Identification and Characterization of β-Glucosidase PpF26G Catalyzing the Conversion of Steroidal Saponins To Improve Quality of Paridis Rhizoma during Postharvest Process

Steroidal saponins are a class of compounds abundant in plants of the Liliaceae and Dioscoreaceae families, and some of them exhibit several bioactivities, such as antitumor, hemostatic, and anti-inflammatory effects. During postharvest processing of products like Paridis rhizoma, furostanosides are partially converted into high-activity spirostanosides, but the underlying mechanism remains unclear. In this study, transcriptomics was employed to identify the key endogenous enzyme mediating this process: PpF26G. Using heterologous expression techniques, we successfully obtained the enzyme and found that it has a high specificity for the glucose at C-26 in furostanosides, hydrolyzing them to generate the corresponding spirostanosides. The enzyme exhibited strong hydrolytic activity toward proto-pennogenyl glycosides. Mutagenesis experiments, together with docking and molecular dynamics simulation, verified that E236 and E450 synergized to perform the "two-step" catalytic mechanism. PpF26G could be activated by the damage of cellular structures during slicing, which resulted in the quality enhancement of Paridis rhizoma during processing. This pioneering study unveils the actual mechanism by which PpF26G mediates the conversion of steroidal saponin in saponin-rich products, such as Paridis rhizoma, and its importance in improving quality.