Purification and Characterization ofα-l-Arabinopyranosidase andα-l-Arabinofuranosidase fromBifidobacteriumbreveK-110, a Human Intestinal AnaerobicBacterium Metabolizing Ginsenoside Rb2 andRc

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Abstract
Two arabinosidases, α-l-arabinopyranosidase (no EC number) and α-l-arabinofuranosidase (EC 3.2.1.55), were purified from ginsenoside-metabolizingBifidobacterium breveK-110, which was isolated from human intestinal microflora. α-l-Arabinopyranosidase was purified to apparent homogeneity, using a combination of ammonium sulfate fractionation, DEAE-cellulose, butyl Toyopearl, hydroxyapatite Ultrogel, QAE-cellulose, and Sephacryl S-300 HR column chromatography, with a final specific activity of 8.81 μmol/min/mg.α -l-Arabinofuranosidase was purified to apparent homogeneity, using a combination of ammonium sulfate fractionation, DEAE-cellulose, butyl Toyopearl, hydroxyapatite Ultrogel, Q-Sepharose, and Sephacryl S-300 column chromatography, with a final specific activity of 6.46 μmol/min/mg. The molecular mass ofα -l-arabinopyranosidase was found to be 310 kDa by gel filtration, consisting of four identical subunits (77 kDa each, measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis [SDS-PAGE]), and that ofα -l-arabinofuranosidase was found to be 60 kDa by gel filtration and SDS-PAGE. α-l-Arabinopyranosidase and α-l-arabinofuranosidase showed optimal activity at pH 5.5 to 6.0 and 40°C and pH 4.5 and 45°C, respectively. Both purified enzymes were potently inhibited by Cu2+andp-chlormercuryphenylsulfonic acid.α -l-Arabinopyranosidase acted to the greatest extent onp-nitrophenyl-α-l-arabinopyranoside, followed by ginsenoside Rb2. α-l-Arabinofuranosidase acted to the greatest extent onp-nitrophenyl-α-l-arabinofuranoside, followed by ginsenoside Rc. Neither enzyme acted onp-nitrophenyl-β-galactopyranoside orp-nitrophenyl-β-d-fucopyranoside. These findings suggest that the biochemical properties and substrate specificities of these purified enzymes are different from those of previously purified α-l-arabinosidases. This is the first reported purification ofα -l-arabinopyranosidase from an anaerobicBifidobacteriumsp.

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