An Engineered Fusion Enzyme-MOF Platform for Enhanced Biotransformation of Ginsenoside Rd to Rare Ginsenoside Rh2

Rare ginsenoside Rh2 has attracted increasing interest for its antitumor, anti-inflammatory, and metabolic regulatory activities. Enzymatic biotransformation provides a green and selective route for Rh2 production, but current systems suffer from inefficient cascade conversion and limited enzyme stability. Here, two β-glucosidases enabling the stepwise and selective conversion of 20(S)-ginsenoside Rd to 20(S)-ginsenoside Rh2 were identified and assembled into a fusion biocatalyst through linker engineering. Compared with the parental two-enzyme cascade under equimolar enzyme loading, the optimized fusion enzyme reduced the accumulation of the intermediate Rg3 and increased 20(S)-Rh2 production rate by 40%. A Zn-Ni metal-organic framework was further introduced to enable simultaneous purification and immobilization. The immobilized fusion enzyme showed enhanced operational stability, retaining 82% of its initial activity after seven successive substrate-feeding intervals. This study presents a multiscale strategy integrating enzyme fusion and MOF immobilization for the efficient, operationally stable, and sustainable production of 20(S)-ginsenoside Rh2.