Purification and characterization of a beta-glucosidase from solid-state cultures of Humicola grisea var. thermoidea.
The thermophilic fungus Humicola grisea var. thermoidea produced beta-glucosidase activity when grown in a solid-state culture on wheat bran as carbon source. A beta-glucosidase was purified to apparent homogeneity by ultrafiltration, gel filtration chromatography on Sephacryl S-100, and ion-exchange chromatography on S-Sepharose, as judged by sodium dodecyl sulfate--polyacrylamide gel electrophoresis (SDS-PAGE) on a 12.5% (w/v) slab gel. The enzyme had a molecular mass of 82 and 156 kDa, as estimated by SDS-PAGE and gel filtration on a high performance liquid chromatographic column, respectively, suggesting that the native enzyme may consist of two identical subunits. The purified enzyme was thermostable at 60 degrees C for 1 h with a half-life of 15 min at 65 degrees C, and displayed optimum activity at 60 degrees C and a pH range. of 4.0-4.5. The Km and Vmax values for p-nitrophenyl beta-D-glucopyranoside were determined to be 0.316 mM and 0.459 IU.mL-1, respectively. D-Glucose, D-gluconic acid lactone, Hg2+, Cu2+, and Mn2+ inhibited beta-glucosidase activity. The enzyme activity was competitively inhibited by D-glucose (ki = 0.6 mM). The purified enzyme was very active against cellobiose and p-nitrophenyl beta-D-glucopyranoside.
Ferreira Filho
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Departmento de Biologia Celular, Universidade de Brasilia, Distrito Federal, Brasil.
D002413:Cations, Divalent / Q000494:pharmacology; D002475:Cellobiose / Q000378:metabolism; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003470:Culture Media; D004043:Dietary Fiber; D005656:Fungal Proteins / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D005942:Gluconates / Q000494:pharmacology; D005947:Glucose / Q000494:pharmacology; D005960:Glucosides / Q000378:metabolism*; D007700:Kinetics; D007783:Lactones; D003904:Mitosporic Fungi / Q000201:enzymology*; D008970:Molecular Weight; D013379:Substrate Specificity; D014462:Ultrafiltration; D001617:beta-Glucosidase / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism
D002413:Cations, Divalent; D003470:Culture Media; D004043:Dietary Fiber; D005656:Fungal Proteins; D005942:Gluconates; D005960:Glucosides; D007783:Lactones; D002475:Cellobiose; C025193:4-nitrophenyl beta-D-glucoside; D001617:beta-Glucosidase; D005947:Glucose; C010730:beta-glucono-1,5-lactone