【摘要】 为开发木霉天冬氨酸蛋白酶的应用潜能，采用实时聚合酶链式反应技术，从棘孢木霉（Trichoderma asperellum）中克隆蛋白酶基因asp，并在毕赤酵母（Pichia pastoris）GS115中成功表达，进一步对重组蛋白酶rAsp进行分离纯化，分析其生化特性及对大豆分离蛋白的水解作用。结果显示，asp基因编码的蛋白酶属于天冬氨酸蛋白酶家族，其序列与本家族成员序列相似性最高为47.74%。在三角瓶中诱导表达时，发酵液中rAsp的酶活力为25.8 U/mL。rAsp最适反应pH值和温度分别为2.5和45℃，在pH 2.0～6.0范围和45℃以下具有较强的稳定性。Cu²⁺和Mn²⁺具有促进rAsp活性的作用，而Fe³⁺、十二烷基硫酸钠和胃蛋白酶抑制剂显著抑制rAsp的活性。rAsp对大豆分离蛋白的水解效率较商业化胃蛋白酶高7.7%；同时，降低β-伴大豆球蛋白和球蛋白致敏性的能力分别是胃蛋白酶的1.4倍和1.8倍。因此，rAsp在大豆蛋白加工应用中具有潜在的开发价值。更多还原

【Abstract】 In order to explore the application potential of aspartic protease（Asp） of Trichoderma sp., the protease gene（asp） was cloned from Trichoderma asperellum by real-time polymerase chain reaction, and was successfully expressed in Pichia Pastoris GS115. The recombinant protease（rAsp） was isolated and purified, and its biochemical properties and its effectiveness in hydrolyzing soy protein isolate（SPI） were studied. The results showed that the protease encoded by the asp gene belonged to the aspartic protease family, and its sequence identity with other members of this family was up to 47.74%. The protease activity of rAsp in the fermentation broth obtained by induced expression in a conical flask was 25.8 U/mL. The optimal reaction pH and temperature of rAsp were 2.5 and 45 ℃, respectively, and rAsp had strong stability in the pH range of 2.0–6.0 and below 45 ℃. The activity of rAsp was promoted by Cu²⁺ and Mn²⁺ but inhibited by Fe³⁺,sodium dodecylsulfate（SDS） and pepstantin. The hydrolysis efficiency of SPI with rAsp was 7.7% higher than that with commercial pepsin. Moreover, the ability of rAsp to reduce the allergenicity of β-conglycinin and glycinin was 1.4 and 1.8 times greater than that of the pepsin, respectively. Therefore, rAsp has potential application in soy protein processing.更多还原