|1301-1||Purification and biochemical characterization of a xylose-stimulated ß-glucosidase from the thermophilic fungus Humicola brevis var. thermoidea|
|Autores:||Douglas Chodi Masui (FFCLRP/USP - Universidade de São Paulo/FFCLRP/Depto Biologia) ; Ana Lucia Ribeiro Latorre Zimbardi (FFCLRP/USP - Universidade de são Paulo/FFCLRP/Depto Química) ; Flavio Henrique Moreira Souza (FFCLRP/USP - Universidade de são Paulo/FFCLRP/Depto Química) ; Rosa dos Prazeres Melo Furriel (FFCLRP/USP - Universidade de são Paulo/FFCLRP/Depto Química) ; João Atílio Jorge (FFCLRP/USP - Universidade de São Paulo/FFCLRP/Depto Biologia) |
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 65oC, 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 55oC, with a half-life of about 27 h. Half-lives of 1.5 h and 0.3 h were determined for enzyme incubation at 60oC and 65oC, respectively. Kinetic parameters for the hydrolysis of pNP-Glc were VM= 23.3 U mg-1 and K0.5= 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 VM of 33.7 U.mg-1, with K0.5= 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.
Palavras-chave: ß-glucosidase purification, Humicola brevis, kinetic characterization, xylose-stimulated enzyme