title: Extracellular enzymes of the white-rot fungus Fomes fomentarius and purification of 1,4-β-glucosidase.
abstract: Production of the lignocellulose-degrading enzymes endo-1,4-β-glucanase, 1,4-β-glucosidase, cellobiohydrolase, endo-1,4-β-xylanase, 1,4-β-xylosidase, Mn peroxidase, and laccase was characterized in a common wood-rotting fungus Fomes fomentarius, a species able to efficiently decompose dead wood, and compared to the production in eight other fungal species. The main aim of this study was to characterize the 1,4-β-glucosidase produced by F. fomentarius that was produced in high quantities in liquid stationary culture (25.9 U ml(-1)), at least threefold compared to other saprotrophic basidiomycetes, such as Rhodocollybia butyracea, Hypholoma fasciculare, Irpex lacteus, Fomitopsis pinicola, Pleurotus ostreatus, Piptoporus betulinus, and Gymnopus sp. (between 0.7 and 7.9 U ml(-1)). The 1,4-β-glucosidase enzyme was purified to electrophoretic homogeneity by both anion-exchange and size-exclusion chromatography. A single 1,4-β-glucosidase was found to have an apparent molecular mass of 58 kDa and a pI of 6.7. The enzyme exhibited high thermotolerance with an optimum temperature of 60 °C. Maximal activity was found in the pH range of 4.5-5.0, and K (M) and V (max) values were 62 μM and 15.8 μmol min(-1) l(-1), respectively, when p-nitrophenylglucoside was used as a substrate. The enzyme was competitively inhibited by glucose with a K (i) of 3.37 mM. The enzyme also acted on p-nitrophenylxyloside, p-nitrophenylcellobioside, p-nitrophenylgalactoside, and p-nitrophenylmannoside with optimal pH values of 6.0, 3.5, 5.0, and 4.0-6.0, respectively. The combination of relatively low molecular mass and low K (M) value make the 1,4-β-glucosidase a promising enzyme for biotechnological applications.
authors.affiliation: Laboratory of Environmental Microbiology, Institute of Microbiology of the ASCR, v.v.i., Vídeňská 1083, 14220 Praha 4, Czech Republic.
authors.forename: Tomáš
authors.initials: T
authors.lastname: Větrovský
authors.forename: Petr
authors.initials: P
authors.lastname: Baldrian
authors.forename: Jiří
authors.initials: J
authors.lastname: Gabriel
mesh_terms: D055453:Coriolaceae / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D004795:Enzyme Stability; D005110:Extracellular Space / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D007700:Kinetics; D021381:Protein Transport; D013379:Substrate Specificity; D014995:Xylosidases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*
chemical_list: D005656:Fungal Proteins; D014995:Xylosidases; C026579:exo-1,4-beta-D-xylosidase