Polyethylene Glycol Crowding Enhances Protease-Mediated Albuminolysis with Distinct Surface Tension Dynamics

Macromolecular crowding profoundly influences biomolecular interactions and enzymatic processes through excluded volume effects, providing opportunities to optimize enzymatic reactions for diverse applications. To this end, albuminolysis is particularly important for various biotechnological applications, including as a regulator of biointerfacial properties, in food processing, in the detergent industry, etc. In this study, we report that protease-mediated albuminolysis is markedly enhanced in the presence of PEG9K (even in the presence of solid-like BSA condensates). Under the respective experimental conditions, proteinase K produced the most extensive BSA fragmentation, followed by trypsin and chymotrypsin. Dynamic light scattering (DLS) measurements demonstrated the formation of submicrometer-sized albumin clusters induced by the depletion effect in crowded media, which probably enhance substrate accessibility for proteases, thereby accelerating cleavage kinetics. Consistent with the surfactant nature of BSA, protease-mediated hydrolysis resulted in a time-dependent evolution of surface tension. Substantially greater changes and distinct protease-specific effects in surface tension dynamics were observed in the PEG-crowded media. Overall, this work links fundamental biochemistry with biointerfacial properties, demonstrating how macromolecular crowding can be harnessed to optimize controlled protein hydrolysis in food processing and bioemulsion development.