초록
<P>Here, we isolated and characterized a new ginsenoside-transforming β-glucosidase (BglQM) from <I>Mucilaginibacter</I> sp. strain QM49 that shows biotransformation activity for various major ginsenosides. The gene responsible for this activity, <I>bglQM</I>, 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 in <I>Escherichia coli</I> BL21(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 Rg<SUB>1</SUB> into (<I>S</I>)-Rg<SUB>2</SUB> and (<I>S</I>)-Rh<SUB>1</SUB>, respectively, by hydrolyzing one glucose moiety attached to the C-20 position at pH 8.0 and 30°C. The <I>K<SUB>m</SUB></I> values for <I>p</I>-nitrophenyl-β-<SMALL>d</SMALL>-glucopyranoside, Re, and Rg<SUB>1</SUB> were 37.0 ± 0.4 μM and 3.22 ± 0.15 and 1.48 ± 0.09 mM, respectively, and the <I>V</I><SUB>max</SUB> values were 33.4 ± 0.6 μmol min<SUP>−1</SUP> mg<SUP>−1</SUP> of protein and 19.2 ± 0.2 and 28.8 ± 0.27 nmol min<SUP>−1</SUP> mg<SUP>−1</SUP> of protein, respectively. A crude protopanaxatriol-type ginsenoside mixture (PPTGM) was treated with BglQM, followed by silica column purification, to produce (<I>S</I>)-Rh<SUB>1</SUB> and (<I>S</I>)-Rg<SUB>2</SUB> at chromatographic purities of 98% ± 0.5% and 97% ± 1.2%, respectively. This is the first report of gram-scale production of (<I>S</I>)-Rh<SUB>1</SUB> and (<I>S</I>)-Rg<SUB>2</SUB> from PPTGM using a novel ginsenoside-transforming β-glucosidase of glycoside hydrolase family 3.</P>