Purification and characterization of a novel sennoside-hydrolyzing beta-glucosidase from Bifidobacterium sp. strain SEN, a human intestinal anaerobe.
A novel beta-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 beta-glucoside (MUG), and p-nitrophenyl beta-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 beta-mercaptoethanal when heated. The final preparation was shown to be nearly homogeneous on SDS-polyacrylamide 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. Km 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 beta-glucoside and incapable of hydrolyzing cellobiose. The beta-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).
Yang
L
L
Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, Japan.
Akao
T
T
Kobashi
K
K
Hattori
M
M
D000880:Anthraquinones / Q000302:isolation & purification / Q000378:metabolism*; D001644:Bifidobacterium / Q000201:enzymology* / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D005243:Feces / Q000382:microbiology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D009598:Nitrophenylgalactosides / Q000494:pharmacology; D017830:Octoxynol / Q000494:pharmacology; D012676:Senna Extract; D000081226:Sennosides; D013379:Substrate Specificity; D001617:beta-Glucosidase / Q000302:isolation & purification* / Q000378:metabolism
D000880:Anthraquinones; D000081226:Sennosides; D009598:Nitrophenylgalactosides; C059200:4-nitrophenylgalactoside; D012676:Senna Extract; D017830:Octoxynol; D001617:beta-Glucosidase