ABSTRACT
Two
arabinosidases, α-l-arabinopyranosidase (no EC
number) and α-l-arabinofuranosidase (EC
3.2.1.55), were purified from ginsenoside-metabolizing
Bifidobacterium breve K-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+ and p-chlormercuryphenylsulfonic acid.α
-l-Arabinopyranosidase acted to the greatest extent
on p-nitrophenyl-α-l-arabinopyranoside,
followed by ginsenoside Rb2. α-l-Arabinofuranosidase
acted to the greatest extent on
p-nitrophenyl-α-l-arabinofuranoside,
followed by ginsenoside Rc. Neither enzyme acted on
p-nitrophenyl-β-galactopyranoside or
p-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 anaerobic
Bifidobacterium
sp.
Studying Some Properties of Amylases Extracted from `d'Anjou' Pears
