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

Steroidal saponins are a class of compounds abundant in plants of the <i>Liliaceae</i> and <i>Dioscoreaceae</i> families, and some of them exhibit several bioactivities, such as antitumor, hemostatic, and anti-inflammatory effects. During postharvest processing of products like <i>Paridis rhizoma</i>, 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 <i>Paridis rhizoma</i> during processing. This pioneering study unveils the actual mechanism by which PpF26G mediates the conversion of steroidal saponin in saponin-rich products, such as <i>Paridis rhizoma</i>, and its importance in improving quality.