前导肽对Aspergillus pseudoglaucus酸性蛋白酶App表达及功能的影响  

Effects of Propeptide on the Expression and Enzyme Function of Aspergillus pseudoglaucus Aspartic Protease App

作　　者：刘海燕 张荣珍[1] 李利宏 周丽仙 徐岩 LIU Haiyan;ZHANG Rongzhen;LI Lihong;ZHOU Lixian;XU Yan(School of Biotechnology,Jiangnan University,Wuxi 214122,China)

机构地区：[1]江南大学生物工程学院,江苏无锡214122

出　　处：《食品与生物技术学报》2020年第3期32-40,共9页Journal of Food Science and Biotechnology

基　　金：国家自然科学基金项目(31370100);江苏省六大人才高峰高层次人才资助项目(2015-SWYY-010);高等学校学科创新引智计划资助项目(111-2-06)。

摘　　要：从Aspergillus pseudoglaucus基因组中克隆酸性蛋白酶App基因,分别将其自身前导肽基因pro、来自Candida tropicalis的candidapepsin前导肽基因CP、Saccharomyces cerevisiae的proteinase A前导肽基因PP、Rhizomucor miehei的proteinase前导肽基因RP、Candida albicans的aspartic proteinase 2前导肽基因SP导入基因的5′端,获得含不同前导肽的App基因片段,于Escherichia coli中表达。SDS-PAGE结果显示,不加前导肽时,蛋白质不表达;酶活测定结果显示,加入自身前导肽时,蛋白质proApp在55℃、pH 2.8条件下有最大酶活质903 U/mg。而其他前导肽能使蛋白质表达但蛋白质无酶活。圆二色谱结果显示,前导肽能够改变蛋白质二级结构组成,使其无法正确折叠。乳蛋白水解结果显示,在55℃,pH 2.2条件下,proApp有最大水解活力252 U/mg。该研究表明特定前导肽对蛋白酶异源表达与水解功能的重要性,同时为酸性蛋白酶水解乳蛋白提供了良好的研究基础。The gene coding aspartic protease App was cloned from the genome of Aspergillus pseudoglaucus.Its self propeptide gene pro,the propeptide gene CP from Candida tropicalis candidapepsin,the gene PP from Saccharomyces cerevisiae proteinase A,the gene RP from Rhizomucor miehei proteinase,and the gene SP from Candida albicans aspartic proteinase 2 were cloned at the 5′-terminal of App gene.The different gene fragments including the different propeptides were obtained,and expressed in Escherichia coli.SDS-PAGE analysis showed that App could not expressed in E.coli without any propeptides at 5'-terminal App gene.When casein was used as substate,proApp with its self propeptide showed the highest specific activity of 903 U/mg at 55℃and pH 2.8.Although the App with propeptides of the other proteases were expressed at a high level in E.coli,they did not present enzyme activity towards casein.The results of circular dichroism showed that the different propeptides changed the constituents of the secondary structure of protease,and thus the protein were not correctly folded.With milk protein as the substrate,proApp showed the highest hyhrolytic activity at 55℃and pH 2.2.This work shows the important of the specific propeptide on heterologous expression and hydrolytic function of protease,and provides a good research basis for aspartic protease catalyzing the hydrolysis of milk protein.

关 键 词：酸性蛋白酶 前导肽 异源表达 蛋白质折叠 假灰绿曲霉 

分 类 号：Q78[生物学—分子生物学]