Mechanistic insights into the synergistic hydrolysis of elastase-trypsin for highly active elastase inhibitory...

Due to the incomplete understanding of enzymatic degradation pathways of complex marine proteins, the production of highly active elastase inhibitory peptides (EIPs) remains limited by low yield, unstable activity, and poor controllability. Therefore, in this study, skipjack tuna cardiac arterial bulbs was used as the substrate, and kinetic analysis of pancreatic enzyme-mediated hydrolysis was performed to clarify the hydrolysis mechanism and obtain highly active EIPs. LC-MS/MS analysis identified 17 parent proteins and 430 unique peptides, with myoglobin, cytochrome C and elastin as the main bioactive precursors. The synergistic action of elastase and trypsin (Ela-Try) enhanced EIPs release, achieving 54.17% elastase inhibitory activity at the optimal enzyme ratio (elastase:trypsin = 1.2:1). Enzyme kinetics, molecular docking, and molecular dynamics simulations show that Ela-Try has lower K_m (2.3751 mg/mL) and higher V_max (0.0199 mg/mL·min) than elastase or trypsin alone, demonstrating synergistic hydrolysis. Ela-Try performs stepwise hydrolysis of proteins (especially cytochrome C), with elastase first cleaving hydrophobic residues (Gly, Ala, Val) according to a pseudo-first-order kinetic model, causing conformational changes and exposing new active sites (Lys, Arg); subsequently, trypsin cooperates with elastase following a pseudo-second-order kinetic model to further cleave the newly exposed sites and remaining hydrophobic residues. This study revealed the hydrolysis mechanism of the cooperative formation of highly active EIPs by Ela-Try, improving the release efficiency, activity and controllability of EIPs, and providing a theoretical basis for the high-value utilization of marine protein resources and the development of functional peptides.