Expression and functional analysis of high-stability hybrid human trypsin

Trypsin is a critical enzyme in industrial applications, where high catalytic activity and stability are essential for optimizing production efficiency. To address the inherent limitations of natural trypsin, this study constructed a recombinant human hybrid trypsin (rhhT) by integrating the auto-activation properties of human anionic trypsin (hT2) and high-stability of human cationic trypsin (hT1). These enzymes were expressed as inclusion bodies in Escherichia coli BL21 (DE3) cells and purified through ion exchange chromatography followed by denaturation-refolding procedures. The purified rhhT exhibited significantly enhanced stability while maintaining catalytic activity comparable to rhT2. Enzymatic characterization identified an optimal reaction pH of 9 and a temperature of 37°C for rhhT. Kinetic analysis revealed no statistically significant differences in Michaelis constant (K_m) or catalytic constant (K_cat) compared to rhT2, confirming preserved catalytic efficiency. Furthermore, rhhT demonstrated superior stability under diverse conditions, including temperature fluctuations, pH variations, and ionic stress, alongside improved degradation and storage resistance. A key structural distinction was observed in calcium ion binding, with rhhT forming a more stable complex than anionic trypsin. Collectively, this study successfully designed a "highly activated and stable" human hybrid trypsin, providing a robust experimental foundation for its industrial applications in medical and food processing sectors.