为了对哈茨木霉酸性蛋白酶（Acid Protease，Ap）的性质研究，采用RT-PCR克隆了哈茨木霉Ap基因，转化至毕赤酵母GS115菌株中获得高效表达，然后对该重组蛋白酶（Recombinant Acid Protease，rAp）的酶学性质和水解大豆分离蛋白的效果进行测定。结果表明，重组毕赤酵母在500 mL三角瓶中诱导表达时，发酵液中rAp酶活力达到21.50 U/mL。该rAp为天冬氨酸蛋白酶，最适温度为55 ℃，在40 ℃处理120 min仍具有较强的热稳定性；最适pH值为2.50，在不同pH缓冲液中处理24 h后，pH值2.00~5.00具有较强稳定性。Cu2+、Ni2+和Mn2+能显著促进活性，相对酶活分别高达116.21%、113.79%和117.44%；而Fe2+、Fe3+、0.50% SDS和5.00% Trion X-100显著抑制活性，其相对酶活分别78.02%、79.26%、2.6%和13.19%。rAp和胃蛋白酶对大豆分离蛋白水解后，水解产物中蛋白相对含量分别为14.67%和3.64%，β-伴大豆球蛋白抗原性分别降低了30.01%和26.10%，球蛋白抗原性分别降低了22.37%和15.63%。从以上结果可知，rAp对大豆分离蛋白具有较强水解和降低抗原性的能力，具有潜在的应用开发价值。

In order to study the properties of acid protease (Ap) from Trichoderma harzianum, the acid protease gene was cloned using RT-PCR and then transferred to the Pichia pastoris GS115 strain to obtain high expression. Subsequently, the enzymatic properties of this recombinant acid protease (rAp) and its performance in hydrolyzing soybean protein isolate were determined. It was observed that the activity of rAp in the fermentation broth reached 21.5 U/mL when the recombinant P. pastoris was induced in a 500 mL triangular flask. This rAp is an aspartic protease with an optimal temperature of 55 ℃ and an optimum pH value of 2.5, which exhibited strong thermal stability after treatment at 40 ℃ for 120 min. After 24 h treatments in buffers of different pH, rAp still demonstrated excellent stability in the pH range of 2.00~5.00. The relative activity of rAp significantly increased to 116.21%, 113.79%, and 117.44% by Cu2+, Ni2+, and Mn2+, respectively. In the meantime, rAp activity was significantly inhibited by Fe2+, Fe3+, 0.50% SDS, and 5.00% Triton X-100, and the resulting relative enzyme activity was 78.02%, 79.26%, 2.6%, and 13.19%, respectively. After hydrolysis of soybean protein isolates by rAp and pepsin, the relative protein contents of the hydrolyzed products were 14.67% and 3.64%, respectively. Moreover, the β-conglycinin antigenicity is reduced by 30.01% and 26.11%, respectively, and the antigenicity of globulin is decreased by 22.37% and 15.63%, respectively. Based on these results, it is clear that rAp has a strong ability to hydrolyze and reduce the antigenicity of soybean isolates, demonstrating its potential development and application value.

广东省自然科学基金项目（2018A0303130108）；韶关学院大学生创新创业训练计划国家级项目（202210576016）；广东大学生科技创新培育专项资金资助项目（pdjh2023b0474）