Compartmentalization of multi-enzymes within designable metal-organic frameworks (MOF): A review of systematic approaches.

Integrating multi-enzyme systems within metal-organic frameworks (MOFs) has garnered significant attention in biocatalysis due to their tunable structural properties and ability to enhance enzyme performance in cascade reactions. The unique features of MOFs, such as well-defined pore apertures, tailorable compositions, and high loading capacity, facilitate the design of robust multi-enzyme bio-composites with enhanced recyclability and specificity. This review explores systematic approaches for the compartmentalization and positional co-immobilization of multiple enzymes within MOFs, focusing on two key strategies: (i) layer-by-layer assembly and (ii) pore-engineered compartmentalization. These methods enable precise spatial organization of enzymes, improving mass transfer of intermediates, catalytic efficiency, and enzyme stability under harsh conditions. This review discusses state-of-the-art examples, properties like proximity effects and microenvironment tuning, and emerging tools like machine learning for optimizing multi-enzyme MOF designs. Despite these advancements, challenges such as incompatible reaction conditions, differing enzyme stabilities, and complex MOF synthesis persist, limiting large-scale applications. By addressing current limitations and future opportunities, this review underscores the potential of systematic enzyme immobilization in MOFs to revolutionize sustainable biocatalytic processes.