RESUMEN
BACKGROUND: The increasing demand and the continuous depletion in fossil fuels have persuaded researchers to investigate new sources of renewable energy. Bioethanol produced from cellulose could be a cost-effective and a viable alternative to petroleum. It is worth note that ß-glucosidase plays a key role in the hydrolysis of cellulose and therefore in the production of bioethanol. This study aims to investigate a simple and standardized method for maximization of extracellular ß-glucosidase production from a novel fungal isolate under solid-state fermentation using agro-industrial residues as the sole source of carbon and nitrogen. Furthermore, purification and characterization of ß-glucosidase were performed to determine the conditions under which the enzyme displayed the highest performance. RESULTS: A fungus identified genetically as a new Aspergillus sp. DHE7 was found to exhibit the highest extracellular ß-glucosidase production among the sixty fungal isolates tested. Optimization of culture conditions improved the enzyme biosynthesis by 2.1-fold (174.6 ± 5.8 U/g of dry substrate) when the fungus grown for 72 h at 35 °C on jojoba meal with 60% of initial substrate moisture, pH 6.0, and an inoculum size of 2.54 × 107 spores/mL. The enzyme was purified to homogeneity through a multi-step purification process. The purified ß-glucosidase is monomeric with a molecular mass of 135 kDa as revealed by the SDS-PAGE analysis. Optimum activity was observed at 60 °C and pH of 6.0, with a remarkable pH and thermal stability. The enzyme retained about 79% and 53% of its activity, after 1 h at 70 °C and 80 °C, respectively. The purified ß-glucosidase hydrolysed a wide range of substrates but displaying its greater activity on p-nitrophenyl-ß-D-glucopyranoside and cellobiose. The values of Km and Vmax on p-nitrophenyl ß-D-glucopyranoside were 0.4 mM and 232.6 U/mL, respectively. Purified ß-glucosidase displayed high catalytic activity (improved by 25%) in solutions contained ethanol up to 15%. CONCLUSION: ß-glucosidase characteristics associated with its ability to hydrolyse cellobiose, underscore its utilization in improving the quality of food and beverages. In addition, taking into consideration that the final concentration of ethanol produced by the conventional methods is about 10%, suggests its use in ethanol-containing industrial processes and in the saccharification processes for bioethanol production.
RESUMEN
A ß-glycoside hydrolase was isolated from cell walls material in Coprinopsis cinerea elongating stipes. By analysis of SDS-PAGE, MALDI-TOF/TOF MS and substrate specificity, this enzyme was characterized as an extracellular ß-glucosidase which is a trimer consisting of three homosubunits. ß-Glucosidase did not degrade ß-glucans with modified ends, whereas it hydrolyzed various ß-glucans with free ends and related oligosaccharides with ß-1,3-, ß-1,4- or ß-1,6-linkages. Although this ß-glucosidase possesses glycosyltransferase activity on laminarioligosaccharides, it did not transfer glucose residues from laminaritriose to ß-glucan in stipe cell walls to produce larger ß-glucan molecules; instead, it caused a decrease in the molecular size of stipe wall ß-glucan by removing glucose. Relatively, the molecular size of wall ß-glucans in the elongating apical stipe was less than that found in the non-elongating basal stipes, and this ß-glucosidase was more highly expressed in the elongating apical stipe than in non-elongating basal regions. Therefore, we propose that ß-glucosidase functions by trimming or cutting the ß-glucan side chains on the ß-1,3-glucan backbone to prevent them from forming longer branches, keeping the wall plastic to promote diffuse wall growth.