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ABSTRACT Silk fibroin self‐assembly is introduced as a fully aqueous, solvent‐ and cross‐linker‐free strategy for noncovalent multienzyme coimmobilization. Salt‐driven structuring enables simultaneous enzyme entrapment within a shared protein matrix, while modulation of ionic strength allows controlled enzyme incorporation and adjustment of cascade stoichiometry. Using the alanine dehydrogenase/formate dehydrogenase redox cascade for l ‐norvaline synthesis as a model system, co‐entrapment within the fibroin matrix enhances operational robustness, sustaining 200 h of cumulative batch operation and achieving a cumulative catalytic turnover exceeding 9000 mol l ‐norvaline per mol of alanine dehydrogenase under the tested conditions. Structural characterization reveals an interconnected microglobular architecture consistent with a confined protein‐based microenvironment capable of influencing diffusion–reaction coupling. Competitive assays indicate reduced NADH accessibility in the coassembled system, consistent with partial cofactor confinement. Extension to additional reductase/glucose dehydrogenase cascades demonstrates compatibility across distinct enzyme classes and reveals substrate‐dependent effects linked to diffusion dynamics and matrix–substrate interactions. Collectively, these results establish salt‐assembled silk fibroin as an active protein matrix that stabilizes multienzyme systems while modulating cofactor accessibility and substrate distribution without covalent modification.
Almada et al. (Mon,) studied this question.