Enzyme immobilization on metal-organic frameworks (MOFs) offers a promising strategy to enhance biocatalytic stability and reusability, yet achieving high loading capacity while maintaining enzymatic activity remains challenging. Here, we report the rational design of functionalized zirconium-based MOFs (ZrBTB-X) for the efficient immobilization of cytochrome c (Cyt c). By systematically modifying ZrBTB with diverse functional groups, we demonstrate that amine-functionalized ZrBTB-NH2 exhibits the highest enzyme loading capacity while significantly improving catalytic activity and stability compared to free Cyt c. Notably, the Cyt c@ZrBTB-NH2 composite maintains high activity across a wide pH range (2-8) and demonstrates exceptional stability under extreme conditions, such as elevated temperatures (25-90 °C) and exposure to non-physiological environments. Experimental and theoretical investigations reveal that the enhanced performance stems from optimized enzyme-MOF interactions, including high specific surface area, strengthened hydrogen bonding and electrostatic complementarity. This work not only provides a practical approach for high-efficiency enzyme immobilization but also elucidates the critical role of MOF surface engineering in modulating enzyme-MOF affinity and catalytic performance, offering valuable insights for the design of advanced biocatalytic systems.
Keywords: Cytochrome c; Enzyme immobilization; Metal-organic frameworks; Surface engineering.
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