Purification and characterization of a geniposide-hydrolyzing beta-glucosidase from Eubacterium sp. A-44, a strict anaerobe from human feces.
A geniposide-hydrolyzing beta-glucosidase was discovered in Eubacterium sp. A-44, a human intestinal anaerobe. The enzyme was intracellularly distributed in the bacterium, and purified to homogeneity from the extract using Butyl-Toyopearl 650M, Sephacryl S-300, hydroxyapatite and chromatofocusing column chromatography. The enzyme was a single polypeptide chain with the molecular weight of 90 kDa and the N-terminal amino acid sequence initiated from methionine up to the 29th residue did not show more than 50% homology against known protein sequences. A broad substrate specificity was shown for the beta-glucosidase to hydrolyze aryl beta-D-glucosides (p-nitrophenyl beta-D-glucopyranoside-pNPG, esculin and salicin), alkyl beta-D-glucosides (geniposide and amygdalin) and cellobiose. The Km values (mM) for various beta-D-glucosides were 0.068 for geniposide, 0.10 for pNPG, 0.21 for esculin, 0.22 for salicin, 2.9 for amygdalin, and 0.91 for cellobiose. The pH optimum with pNPG and geniposide as the substrates was 6.0. The enzyme was inhibited by sulfhydryl reagents, Cu2+, and nojirimycin bisulfite.
Yang
L
L
Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, Japan.
Akao
T
T
Kobashi
K
K
D000595:Amino Acid Sequence; D005051:Eubacterium / Q000201:enzymology*; D005243:Feces / Q000382:microbiology*; D006801:Humans; D006868:Hydrolysis; D039823:Iridoids; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011714:Pyrans / Q000378:metabolism*; D001617:beta-Glucosidase / Q000302:isolation & purification*
D039823:Iridoids; D011714:Pyrans; C007835:geniposide; D001617:beta-Glucosidase