Purification and characterization of alpha-L-arabinopyranosidase and alpha-L-arabinofuranosidase from Bifidobacterium breve K-110, a human intestinal anaerobic bacterium metabolizing ginsenoside Rb2 and Rc.
Two arabinosidases, alpha-L-arabinopyranosidase (no EC number) and alpha-L-arabinofuranosidase (EC 3.2.1.55), were purified from ginsenoside-metabolizing Bifidobacterium breve K-110, which was isolated from human intestinal microflora. alpha-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 micro mol/min/mg. alpha-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 micro mol/min/mg. The molecular mass of alpha-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 of alpha-L-arabinofuranosidase was found to be 60 kDa by gel filtration and SDS-PAGE. alpha-L-Arabinopyranosidase and alpha-L-arabinofuranosidase showed optimal activity at pH 5.5 to 6.0 and 40 degrees C and pH 4.5 and 45 degrees C, respectively. Both purified enzymes were potently inhibited by Cu(2+) and p-chlormercuryphenylsulfonic acid. alpha-L-Arabinopyranosidase acted to the greatest extent on p-nitrophenyl-alpha-L-arabinopyranoside, followed by ginsenoside Rb2. alpha-L-Arabinofuranosidase acted to the greatest extent on p-nitrophenyl-alpha-L-arabinofuranoside, followed by ginsenoside Rc. Neither enzyme acted on p-nitrophenyl-beta-galactopyranoside or p-nitrophenyl-beta-D-fucopyranoside. These findings suggest that the biochemical properties and substrate specificities of these purified enzymes are different from those of previously purified alpha-L-arabinosidases. This is the first reported purification of alpha-L-arabinopyranosidase from an anaerobic Bifidobacterium sp.
Shin
Ho-Young
HY
College of Pharmacy, Kyung Hee University, Dongdaemun-ku, Seoul, Korea.
Park
Sun-Young
SY
Sung
Jong Hwan
JH
Kim
Dong-Hyun
DH
D000693:Anaerobiosis; D001644:Bifidobacterium / Q000201:enzymology* / Q000254:growth & development*; D036145:Ginsenosides / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D007422:Intestines / Q000382:microbiology*; D008297:Male
D036145:Ginsenosides; C044462:ginsenoside Rc; C048026:ginsenoside Rb2; D006026:Glycoside Hydrolases; C019713:alpha-N-arabinofuranosidase