Rigid‐Flexible Layered Immobilization Enables Precise Confinement and Dynamic Activation of Small Enzymes

Small enzymes (10-50 kDa) encounter challenges of unstable enzyme-carrier interactions and low activity retention during immobilization, with existing strategies lacking specificity. Unlike previous industrial enzyme-focused studies, a rigid-flexible layered immobilization strategy integrating networked metal-organic frameworks (MOFs) into flexible hydrogels is proposed. This innovative strategy stabilizes enzyme conformation, strengthens enzyme-matrix interactions, and prevents enzyme leakage via a dual-pore system. This synergistic system, composed of MOF micropores (50-150 nm, confinement) and hydrogel macropores (800-900 nm, mass transfer), resolves the stability-accessibility trade-off. Molecular docking shows MOFs reduce substrate-enzyme distance by 34.2% and enhance rigidity. In industrial biocatalysis, immobilized horseradish peroxidase maintains robust conversion efficiency for the reaction of 1 M o-phenylenediamine over 40 cycles, effectively promoting the production efficiency of related chemical products. In the biomedical field, immobilizing thrombin reduces murine wound bleeding by 55%, providing a new solution to wound hemostasis. This scalable platform integrates nanoscale precision with macroscale responsiveness, holding great promise for advancing sustainable biocatalysis and biomedicine.