RESUMO
Abstract Obtaining low cost lignocellulolytic enzymes and efficient biomass pretreatment are key to increase the competitiveness of second-generation ethanol in comparison with fossil fuels. The enzymatic cocktail produced by the Chrysoporthe cubensis fungus as well as the mixture prepared with the cocktails of the Chrysoporthe cubensis and Penicillium pinophilum fungi have already proven to be efficient for hydrolyzing biomass pretreated with alkali. In this study, they were evaluated in saccharification of sugarcane bagasse pretreated with dilute acid or hot water at 121°C using an enzyme loading equal to 8 filter paper units per gram of biomass. The most promising results were obtained from the hydrolysis of biomass pretreated with hot water by the C. cubensis-P. pinophilum enzymes blend. In this condition, the glucose and xylose production were 25.2 g.L-1 and 4.6 g.L-1, respectively, that resulted in the conversion of 68% of glucan and 23% of xylan in only 48 hours. This study shows that the hydrothermal pretreatment is a promising alternative to improve the enzymes performance, produced by the fungi C. cubensis and P. pinophilum, in the sugarcane bagasse hydrolysis without the need of chemical compounds, generally used in the acid and alkali pretreatments. Furthermore, the hydrothermal pretreatment for 60 min allowed all cocktails applied to convert the cellulose efficiently with only 24 h of saccharification, which contributes to the energy savings employed in the process.
RESUMO
Cellulases find increasing prominence in sustainable production of fuel and feedstock from lignocellulosic biomass. The purification and biochemical characterization of individual components of cellulase complex is important to understand the mechanism of their action for the solubilization of crystalline cellulose. In this study, an extra-cellular endoglucanase isolated from culture filtrate of Penicillium pinophilum MS 20 was purified to homogeneity by ammonium sulphate precipitation, ion-exchange chromatography and gel filtration. The purified endoglucanase (specific activity 69 U/mg) was a monomeric protein with molecular mass of 42 kDa, as determined by SDS-PAGE. The endoglucanase was active over a broad range of pH (4-7) with maximum activity at pH 5 and showed optimum temperature of 50°C. It retained 100% activity at 50°C for 6 h and half- lives of 4 h and 3 h at 60°C and 70°C, respectively. The kinetic constants for the endoglucanase determined with carboxymethyl cellulose as substrate were Vmax of 72.5 U/mg and apparent Km of 4.8 mg/ml. The enzyme also showed moderate activity towards H3PO4 swollen cellulose and p-nitrophenyl β-D-glucoside, but no activity towards filter paper, Avicel and oat spelt xylan. The activity was positively modulated by 47, 32 and 25% in the presence of Co2+, Zn2+ and Mg2+, respectively to the reaction mixture. The wide pH stability (4-7) and temperature stability up to 50°C of endoglucanase makes the enzyme suitable for use in cellulose saccharification at moderate temperature and pH.