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

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, α₈-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 °C was increased approximately 39-fold (from 1.6 to 62.4 min). The optimal temperature and specific activity of M9 increased by 5 °C (from 60 to 65 °C) and 62% (4985 vs 3078 U/mg), respectively. M9 showed significant advantages in shrimp shell deproteinization.