Identification and Characterization of a Mucilaginibacter sp. Strain QM49 β-Glucosidase and Its Use in the Production of the Pharmaceutically Active Minor Ginsenosides (S)-Rh1and (S)-Rg2

Abstract

ABSTRACTHere, we isolated and characterized a new ginsenoside-transforming β-glucosidase (BglQM) fromMucilaginibactersp. strain QM49 that shows biotransformation activity for various major ginsenosides. The gene responsible for this activity,bglQM, consists of 2,346 bp and is predicted to encode 781 amino acid residues. This enzyme has a molecular mass of 85.6 kDa. Sequence analysis of BglQM revealed that it could be classified into glycoside hydrolase family 3. The enzyme was overexpressed inEscherichia coliBL21(DE3) using a maltose binding protein (MBP)-fused pMAL-c2x vector system containing the tobacco etch virus (TEV) proteolytic cleavage site. Overexpressed recombinant BglQM could efficiently transform the protopanaxatriol-type ginsenosides Re and Rg1into (S)-Rg2and (S)-Rh1, respectively, by hydrolyzing one glucose moiety attached to the C-20 position at pH 8.0 and 30°C. TheKmvalues forp-nitrophenyl-β-d-glucopyranoside, Re, and Rg1were 37.0 ± 0.4 μM and 3.22 ± 0.15 and 1.48 ± 0.09 mM, respectively, and theVmaxvalues were 33.4 ± 0.6 μmol min−1mg−1of protein and 19.2 ± 0.2 and 28.8 ± 0.27 nmol min−1mg−1of protein, respectively. A crude protopanaxatriol-type ginsenoside mixture (PPTGM) was treated with BglQM, followed by silica column purification, to produce (S)-Rh1and (S)-Rg2at chromatographic purities of 98% ± 0.5% and 97% ± 1.2%, respectively. This is the first report of gram-scale production of (S)-Rh1and (S)-Rg2from PPTGM using a novel ginsenoside-transforming β-glucosidase of glycoside hydrolase family 3.