Cloning and characterization of a novel β-glucosidase from Bifidobacterium breve 689b and its application in the preparation of rare ginsenoside F2

The rare ginsenoside F2, exhibiting a wide spectrum of pharmacological activities, is present in the human body and is readily absorbed. However, the extremely low content of ginseng has hindered its development and application. In this study, a glycoside hydrolase gene (BbGlu1B) encoding a Bifidobacterium breve 689b was cloned and expressed in Escherichia coli BL21. BbGlu1B was purified, exhibiting a molecular weight of 44.6 kDa, with optimal activity at 40 °C and pH 6. The purified BbGlu1B exhibited β-glucosidase activity and was able to highly selectively hydrolyse the outer Glu residue at the C-3 position of ginsenoside Rd, yielding the rare ginsenoside F2. The optimal reaction conditions for the preparation of ginsenoside F2 were determined through a one-factor experiment: at 35 °C, pH 6.5, a substrate concentration of 6 mg/mL, and an enzyme addition of 0.29 U/mg, the conversion rate reached over 90%. The structure was characterized using TLC, HPLC,13C-NMR, MALDI-TOF-MS, FTIR, and UV spectroscopy. Molecular docking results revealed that hydrogen bonds formed by key amino acid residues ARG-35, GLY-280, and THR-260 with ginsenoside Rd play a dominant role in mediating the interaction between Rd and BbGlu1B, which is of crucial importance for maintaining the binding stability of Rd and enhancing its catalytic efficiency. In this study, a novel β-glucosidase BbGlu1B possessing exceptional biotransformation activity towards ginsenosides Rd was explored, and a feasible and efficient method for the enzymatic preparation of rare ginsenoside F2 was developed.