Efficient d -Galactose Conversion and Functional Rare Sugar Production via Scaffold-Based Enzyme Complex Platforms

d-Sorbose, a health-promoting rare sugar with growing demand in food and pharmaceutical applications, faces commercial production challenges due to its low conversion efficiency and thermodynamic equilibrium constraints in conventional enzymatic processes. In this study, a Catcher–Tag-scaffolded dual-enzyme cascade was developed for the conversion of d-galactose to d-sorbose, with d-tagatose as an intermediate. Lactobacillus fermentum l-arabinose isomerase (LfAraA) and Pseudomonas cichorii d-tagatose 3-epimerase (PcDTEase) were assembled onto SpyCatcher/DogCatcher scaffolds to enhance enzyme proximity and continuous cascade reactions. Compared to free enzymes, the scaffolded complex increased d-sorbose yield by 21%, exhibiting lower Km and higher kcat/Km, demonstrating improved substrate affinity and catalytic efficiency. These results establish scaffold-guided enzyme assembly as a robust and scalable platform for rare sugar biosynthesis with strong industrial potential.