酿酒酵母表面展示技术表达酸性蛋白酶  

Expression of Acid Protease Using Surface Display Technology of Saccharomyces cerevisiae

作　　者：黄蓉 董超 余鸿飞 秦义[1] 刘延琳[1] 宋育阳[1] Huang Rong;Dong Chao;Yu Hongfei;Qin Yi;Liu Yanlin;Song Yuyang(College of Enology,Northwest A&F University,Yangling 712100,Shaanxi)

机构地区：[1]西北农林科技大学葡萄酒学院,陕西杨凌712100

出　　处：《中国食品学报》2022年第2期114-122,共9页Journal of Chinese Institute Of Food Science and Technology

基　　金：国家重点研发计划项目(2019YFD1002500);宁夏回族自治区重大研发计划项目(2020BCF01003);财政部和农业农村部:国家现代农业产业技术体系资助。

摘　　要：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,利用同源重组定点整合酸性蛋白酶基因到酿酒酵母的基因位点。经过PCR和测序验证,获得两株可细胞表面展示酸性蛋白酶的单倍体和二倍体的重组菌株,其最高酶活分别是285.71 U/mL和495.24 U/mL。本研究成功构建了利用酿酒酵母细胞壁蛋白SED1作为锚定蛋白的展示系统,为解决葡萄酒中蛋白浑浊的问题提供新思路,为实现pepA酸性蛋白酶全细胞催化剂工业化应用奠定理论基础。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.

关 键 词：酸性蛋白酶 酵母表面展示 蛋白沉淀 酿酒酵母 稳定性 

分 类 号：TS261.1[轻工技术与工程—发酵工程]