title: Characterization of a GH3 family β-glucosidase from Dictyoglomus turgidum and its application to the hydrolysis of isoflavone glycosides in spent coffee grounds.
abstract: A recombinant β-glucosidase from Dictyoglomus turgidum was purified with a specific activity of 31 U/mg by His-Trap affinity chromatography. D. turgidum β-glucosidase was identified as a memmber of the glycoside hydrolase (GH) 3 family on the basis of its amino acid sequence. The native enzyme existed as an 86 kDa monomer with an activity maximum at pH 5 and 85 °C with a half-life of 334 min. The hydrolytic activity of the enzyme with aryl-glycoside substrates was the highest for p-nitrophenyl (pNP)-β-D-glucopyranoside (with a K(m) of 1.3 mM and a k(cat) of 13900 1/s), followed by oNP-β-D-glucopyranoside, pNP-β-D-xylopyranoside, pNP-β-D-fucopyranoside, and pNP-β-D-galactopyranoside. However, no activity was observed for oNP-β-D-galactopyranoside, pNP-α-D-glucopyranoside, pNP-α-D-glucopyranoside, pNP-β-D-mannopyranoside, pNP-β-L-arabinopyranoside, and pNP-α-L-rhamnopyranoside. The hydrolytic activity of the β-glucosidase for coffee isoflavones followed the order genistin (with a K(m) of 0.67 mM and a k(cat) of 5750 1/s) > daidzin > ononin > glycitin. The concentrations of daidzin in ground coffee and spent coffee grounds were 160 and 107 μg/g, respectively, but other isoflavones were present at low concentrations or absent. The enzyme completely hydrolyzed 1.2 mM daidzin in spent coffee grounds after 2 h, with a productivity of 0.6 mM/h. This is the first report concerning the enzymatic hydrolysis of isoflavone glycosides in spent coffee grounds.
authors.affiliation: Department of Bioscience and Biotechnology, Konkuk University, Seoul 143-701, Republic of Korea.
authors.forename: Yeong-Su
authors.initials: YS
authors.lastname: Kim
authors.forename: Soo-Jin
authors.initials: SJ
authors.lastname: Yeom
authors.forename: Deok-Kun
authors.initials: DK
authors.lastname: Oh
mesh_terms: D000595:Amino Acid Sequence; D001419:Bacteria / Q000737:chemistry / Q000145:classification / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D040503:Coffea / Q000737:chemistry*; D004795:Enzyme Stability; D006027:Glycosides / Q000737:chemistry*; D006868:Hydrolysis; D007529:Isoflavones / Q000737:chemistry*; D007700:Kinetics; D008969:Molecular Sequence Data; D013379:Substrate Specificity; D014866:Waste Products / Q000032:analysis*; D001617:beta-Glucosidase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D006027:Glycosides; D007529:Isoflavones; D014866:Waste Products; D001617:beta-Glucosidase