Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute , Chinese Academy of Agricultural Sciences , No. 18 12 Zhongguancun South Street , Beijing 100081 , P. R. China.
J Agric Food Chem. 2019 Sep 18;67(37):10505-10512. doi: 10.1021/acs.jafc.9b03959. Epub 2019 Sep 10.
An aspartic protease gene () was cloned from sp. MEY-1 and expressed in . The recombinant APA showed maximal activity at pH 3.0 and 75 °C and remained stable at 70 °C and below, indicating the thermostable nature of APA. However, heat inactivation still limits the application of APA. To further improve its thermostability, an autocatalysis site (L205-F206) in APA was identified and three mutants (F193W, K204P, and A371V) were generated based on the analysis of the structure neighboring the autocatalysis site. These mutants have improved thermostability, and their half-life at 75 °C increased by 0.5-, 0.2-, and 0.3-fold, respectively. A triple-site mutant (F193W/K204P/A371V) was generated, with 1.5-fold increased half-life at 80 and a 10.7 °C increased , compared with those of the wild-type. These results indicate that autocatalysis of aspartic protease reduces enzyme thermostability. Furthermore, site-directed mutagenesis at regions near the autocatalysis site is an efficient approach to improve aspartic protease thermostability.
从 sp. MEY-1 中克隆了一个天冬氨酸蛋白酶基因 ()，并在 中表达。重组 APA 在 pH 3.0 和 75°C 下表现出最大活性，在 70°C 及以下保持稳定，表明 APA 具有热稳定性。然而，热失活仍然限制了 APA 的应用。为了进一步提高其热稳定性，鉴定了 APA 中的一个自催化位点 (L205-F206)，并根据紧邻自催化位点的结构分析生成了三个突变体 (F193W、K204P 和 A371V)。这些突变体提高了热稳定性，其在 75°C 下的半衰期分别延长了 0.5 倍、0.2 倍和 0.3 倍。生成了一个三突变体 (F193W/K204P/A371V)，与野生型相比，在 80°C 下半衰期延长了 1.5 倍， 提高了 10.7°C。这些结果表明，天冬氨酸蛋白酶的自催化降低了酶的热稳定性。此外，在自催化位点附近区域进行定点突变是提高天冬氨酸蛋白酶热稳定性的有效方法。
