A novel β-glucosidase from Lacticaseibacillus rhamnosus: Catalytic specificity and efficient production of high-value...

Rare ginsenosides, especially compound K (CK), have attracted interest due to their notable bioactivities and potential applications in pharmaceuticals and functional foods. Here, a novel β-glucosidase (Lrbgl) was identified from Lacticaseibacillus rhamnosus. Lrbgl exhibited optimal activity at weakly acidic pH (5.0-7.0) and remained stable at temperatures up to 40 °C. The enzyme remained stable in the presence of organic solvents such as isopropanol and ethanol, and its catalytic activity was significantly enhanced by the addition of 0.2-1 M NaCl. Site-directed mutagenesis confirmed aspartate 232 (Asp232) and glutamate 417 (Glu417) as critical catalytic residues for β-glycosidic bond hydrolysis, while molecular docking revealed that Glu719, Arg515, Ser166, and Arg164 mediate key hydrogen bonding interactions with the C-3/C-20 sugar moieties of the ginsenoside substrate, stabilizing the substrate in the active pocket. Functional characterization established Lrbgl as a type I ginsenoside hydrolase that specifically cleaves the outer β-(1 → 2) and β-(1 → 6) glucosidic bonds at C-3 and C-20 positions of protopanaxadiol-type ginsenosides. Lrbgl achieved near-complete conversion of ginsenoside Rb1 (>99.9% consumption) via the sequential pathway Rb1 → Rd → F2 → CK, yielding CK at a molar efficiency of 60%-distinguishing it from typical probiotic-derived β-glucosidases that only hydrolyze Rb1 to Rd. When applied to Panax notoginseng saponin, Lrbgl demonstrated its adaptability by transforming 1.75 g/L protopanaxadiol-type ginsenosides into 1.15 g/L rare ginsenosides (primarily CK and F2), with a 76% molar yield for CK. This work establishes an efficient enzymatic platform for the production of high-value ginsenosides, suggesting its potential in bioorganic catalysis for food and pharmaceutical manufacturing.