Elucidating Molecular Confinement Mechanisms of Glucose Oxidase in Hydrogen-Bonded Organic Frameworks

Hydrogen-bonded organic frameworks (HOFs) have emerged as promising scaffolds for enzyme immobilization under mild, aqueous conditions, yet the molecular mechanism of enzyme encapsulation remains poorly understood. Here, we combine experimental characterization with all-atom molecular dynamics simulations to elucidate the one-pot encapsulation of glucose oxidase (GOx) within hydrogen-bonded organic frameworks (HOF-1). Confocal imaging and spectroscopy confirm enzyme confinement with preserved structural integrity, while NMR and SAXS reveal that GOx actively modulates the local chemical environment and pore architecture during framework assembly. Simulations uncover a stepwise mechanism in which HOF-1 monomers nucleate on the enzyme surface via synergistic hydrogen-bonding and electrostatic interactions, directing ordered crystal growth without perturbing the active site. These results show GOx as an active molecular model template for its own encapsulation, providing mechanistic design principles for next-generation HOF-based biocatalysts.