Purification and characterization of beta-D-glucosidase (beta-D-fucosidase) from Bifidobacterium breve clb acclimated to cellobiose.
The beta-D-glucosidase (EC. 3.2.1.21) activity of Bifidobacterium breve 203 was increased by acclimation with cellobiose, and the enzyme was purified to homogeneity from cell-free extracts of an acclimatized strain of B. breve clb, by ammonium sulfate fractionation and column chromatographies of anion-exchange, gel filtration, Gigapaite, and hydrophobic interaction. This enzyme had not only beta-D-glucosidase activity but also beta-D-fucosidase activity, which is specific to Bifidobacteria in intestinal flora. The molecular weight of the purified enzyme was estimated to be 47,000-48,000 and the enzyme was assumed to be a monomeric protein. The optimum pH and temperature of the enzyme were around 5.5 and 45 degrees C, respectively. The enzyme was stable up to 40 degrees C and between pH 5 and 8. The isoelectric point of the enzyme was 4.3 and the Km values for p-nitrophenyl-beta-D-glucoside and p-nitrophenyl-beta-D-fucoside were 1.3 mM and 0.7 mM, respectively. This enzyme had also transferase activity for the beta-D-fucosyl group but not for the beta-D-glucosyl group. The N-terminal amino acid sequence of this enzyme was similar to those of beta-D-glucosidase from other bacteria, actinomycetes, and plants.
Nunoura
N
N
Department of Food Science and Technology, Kyoto University, Kitashirakawa, Japan.
Ohdan
K
K
Yano
T
T
Yamamoto
K
K
Kumagai
H
H
D000328:Adult; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D001644:Bifidobacterium / Q000201:enzymology*; D002412:Cations; D002475:Cellobiose / Q000378:metabolism / Q000494:pharmacology*; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D006031:Glycosylation; D006801:Humans; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D013379:Substrate Specificity; D001617:beta-Glucosidase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*
D001426:Bacterial Proteins; D002412:Cations; D008670:Metals; D002475:Cellobiose; D001617:beta-Glucosidase