酿酒酵母表面展示技术表达酸性蛋白酶

为解决白葡萄酒蛋白不稳定性问题,实现酸性蛋白酶的利用与酒精发酵的同步进行,本研究以酿酒酵母单倍体菌株BY4741和二倍体菌株82-9-35为宿主菌,通过酵母表面展示系统中的启动子和锚定蛋白的作用在酵母细胞表面表达酸性蛋白酶.将来源于宇佐美曲霉酸性蛋白酶基因(pepA)克隆,构建表达酸性蛋白酶的细胞展示盒,利用同源重组定...

Expression of Acid Protease Using Surface Display Technology of Saccharomyces cerevisiae

In order to solve the protein instability problem of white wine and to realize the utilization of acid protease and alcohol fermentation simultaneously,this study used Saccharomyces cerevisiae haploid strain BY4741 and diploid strain 82-9-35 as hosts to express acid protease on the yeast cell surface by the action of promoter and anchor protein in the yeast surface display system.The acid protease gene(pepA)from Aspergillus usamii was cloned,a cell display cassette expressing acid protease was constructed,and homologous recombination was used to integrate the acid protease gene into the gene locus of S.cerevisiae.Through PCR and sequencing validation,two haploid and diploid recombinant strains with the highest enzyme activity of 285.71 U/mL and 495.24 U/mL,respectively,were obtained for the cell surface display of acid protease.This study successfully constructed a new idea of using SED1 as an anchoring protein display system to solve the protein turbidity problem in wine,and laid the theoretical foundation for the industrial application of pepA acidic protease whole cell catalyst.