Resumo

Renewable energy achievement is now a major concern and celulosic ethanol is a promising option to replace fossil fuels. β-glucosidases act at the final step of cellulose hydrolysis, releasing endo- and exoglucanases from product inhibition. However, they are usually inhibited by glucose, limiting the process efficiency. Although five β-glucosidases stimulated by glucose have been described, little is known about the stimulation mechanisms. Here we present a kinetic and biophysical study of the stimulation of a β-glucosidase from H. insolens by glucose and xylose. pNP-Glu hydrolysis occurred with positive cooperativity, V_M= 22.4 U/mg and K_0.5= 0.22 mM. Maximal stimulation by glucose (1.8-fold) or xylose (2.0-fold) occurred at 40-50 mM. Higher concentrations were inhibitory and above 400 mM glucose or 700 mM xylose the activity was lower than in the absence of monosaccharides. Glucose and xylose compete for the same modulatory site(s) in the enzyme molecule, and compete with pNP-Glu at the catalytic site(s) with K_i= 30 mM and 260 mM, respectively. Stimulation by glucose in the presence of fixed concentrations of pNP-Glu (0.2-2.0 mM) occurred with positive cooperativity, and V_M (17.0-48.9 U/mg) and K_0.5 (1.7-12.0 mM) increased with raising pNP-Glu concentrations. Similarly, for xylose stimulation V_Mvaried from 18.3 to 50.5 U/mg and K_0.5 from 2.0-17.0 mM. Stimulation by pNP-Glu was also modulated by fixed concentrations of glucose (5-20 mM), and V_M (32.0-43.7 U/mg) and K_0.5 (0.22-0.37 mM) increased as glucose concentration raised. Equally, fixed concentrations of xylose (10-30 mM) resulted in V_M (39.6-54.2 U/mg) and K_0.5 (0.22-0.35 mM) increases. Substrate stimulation occurred with positive cooperativity in all conditions. Above 5 mM pNP-Glu inhibited the enzymatic activity independent of the presence of monosaccharides. Circular dichroism (CD) analyses showed that the enzyme secondary structure was nearly unchanged at saturating concentrations of cellobiose, but the fluorescence was significantly altered. Evident alterations in CD spectra occurred at saturating concentrations of glucose or xylose, but minor changes were detected at saturating conditions of both cellobiose and glucose or xylose. Data suggest that H. insolens ß-glucosidase shows at least a catalytic and a modulatory site, which may be both occupied by the substrate and modulators. Cooperative effects suggest interactions among substrate and modulator sites, and enzyme conformation is altered in response to substrate/modulators addition. Thus, the modulation of activity results from a fine balance of substrate and modulators concentrations.