Enhanced catalytic activity of a novel trypsin by semi-rational design with mechanistic insights from molecular simulations.

Trypsin is widely used in the food industry for meat processing, dairy production and seafood treatment. However, the industrial application of trypsin is constrained by the pathogenic risks associated with animal-derived trypsin and the low enzymatic activity of microbial-derived trypsin. This study aimed to enhance the catalytic activity of a novel trypsin heterologously expressed in SCK6. Given the catalytic specificity of trypsin, numerous lysine and arginine residues within the trypsin are susceptible to autolytic cleavage, which may compromise the integrity and stability of its tertiary structure, thereby affecting its catalytic efficiency. To address this, a semi-rational design strategy was employed to introduce mutations at lysine and arginine residues. As a result, a trypsin variant with a 2.2-fold increase in enzymatic activity was obtained, reaching 93.9 U/ml. Further optimization of the fermentation process elevated the enzymatic activity to 132.8 U/ml. Additionally, this study pioneered molecular docking and molecular dynamics simulations in trypsin engineering, revealing that the introduced mutations stabilize the catalytic pocket and enhance enzyme activity. These findings demonstrate that structure-guided mutagenesis of autolysis-prone lysine and arginine residues can significantly improve the catalytic performance of microbial trypsin. This strategy provides a rational framework for the targeted engineering of trypsin variants and offers a practical approach for developing safer, high-activity preparations suitable for industrial food processing applications.