Glucosidases from Flavobacterium saccharophilum: characterization and application in the preparation of minor ginsenosides

Abstract:

[Background] Some minor ginsenosides possess valuable pharmacological activities. Mining glycosidases with high activity and specificity can realize the directional preparation of minor ginsenosides.Flavobacterium saccharophilum carries abundant and uncharacterized glycosidase genes, which are potential sources for excavating new glycosidases.[Objective] To obtain highly active and specific glycosidases from F. saccharophilum for the preparation of minor ginsenosides. [Methods] Fifteen putative glucosidase genes were cloned from F. saccharophilum and expressed. To screen out the enzymes for the preparation of minor ginsenosides, we fully characterized the recombinants and identified the biotransformation products by employing thin layer chromatography and high performance liquid chromatography. [Results] Three β-glucosidases (SA2629, SA0236, and SA2851) with high activities were obtained from F. saccharophilum. SA2629 showed the highest specific activity (78.7 U/mg) and catalytic efficiency [k_cat=(27.38±1.40) s⁻¹]. Furthermore, it could simultaneously hydrolyze the β-1,6-glucosidic bond at the C−20 position and the glucosidic bond directly connected to the aglycone at the C-3 position. SA2851 and SA0236 only had hydrolysis activity on the β-1,6 glucosidic bond at the C-20 position, and SA0236 had higher activity. Furthermore, we completely transformed ginsenoside Rb1 to the minor ginsenosides CK and F2 by using SA2629 and SA0236, respectively, with a β-1,2-glucosidase obtained in our lab. [Conclusion] The glycosidases that can be employed to prepare minor ginsenosides were obtained, which filled the gap in the study of β-glucosidases from F. saccharophilum.