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Cloning and Characterization of Ginsenoside Ra1-Hydrolyzing ${\beta}$-D-Xylosidase from Bifidobacterium breve K-110

  • Hyun, Yang-Jin (Department of Life and Nanopharmaceutical Sciences and Department of Pharmaceutical Science, Kyung Hee University) ;
  • Kim, Bo-Mi (Department of Life and Nanopharmaceutical Sciences and Department of Pharmaceutical Science, Kyung Hee University) ;
  • Kim, Dong-Hyun (Department of Life and Nanopharmaceutical Sciences and Department of Pharmaceutical Science, Kyung Hee University)
  • 투고 : 2011.10.04
  • 심사 : 2011.11.28
  • 발행 : 2012.04.28

초록

${\beta}$-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 ($His_6$)-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-${\beta}$-D-xylopyranoside (pNPX) were 1.45mM and 10.75 ${\mu}mol/min/mg$, respectively. This enzyme had pH and temperature optima at 6.0 and $45^{\circ}C$, respectively. XylBK-110 acted to the greatest extent on xyloglucosyl kakkalide, followed by pNPX and ginsenoside Ra1, but did not act on p-nitrophenyl-${\alpha}$-L-arabinofuranoside, p-nitrophenyl-${\beta}$-D-glucopyranoside, or p-nitrophenyl-${\beta}$-D-fucopyranoside. In conclusion, this is the first report on the cloning and expression of ${\beta}$-D-xylosidase-hydrolyzing ginsenoside Ra1 and kakkalide from human intestinal microflora.

키워드

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