Advances in trypsin research: sources, structural insights, applications and protein engineering for industrial use.

Trypsin (E.C. 3.4.21.4) is a serine protease that specifically cleaves peptide bonds at the carboxyl termini of arginine and lysine residues. It serves as an important biocatalyst with extensive applications in various fields. Historically sourced from animal pancreas, trypsin production is associated with risks of pathogen contamination and batch-to-batch variability, prompting the adoption of microbial recombinant expression systems as preferred platforms for standardized, large-scale production. The enzymatic activity and substrate specificity of trypsin are determined by the conserved catalytic triad (H57, D102, S195), the substrate-binding S1 pocket and the calcium-binding loop essential for structural stability. However, its inherent susceptibility to self-degradation and weak thermal instability limit its broader industrial utility. Protein engineering approaches, particularly site-directed mutagenesis targeting autolytic sites and flexible regions, have significantly enhanced resistance to self-degradation and improved thermostability. Despite significant advances in recombinant protein expression and enzyme engineering, challenges remain in elucidating dynamic enzyme-substrate interactions, leveraging natural sequence diversity, and achieving optimal yields under industrial-scale conditions. Future research should focus on integrating AI-driven protein design with synthetic biology tools to accelerate sustainable manufacturing and expand the biocatalytic applications of trypsin.