Synergistic bimetallic competition and defect engineering in hierarchically porous MOF-enzyme composites to enhance the enzymatic synthesis of rare ginsenoside CK.

The confinement effect of microporous MIL-88B(Fe) significantly limits enzyme immobilization efficiency and mass transfer, particularly for bulky ginsenoside substrates. To address this challenge, we propose a synergistic strategy that integrates bimetallic competitive coordination and defect engineering to construct a hierarchically porous MOF-enzyme composite (Sna/M8Co(Fe)-Et) for the enhanced enzymatic synthesis of the rare ginsenoside CK. This approach involves generating oxygen vacancies via Fe3+/Co2+ bimetallic competitive coordination, which guides propionic acid to selectively etch the MOF framework, thereby creating interconnected mesopores without compromising structural integrity. This hierarchical porosity substantially enhances mass transfer efficiency. Snailase (Sna) was then covalently immobilized via carbodiimide-mediated crosslinking to optimize the catalytic microenvironment. The resulting Sna/M8Co(Fe)-Et exhibited superior catalytic efficiency, with a Vmax of 47.36 mM·min-1 and a Km of 1.42 mM. The Vmax/Km ratio of 33.22 min-1 was 1.3-fold higher than that of the free enzyme (25.51 min-1). Under optimal conditions (pH 4.5, 55 °C, 96 h), the composite achieved a 66.2% CK conversion rate (94.02% purity) and retained 38.5% of its initial productivity after six catalytic cycles, thus demonstrating robust operational stability. This study presents a novel and scalable design strategy for hierarchically porous MOFs, thereby opening new possibilities for the efficient biocatalysis of macromolecular substrates.