A halotolerant marine β-glucosidase from Celeribacter halophilus for efficient bioconversion of soybean isoflavone glycosides to bioactive aglycones.

Soybean isoflavones exist predominantly as glycosides (e.g., daidzin, genistin), requiring enzymatic hydrolysis by β-glucosidases (EC 3.2.1.21) to release bioactive aglycones (e.g., daidzein, genistein) with enhanced bioavailability. In this study, a novel glycoside hydrolase family 1 (GH1) β-glucosidase gene (ChaBGL) derived from the halophilic marine bacterium Celeribacter halophilus was successfully cloned and heterologously overexpressed in Escherichia coli BL21(DE3). The purified recombinant enzyme (ChaBGL) displayed maximal activity at 45 °C and pH 6.0. ChaBGL exhibited broad halotolerance across multiple salt types, retaining 97.5 ± 4.4% activity at 1000 mM NaCl, 76.5 ± 5.6% at 1000 mM KCl, 79.5 ± 4.5% at 1000 mM Na2SO4, and 83.2 ± 5.2% at 500 mM K2SO4. ChaBGL demonstrated high catalytic efficiency towards the substrate p-nitrophenyl-β-D-glucopyranoside (pNPG), achieving a specific activity of 101.3 ± 4.0 U/mg. Kinetic analysis revealed a Km of 0.17 ± 0.02 mM and an apparent kcat of 87.8 ± 3.5 s-1. Notably, ChaBGL demonstrated high efficacy in hydrolyzing natural soybean isoflavone glycosides. Within 60 min at 30 °C, it achieved near-complete hydrolysis of daidzin (95.4 ± 0.34%) and genistin (∼100%), resulting in substantial increases in the yields of bioactive daidzein (6.73 ± 0.15-fold) and genistein (9.78 ± 0.31-fold). Consequently, the percentage of aglycones in the total isoflavonoids surged from an initial 10.3 ± 0.8% to 96.5 ± 0.07%. These findings collectively highlight ChaBGL's high catalytic efficiency in hydrolyzing glycosidic bonds and its significant potential for diverse industrial applications, particularly in the nutritional, veterinary, and pharmaceutical sectors.