title: Cloning and characterization of ginsenoside Ra1-hydrolyzing beta-D-xylosidase from Bifidobacterium breve K-110.
abstract: beta-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 (His6)-tagged recombinant enzyme, designated as XlyBK- 110, was efficiently purified using Ni²⁺-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 Km and Vmax values toward p-nitrophenyl-beta-D-xylopyranoside (pNPX) were 1.45mM and 10.75 micromol/min/mg, respectively. This enzyme had pH and temperature optima at 6.0 and 45 degrees 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-alpha-Larabinofuranoside, p-nitrophenyl-beta-D-glucopyranoside, or p-nitrophenyl-beta-D-fucopyranoside. In conclusion, this is the first report on the cloning and expression of beta-Dxylosidase- hydrolyzing ginsenoside Ra1 and kakkalide from human intestinal microflora.
authors.affiliation: Department of Life and Nanopharmaceutical Sciences and Department of Pharmaceutical Science, Kyung Hee University, Seoul 130-701, Korea.
authors.forename: Yang-Jin
authors.initials: YJ
authors.lastname: Hyun
authors.forename: Bomi
authors.initials: B
authors.lastname: Kim
authors.forename: Dong-Hyun
authors.initials: DH
authors.lastname: Kim
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D001644:Bifidobacterium / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D003001:Cloning, Molecular; D005243:Feces / Q000382:microbiology; D036145:Ginsenosides / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D014995:Xylosidases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D036145:Ginsenosides; D014995:Xylosidases; C026579:exo-1,4-beta-D-xylosidase