Brazil is a major producer of agro-industrial residues, such as sugarcane bagasse, which could be used as raw material for microbial production of cellulases as an important strategy for the development of sustainable processes of second generation ethanol production. For this purpose, this work aimed at screening for glycosyl hydrolase activities of fungal strains isolated from the Brazilian Cerrado. Among 13 isolates, a Trichoderma harzianum strain (L04) was identified as a promising candidate for cellulase production when cultured on in natura sugarcane bagasse. Strain L04 revealed a well-balanced cellulolytic complex, presenting fast kinetic production of endoglucanases, exoglucanases and β-glucosidases, achieving 4,022, U.L-1 (72 h), 1,228 U.L-1 (120 h) and 1,968 U.L-1 (48 h) as the highest activities, respectively. About 60% glucose yields were obtained from sugarcane bagasse after 18 hours hydrolysis. This new strain represents a potential candidate for on-site enzyme production using sugarcane bagasse as carbon source.
A beta-glucosidase gene (bgl4) from Humicola grisea var thermoidea was successfully expressed in Saccharomyces cerevisiae. The recombinant protein (BGL4(Sc)) was initially detected associated with yeast cells and later in the culture medium. BGL4(Sc) showed optimal pH and temperature of 6.0 and 40 degrees C, respectively, and an apparent molecular mass of 57 kDa. The enzyme showed activity against cellobiose and synthetic substrates, and was inhibited more than 80% by Fe2+, Cu2+, Zn2+, and Al3+. Using p-nitrophenyl-beta-D-glucopyranoside (pNPG) as substrate, BGL4(Sc) presented a V(max) of 6.72 micromol min(-1) mg total protein(-1) and a K (m) of 0.16 mM under optimal conditions. Most important, BGL4(Sc) is resistant to inhibition by glucose and the calculated K (i) value for this sugar is 70 mM. This feature prompts BLG4(Sc) as an ideal enzyme to be used in the saccharification process of lignocellulosic materials for ethanol production.
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