Mechanochemical Nanoarchitectonics for the Synthesis of Enzyme-Based Hydrogen-Bonded Organic Frameworks

Traditional methods for synthesizing functional materials are often energy-intensive and time-consuming and generate significant solvent waste. In this study, we report a sustainable mechanochemical strategy for the direct immobilization of enzymes into hydrogen-bonded organic frameworks (HOF-1), offering a green and scalable route to functional biocomposites. Using ball milling, a solvent-free and efficient technique, we successfully coassembled HOF-1 with either glucose oxidase (GOx) or a bienzyme system (GOx and horseradish peroxidase, HRP) during in situ framework formation. The resulting enzyme@HOF-1 composites retained high enzymatic activity, with GOx@HOF-1 exhibiting enhanced stability under harsh conditions, including extreme pH, high temperature, denaturants, and organic solvents. Notably, the GOx/HRP@HOF-1 bienzyme system achieved a transient response time of nearly zero seconds in cascade reactions, demonstrating a 2.25-fold increase in catalytic efficiency over free enzymes, attributed to improved substrate channeling within the confined framework. This work highlights the potential of mechanochemical synthesis for creating robust, ecofriendly enzyme-based materials with high catalytic performance.