Purification and Characterization of a Novel Sennoside-Hydrolyzing β-Glucosidase from Bifidobacterium Sp. Strain SEN, a Human Intestinal Anaerobe

Abstract

A novel β-glucosidase, which is inducible and capable of catalyzing the hydrolysis of sennosides, was purified from Bifidobacterium sp. strain SEN with Triton X-100 solubilization and DEAE-cellulose column chromatography, by which hydrolytic activities toward sennoside B, 4-methylumbelliferyl β-glucoside (MUG), and p-nitrophenyl β-glucoside (pNPG) were obtained together in the same eluted fractions. The activity was stable against detergents such as sodium dodecyl sulfate (SDS) and Triton X-100, but was denatured by SDS and β-mercaptoethanal when heated. The final preparation was shown to be nearly homogeneous on SDS-polycrylamide gel electrophoresis (PAGE) either after the enzyme was denatured or when it was not denatured. In the non-denaturing SDS-PAGE, a single protein band hydrolyzed MUG on the gel. In the denaturing SDS-PAGE, the subunit mass of the enzyme was estimated to be 110 kDa.The enzyme was optimally active at pH 6.0 for hydrolysis of sennoside B and MUG. K_m values for sennoside B and MUG are 0.94 and 0.53 mM, respectively. The enzyme also catalyzed the hydrolysis of pNPG, amygdalin, geniposide and salicin. It was less active against methyl β-glucoside and incapable of hydrolyzing cellobiose. The β-glucosidase activity was inhibited by deoxynojirimycin and p-chloromercuribenzenesulfonic acid, but was less susceptible to several metals (FeSO₄, ZnCl₂, and CuSO₄), and 5, 5'-dithio-bis(2-nitrobenzoic acid).