Functional characterization of a novel β-glucosidase converting trillin to diosgenin from Enterococcus faecalis.

BACKGROUND: Diosgenin, known as “medicinal gold”, is the main precursor for the synthesis of steroid hormone drugs. Currently, diosgenin is primarily produced industrially via the extraction of plant-derived steroidal saponins followed by acid hydrolysis, which generates substantial acidic wastewater. RESULTS: To address this environmental challenge, we sought to discover efficient glycosidases capable of converting steroidal saponins to diosgenin as a sustainable alternative to conventional acid hydrolysis. Through selective enrichment using dioscin as the sole carbon source, we isolated a strain Enterococcus faecalis D1 from rat gut microbiota. Subsequent genomic sequencing and functional annotation identified a potent steroid saponin β-glucosidase (designated EfD08) from E. faecalis D1, which represents the first reported bacterial β-glucosidase that efficiently converts trillin to diosgenin. EfD08 achieved soluble expression in Escherichia coli with an exceptionally high yield exceeding 200 mg/L, overcoming the low-expression bottleneck typical of reported steroid saponin glycosidases. Enzymatic characterization demonstrated that EfD08 exhibits robust activity with a half-life of approximately 100 h at its optimal temperature of 30 °C, which further extended to 355 h and 154 h in the presence of 5 mM Mg²⁺ or Mn²⁺, respectively. Furthermore, EfD08 displayed broad substrate specificity, efficiently hydrolyzing diverse steroid saponins (e.g., zingiberensis newsaponin, deltonin) and ginsenosides (e.g., Rb1, Rc). CONCLUSIONS: Given its high protein yield, catalytic efficiency, and stability, EfD08 emerges as a promising biocatalyst for the environmentally sustainable industrial production of diosgenin, other secondary saponins or sapogenins from natural saponins.