This cycle was repeated until the enzymatic activity become null. The influences of pH and temperature on β-glucosidase activities were determined using the standard assay for the free and immobilised enzymes, except that the pH values were modified to a range of 2.0–8.0 (Mcllvaine, 1921) and the temperature values Selleck MAPK Inhibitor Library ranged from 10 to 60 °C. The pH stability

of β-glucosidase was determined by incubating the free enzyme solution or the alginate beads in the pH range of 2.0–8.0 for 30 min, on ice. After incubation, the mixture was used for determining residual activity, according to standard assay, using pNPβGlc as the substrate. Thermal stability was investigated by incubating the enzymatic solution or the alginate beads in 50 mM sodium phosphate buffer, pH 6.0 or 5.5, respectively, at temperatures of 45 and 50 °C for different times. After pre-incubation, aliquots of the enzymes or 4 alginate beads were collected and submitted to the standard DZNeP concentration assay, measuring the remaining activity. The relative activities were calculated in

relation to β-glucosidase activity without pre-incubation, which was considered to be 100%. Results of the analyses are presented as mean ± SD for three measurements. The Michaelis–Menten constant (KM) and Vmax for substrate hydrolysis by the free enzyme and the KMapp value for the immobilised enzyme were calculated by the Michaelis–Menten plot. Concentrations of pNPβGlc varied from 0.2 to 5.0 mM. The inhibition Dipeptidyl peptidase constant (Ki) for the free enzyme using glucose as inhibitor was determined by varying the pNPβGlc concentrations from 0.05 to 1.2 mM in the presence of 50, 100 or 120 mM of glucose. Enzymatic assays were performed with various synthetic, natural and polymeric substrates. The reaction mixtures contained 650 μL of 50 mM sodium phosphate buffer pH 6.0, 0–100 μL of enzyme solution and 250 μL of synthetic substrates (0.5 mM)

or celobiose, lactose, maltose, gentiobiose, melibiose and sucrose (2.5 mM) or cellulose (0.025%). Activities were measured under standard assay conditions at 40 °C. The data presented for all enzyme activity determinations are mean values ± SD of three measurements. The effects of ions, simple sugars and reducing agents on enzyme activity were assayed by the standard methods. Reaction mixtures contained 450 μL of 50 mM sodium phosphate buffer pH 6.0, 0–100 μL of the enzyme solution and 200 μL of the compounds (0.2 and 2 mM). The data presented for all enzyme activity assays are mean values ± SD of measurements performed in triplicate. The soy molasses samples were kindly donated by Melaços Brasileiros Ltda., Saltinho, São Paulo, Brazil. One gram samples of soy molasses were incubated with either 10 U of free β-glucosidase in 50 mM sodium phosphate buffer pH 6.0 (10 mL) or with a calculated number of beads corresponding to 10 U of β-glucosidase in 50 mM sodium phosphate buffer pH 5.