Improving the Activity and Stability of Serine Protease Th APT3 by Alleviating Self-Cleavage and Its Application in Deproteinization of Shrimp Shells.

The self-cleavage properties of proteases result in low activity and instability, which limit their industrial application. In this study, the serine protease Th APT3 from Torrubiella hemipterigena was successfully expressed in Komagataella phaffii . We investigated the self-degradation mechanism of Th APT3 and presented a rational strategy to alleviate self-cleavage. A major self-degradation site (Leu 238 -Met 239 ) and a primary autolysis region were identified. The autolysis regions (loop 18 , α 8 -helix, and loop 19 ) were redesigned and optimized using loop transplantation, energy calculations, surface cavity optimization, and loop anchoring. A triple-superposition mutant, Th APT3-M9 (M 239 GKDGAVAAGLC 250 → M 239 TLNRTTAANAC 250 /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.