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Applied and Environmental Microbiology, December 2003, p. 7116-7123, Vol. 69, No. 12
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.12.7116-7123.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Purification and Characterization of -L-Arabinopyranosidase and -L-Arabinofuranosidase from Bifidobacterium breve K-110, a Human Intestinal Anaerobic Bacterium Metabolizing Ginsenoside Rb2 and Rc

Ho-Young Shin,¹ Sun-Young Park,¹ Jong Hwan Sung,² and Dong-Hyun Kim^1*

College of Pharmacy, Kyung Hee University, Dongdaemun-ku, Seoul,¹ Central Research Institute, Il Hwa Co., Ltd., Guri, Kyonggi-do,Korea²

Received 30 May 2003/ Accepted 14 September 2003

Two arabinosidases, -L-arabinopyranosidase (no EC number) and -L-arabinofuranosidase (EC 3.2.1.55), were purified from ginsenoside-metabolizing Bifidobacterium breve K-110, which was isolated from human intestinal microflora. -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. -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 of -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 -L-arabinofuranosidase was found to be 60 kDa by gel filtration and SDS-PAGE. -L-Arabinopyranosidase and -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. -L-Arabinopyranosidase acted to the greatest extent on p-nitrophenyl--L-arabinopyranoside, followed by ginsenoside Rb2. -L-Arabinofuranosidase acted to the greatest extent on p-nitrophenyl--L-arabinofuranoside, followed by ginsenoside Rc. Neither enzyme acted on p-nitrophenyl-ß-galactopyranoside or p-nitrophenyl-ß-D-fucopyranoside. These findings suggest that the biochemical properties and substrate specificities of these purified enzymes are different from those of previously purified -L-arabinosidases. This is the first reported purification of -L-arabinopyranosidase from an anaerobic Bifidobacterium sp.

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* Corresponding author. Mailing address: College of Pharmacy, Kyung Hee University, 1, Hoegi, Dongdaemun-ku, Seoul 130-701, Korea. Phone: 82-2-961-0374. Fax: 82-2-957-5030. E-mail: dhkim{at}khu.ac.kr.

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Applied and Environmental Microbiology, December 2003, p. 7116-7123, Vol. 69, No. 12
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.12.7116-7123.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.