Purification and Characterization of β-D-Glucosidase (β-D-Fucosidase) from Bifidobacterium breve clb Acclimated to Cellobiose

Abstract

The β-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 β-D-glucosidase activity but also β-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°C, respectively. The enzyme was stable up to 40°C and between pH 5 and 8. The isoelectric point of the enzyme was 4.3 and the K_m values for p-nitrophenyl-β-D-glucoside and p-nitrophenyl-β-D-fucoside were 1.3 mM and 0.7 mM, respectively. This enzyme had also transferase activity for the β-D-fucosyl group but not for the β-D-glucosyl group. The N-terminal amino acid sequence of this enzyme was similar to those of β-D-glucosidase from other bacteria, actinomycetes, and plants.