的高效展示进而提高展示酶的催化效率提供了理论指导。

In this research, the activities of enzyme displaying on the surface ofpastorispichia and enzyme in the fermentation supematant of two recombinant displaying yeast were studied, which were based on two surface yeast display systems with the flocculation functional domain of FLOIp and α-agglutinin functional domain respectively. In the process of displaying the lipase on the yeast cell surface, some secreted lipases were found in supematants. With the identification of active lipase, the glycosylation assay and the mounts increase of foreign pmteirg the reason that interest lipase were secreted into the supematant to the great extent was restdted from unstable non-covalence bond between the flocculation functional domain of Flolp and mannose in the cell wall. The study provided theoretical guide for efficient display of lipase on a pastoris pichia and improve the catalytic activity of displaying enzymes.