Abstract
Protein-based materials are emerging as versatile platforms for biocatalysis and biomedical applications due to their structural tunability and intrinsic catalytic capabilities. Here, we present a light-activated strategy for the scalable fabrication of enzymatically active sponges via covalent cross-linking of trypsin within a bovine serum albumin (BSA) matrix. This method leverages photoinitiated Tyr-Tyr coupling, creating a nanoscale enzyme distribution that addresses critical limitations observed in conventional enzyme immobilization methods─namely, instability, autolysis, and restricted reusability. By modulating trypsin concentration and acetic acid (AA) during synthesis, we achieve precise control over cross-link density, enhancing both mechanical flexibility and catalytic accessibility. The sponges retain over 50% of their enzymatic activity after 30 days of storage and maintain ∼60% functionality across ten reuse cycles. Structural integrity and enzyme distribution were validated by attenuated total reflection-Fourier transform infrared (ATR-FTIR) and fluorescence resonance energy transfer (FRET) microscopy, revealing preserved secondary structure and uniform spatial embedding. Proteolytic performance was benchmarked against Cytochrome c, Concanavalin A, and Fetal Bovine Serum, demonstrating enhanced cleavage efficiency and substrate accessibility. This light-activated, reusable platform introduces a scalable approach for stable enzyme immobilization with broad implications for proteomics, biocatalysis, therapeutic devices, and advanced biomedical diagnostics.