Characterization of a β-Glucosidase from Fragaria × ananassa: Substrate Specificity, Catalytic Efficiency, and Structural Insights.

β-Glucosidases (βGLs) play important roles in plant secondary metabolism by hydrolyzing glycosylated compounds involved in defense, signaling, and the formation of bioactive and flavor-related metabolites. In this study, a β-glucosidase from Fragaria × ananassa (Fra-βGL; FxaC_10g48920.t1) was identified and biochemically characterized in comparison with a commercial β-glucosidase from Prunus dulcis (Pru-βGL). The Fra-βGL cDNA was cloned and heterologously expressed in Escherichia coli, and the recombinant protein was purified for biochemical characterization. Substrate screening demonstrated that Fra-βGL hydrolyzed the 7-O-glucosides of quercetin, kaempferol, and luteolin, whereas no detectable activity was observed toward the tested 3-O-glucosides, anthocyanin glucosides, arbutin, or phloridzin under the assay conditions. Kinetic analysis revealed higher catalytic efficiencies of Fra-βGL than Pru-βGL toward all four substrates examined. Fra-βGL showed its highest turnover rate and catalytic efficiency toward the synthetic substrate p-nitrophenyl-β-D-glucopyranoside (pNP-Glc), with a Kcat of 60.33 s-1 and a Kcat/Km of 10.18 mM-1·s-1. Among the naturally occurring flavonoid glucosides, Fra-βGL exhibited the highest catalytic efficiency toward quercetin 7-O-glucoside (4.14 mM-1·s-1), approximately fivefold higher than that of Pru-βGL (0.824 mM-1·s-1). Thermal stability analysis showed progressive loss of activity with increasing temperature, with Pru-βGL retaining greater residual activity than Fra-βGL at elevated temperatures. Molecular docking suggested structural features that may contribute to the observed preference of Fra-βGL for flavonoid 7-O-glucosides over the corresponding 3-O-glucosides. Collectively, these findings demonstrate the substrate selectivity and distinct catalytic properties of Fra-βGL and identify Fra-βGL as a candidate for further investigation in the context of flavonoid glycoside metabolism in strawberry.