Resumo

ß-glucosidases are key enzymes for efficient cellulose saccharification, releasing glucanases from product inhibition. However, these enzymes are strongly inhibited by glucose, raising the interest on cheap forms to be used at high loads per g cellulosic substrate. On the other hand, recent studies revealed that xylan and short xylooligosaccharides are also potent inhibitors of endo- and exoglucanases and thus efficient enzyme cocktails for the hydrolysis of lignocellulosic materials must include hemicellulases and ß-xylosidases, releasing xylose. In this study we purified and biochemically characterized a xylose-stimulated ß-glucosidase from Humicola brevis var. thermoidea cultured under solid state fermentation in wheat bran and water. Enzyme purification involved (NH₄)₂SO₄ precipitation followed by DMAE-Fractogel, Sephacryl S200 and Phenyl-Sepharose chromatography. Enzymatic activity was assayed in 50 mmol.L^-1 sodium acetate buffer, pH 5.5, containing 1 mmol.L^-1 p-nitrophenyl-ß-D-glucopyranoside (pNP-Glc). The enzyme was purified 5.1-fold with 1.2% yield and specific activity of 20.7 U.mg^-1. A single protein band in 7% PAGE was coincident with an activity band using esculin as substrate. SDS-PAGE analysis of the purified enzyme also revealed a single protein band with apparent molecular mass of 100 kDa. Optimal pH and temperature of activity were 5.5 and 65^oC, respectively. The purified ß-glucosidase was fully stable from pH 5.0-8.0, and retained about 85% of its initial activity after 8 h incubation in water at 55^oC, with a half-life of about 27 h. Half-lives of 1.5 h and 0.3 h were determined for enzyme incubation at 60^oC and 65^oC, respectively. Kinetic parameters for the hydrolysis of pNP-Glc were V_M= 23.3 U mg^-1 and K_0.5= 69.9 μmol.L^-1. Increasing concentrations of xylose up to 200 mmol.L^-1 stimulated the enzymatic activity about 1.45-fold, reaching a V_M of 33.7 U.mg^-1, with K_0.5= 1.6 mmol.L^-1. Moreover, the enzyme maintained about 65% of its activity in the presence of 700 mmol.L^-1 xylose. The high thermal and pH stability, allied to the strong stimulation by xylose of H. brevis ß-glucosidase suggest a good potential for application in lignocellulosic biomass saccharification processes.