Cloning and characterization of ginsenoside Ra1-hydrolyzing β-D-xylosidase from Bifidobacterium breve K-110

β-D-Xylosidase (E.C. 3.2.1.37) from Bifidobacterium breve K-110, which hydrolyzes ginsenoside Ra1 to ginsenoside Rb2, was cloned and expressed in Escherichia coli. The (His ₆)-tagged recombinant enzyme, designated as XlyBK- 110, was efficiently purified using Ni ²⁺-affinity chromatography (109.9-fold, 84% yield). The molecular mass of XylBK- 100 was found to be 55.7 kDa by SDS-PAGE. Its sequence revealed a 1,347 bp open reading frame (ORF) encoding a protein containing 448 amino acids, which showed 82% identity (DNA) to the previously reported glycosyl hydrolase family 30 of Bifidobacterium adolescentis ATCC 15703. The K _m and V _max values toward p-nitrophenyl-β-D-xylopyranoside (pNPX) were 1.45mM and 10.75 μmol/min/mg, respectively. This enzyme had pH and temperature optima at 6.0 and 45°C, respectively. XylBK-110 acted to the greatest extent on xyloglucosyl kakkalide, followed by pNPX and ginsenoside Ra1, but did not act on p-nitrophenyl-α-Larabinofuranoside, p-nitrophenyl-β-D-glucopyranoside, or p-nitrophenyl-β-D-fucopyranoside. In conclusion, this is the first report on the cloning and expression of β-Dxylosidase- hydrolyzing ginsenoside Ra1 and kakkalide from human intestinal microflora.