Improving the Activity and Stability of Serine Protease ThAPT3 by Alleviating Self-Cleavage and Its Application in Deproteinization of Shrimp Shells

文献类型： 外文期刊

第一作者： Wang, Xiao

作者： Wang, Xiao;Dong, Tao;Zhou, Qiao;Tong, Lige;Zheng, Jie;Qin, Xing;Wang, Xiaolu;Wang, Yaru;Yao, Bin;Luo, Huiying;Huang, Huoqing

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关键词： serine protease; self-cleavage; stability; rational design; shrimp shell

期刊名称：JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY （ 影响因子：6.1； 五年影响因子：6.3 ）

ISSN： 0021-8561

年卷期： 2023 年 71 卷 20 期

页码：

收录情况： SCI

摘要： The self-cleavage properties of proteases result in low activity and instability, which limit their industrial application. In this study, the serine protease ThAPT3 from Torrubiella hemipterigena was successfully expressed in Komagataella phaffii. We investigated the self-degradation mechanism of ThAPT3 and presented a rational strategy to alleviate self-cleavage. A major self degradation site (Leu238-Met239) and a primary autolysis region were identified. The autolysis regions (loop18, alpha 8-helix, and loop19) were redesigned and optimized using loop transplantation, energy calculations, surface cavity optimization, and loop anchoring. A triple-superposition mutant, ThAPT3-M9 (M239GKDGAVAAGLC250 -> M239TLNRTTAANAC250/A251E/A254Q/R259L/ A267E/S280N), was obtained. Compared to the wild type, the autolysis of M9 was significantly alleviated, and its half-life at 60 degrees C was increased approximately 39-fold (from 1.6 to 62.4 min). The optimal temperature and specific activity of M9 increased by 5 degrees C (from 60 to 65 degrees C) and 62% (4985 vs 3078 U/mg), respectively. M9 showed significant advantages in shrimp shell deproteinization.

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