A novel GH66 family glycoside hydrolase pseudo-dextranase from Streptococcus thermophilus 17140 in catalyzing multi-step deglycosylation of ginsenoside Rb1.

Rare ginsenosides exhibit enhanced bioavailability and strong pharmacological activities such as anti-aging and immunomodulatory effects, making them valuable in health care food and medicine. We previously identified a Streptococcus thermophilus strain 17140 (St17140) capable of converting rare ginsenosides. In this study, we cloned and expressed a novel GH66 family glycoside hydrolase gene, pseudo-Dextranase from St17140 (PStDex). Despite lacking canonical catalytic residues for β-glucan hydrolysis, PStDex specifically cleaves the glycosidic bonds of ginsenoside Rb1 to produce rare ginsenosides Gypenoside XVII (GypXVII), F2, Rg3, Rh2, and CK. This is the first identification of a pseudo-Dextranase with capability of ginsenoside transformation. The enzyme exhibits optimal activity at pH 6.0 and 50 °C with apparent kinetic parameters: kcat = 1.05 × 10-2 s-1, Km = 0.16 × 10-2 mM, and kcat/Km = 6.6 × 10-2 s-1·mM-1. Through molecular dynamics, structural optimization, molecular docking, and site-directed mutagenesis, we identified Asp34 and Asp59 as critical catalytic residues. This study provides new insights into glycosidic bond hydrolysis mechanisms and offers a novel enzymatic tool for efficient biotransformation of ginsenosides.