Enhanced Enzyme Cascade Reactions Through Coencapsulation in Biocompatible Silica Nanoconfinement.

Enzyme cascade reactions, where the product of one enzyme serves as the substrate for another, are fundamental to cellular metabolism and essential for constructing efficient multi-enzyme systems. Spatial confinement of sequential enzymes can enhance cascade efficiency through proximity effects and substrate channeling, mimicking the organization of natural metabolic pathways. Here, we coencapsulated glucose oxidase (GOX) and horseradish peroxidase (HRP) within silica nanocapsules (SiNCs) using an enzyme-friendly synthesis method. The encapsulated enzymes retained high catalytic activity, and Michaelis-Menten kinetics analysis revealed decreased Km for GOX, indicating enhanced substrate affinity under confinement. The coencapsulated system demonstrated faster cascade kinetics compared to spatially separated enzyme configurations, providing direct evidence for the beneficial effects of enzyme proximity on intermediate transfer. Enhanced thermal stability of confined enzymes was also observed, presumably due to preserved hydration shells. This platform enables quantitative studies on enzyme behavior under nanoscale confinement, offering insights into the biochemical principles governing multi-enzyme cascade systems in both natural and artificial environments.