Transforming fragile enzymes into robust nanoreactors via Sol–Gel design

Aqueous one-pot sol–gel encapsulation of fragile NADH oxidase creates silica nanoreactors with high activity, recyclability, and proteolytic stability. • A one‑step silica formation process enables the gentle encapsulation of fragile enzymes in water. • The hybrid nanoparticles containing the nicotinamide cofactor oxidase show high enzyme incorporation and catalytic activity. • The nanoparticles maintain efficient catalytic performance across repeated reaction cycles. • Encapsulation provides exceptional resistance against operational and proteolytic degradation, supporting long‑term operation. The operational stability of fragile enzymes remains a key challenge for integration into industrial and biotechnological systems. Sol‑gel chemistry offers a solution by entrapping enzymes within protective inorganic networks; however, conventional approaches rely on harsh conditions (strong acids, alcohol‑rich solvents, elevated temperatures) incompatible with sensitive enzymes. We design a reproducible, one‑pot sol‑gel tailored for fragile enzymes, enabling encapsulation of a thermally and chemically labile NADH oxidase (NOX) from Lactobacillus pentosus under fully aqueous conditions. By merging standard coupling chemistry with ionic liquid‑assisted silica polymerization, hybrid NOX@SiO 2 nanostructures are produced with high protein loading (up to 40 mg g −1 silica) and high enzymatic activity (up to 37 U g −1 silica). Computational analysis identifies solvent‑accessible Asp and Glu residues as preferred anchoring sites, guiding an efficient encapsulation mechanism. The nanoreactors retain catalytic performance comparable to that of the free enzyme in NAD + regeneration cascades and remain active across multiple cycles, achieving up to 8 mmol h −1 g −1 catalyst productivity. NOX@SiO 2 exhibits exceptional operational and proteolytic stability, enabling controlled partial degradation that enhances activity and underscoring potential for sustainable catalysis and targeted delivery systems in practical applications. This platform provides a reproducible, biocompatible, recyclable route for integrating fragile enzymes into robust nanostructured systems.