Engineering AcidicResistance in UDP-Galactose 4‑EpimeraseEnables Efficient UDP-Galactose Synthesis from Sucrose

Uridine diphosphate galactose (UDP-Gal) provides galactosyl units for active carbohydrate biosynthesis; however, limited availability and high costs hamper large-scale applications. In the two-enzyme cascade system of UDP-Gal synthesis, the pH conflict between UDP-galactose 4-epimerase (GALE) and sucrose synthase (Susy) blocks UDP-Gal production. Therefore, surface charge engineering was conducted to obtain a variant (GALE_M2) with improved acid resistance. GALE_M2 enzyme activity reached 214.26 ± 0.20% that of wild-type GALE at pH 6.5. Its half-life time increased by 2 h at pH 6.5, and the pH resistance range was widened effectively with local surface charge reshaping and a decreased isoelectric point. An improved flexibility of the substrate entrance enhanced the catalytic performance under acidic conditions. Cascading GALE_M2 and Susy_M6 yielded UDP-Gal (24.5 mM) with a space–time yield of 12 g/L/h within 1.25 h, demonstrating the robust route of short reaction time and high efficiency, for rapid UDP-Gal synthesis from readily available sucrose via cascade catalysis.