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 ofAspergillus pseudoglaucusAspartic Protease App

DOI：10.3969/j.issn.1673-1689.2020.03.005

中文关键词: 酸性蛋白酶 前导肽 异源表达 蛋白质折叠 假灰绿曲霉

英文关键词: aspartic protease,propeptide,heterologous expression,protein folding,Aspergillus pseudoglaucus

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中文摘要:

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。

英文摘要:

The gene coding aspartic protease App was cloned from the genome ofAspergillus pseudoglaucus.Its self propeptide gene pro，the propeptide geneCPfrom Candida tropicalis candidapepsin，the gene PP from Saccharomyces cerevisiae proteinase A，the gene RP fromRhizomucormiehei proteinase，and the geneSPfromCandida albicansaspartic proteinase 2 were cloned at the 5′-terminal of App gene. The different gene fragments including the different propeptides were obtained，and expressed inEscherichia 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.

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