白曲霉酸性蛋白酶在白酒酿造发酵过程中具有溶解发酵原料的颗粒，促进微生物繁殖，分解蛋白质生成香味物质，降解酵母菌体蛋白等多种功能，可以提高白酒风味，因此被广泛应用于白酒生产中。本研究利用经基因工程改造的低内源蛋白背景的黑曲霉宿主SH-2，表达白曲霉酸性蛋白酶基因pepB。通过PCR技术获得pepB以及表达元件糖化酶启动子PglaA、糖化酶终止子TglaA、乳清酸核苷-5-磷酸脱羧酶标记基因pyrG，在pMD18-T载体基础上，构建了pepB表达载体，通过PEG介导转化法转化无孢黑曲酶SH-2。重组菌株经发酵罐发酵240 h，发酵粗酶液酶活达9722 U/mL，是报道的白曲原始菌株酶活的8.5倍，SDS-PAGE结果显示表达产物分子量约为47 ku。酶学性质分析结果表明，该酸性蛋白酶最适反应温度为35 ℃，最适pH为4.0，Mn2+、Cu2+对酶活有显著地激活作用。最后，探究了在不同发酵初始pH下重组菌株酶活的变化，结果显示，在pH 4.5~6.5范围内，适当提高发酵初始pH，酸性蛋白酶酶活会提高。以上结果表明，本研究成功构建了一株能胞内高效表达白曲酸性蛋白酶的菌株。

Acid protease of Aspergillus kawachii, which has the functions of dissolving the particles of the fermentation raw material , promoting microbial propagation, degrading yeast cell protein and other functions to improve liquor flavor, has been widely used in liquor production in the process of fermentation. Aspergillus niger SH-2, which had the property of low endogenous protein expression after genetic engineering transformation, was used in this study to express acid protease gene pepB from Aspergillus kawachii.. The gene pepB and expression elements glucoamylase promoter PglaA, glucoamylase terminator TglaA, orotic acid nucleoside-5-phosphate decarboxylase marker gene pyrG were obtained by PCR., and the pepB expression vector was constructed on the basis of pMD18-T vector. The recombinant strains were fermented for 240 h in the fermentor and the enzyme activity of the fermented crude enzyme was 9722 U/g, which was 8.5 times as much as that of the reported original enzyme strain, and SDS-PAGE pattern showed that the molecular weight of the expressed product was about 47 ku. The results of enzyme properties analysis showed that the optimum reaction temperature of the acidic protease was 35 oC and the optimum pH was 4.0. Mn2+ and Cu2+ had significant activation effect on the enzyme activity. Finally, the changes in the enzyme activity of recombinant strains under different fermentation pH were investigated. The results showed that in the range of pH 4.5-6.5, the initial pH of the fermentation was properly increased, and the activity of acid protease increased. Consequently, this study has successfully constructed a strain that can express the acid protease of Aspergillus kawachii.

广东省科技计划项目-粤港联合创新项目（2016A050503016）；广东省自然科学基金项目（2017A030313097）；华南理工大学中央高校基本科研业务费资助项目（2015ZP032）