A Rhizosphere-Derived β-glycosidase with Intrinsic Sequential Regioselectivity for Highly Efficient Production of Siamenoside I from Mogroside V.

Siamenoside I (SI), a high-intensity sweetener, is limited in natural abundance. A β-glycosidase (CoExg1) from Candida orthopsilosis L7 (isolated from the Siraitia grosvenorii rhizosphere) exhibits intrinsic sequential regioselectivity, enabling efficient SI production from mogroside V (MGV). Fermentation optimization increased SI yield from 70.34% (7 d) to 84.06% (72 h). CoExg1 rapidly cleaves the C-3 β-1,6 bond (89.35% SI in 20 min), then slowly hydrolyzes the C-24 bond to mogroside IIIE (MIIIE, 12 h)─a time-controlled process requiring no external regulation. Kinetic analysis showed a ∼50-fold higher catalytic efficiency toward MGV than toward SI. Molecular docking and dynamics revealed that the C-3 glycan of MGV binds in a catalytically competent conformation with stable hydrogen-bond networks, whereas SI's C-24 glycan hardly forms a productive binding mode, explaining the sequential selectivity. This work provides a sustainable SI production platform and unveils the structural basis of sequential regioselectivity in glycoside hydrolases.