Four strains of Aspergillus (Aspergillus niger CDBB-H-176, A. niger CDBB-H-175, A. niger ATCC 9642, and Aspergillus terreus CDBB-H-194) were used to produce extracellular β-glucosidase. Using an orthogonal experimental design (L9), we optimized the parameters of culture medium to maximize the activity of β-glucosidase. The optimal conditions (same for the four strains) were as follows: temperature, 30°C; pH, 6.0; orbital agitation, 200 rpm; concentration of sucrose, 0.5% (w/v). The most productive strain was A. niger CDBB-H-175, with a yield of 701.2 U/mL. In a second stage, we optimized (L18) the concentration of nutrients in the culture medium to determine whether this modification would increase the production of β-glucosidase. The optimal conditions for A. niger CDBB-H-175 were as follows (%, w/v): NaNO3, 0.3; KCl, 0.3; KH2PO4, 0.15; NH4NO3, 0.1; NH4H2PO4, 0.1; MgSO4 · 7H2O, 0.05; yeast extract, 0.1. The production of β-glucosidase under these conditions was 1207.9 U/mL. Enzymatic assays were used to characterize the enzyme; the optimum temperature and pH of β-glucosidase produced by the four selected micro-organisms were found to be 65°C and 5.0, respectively. We determined the Michaelis-Menten constants (Km) only for A. niger CDBB-H-175 and CDBB-H-176; the values were 2.7 and 2.2 mM, respectively.
Keywords: Aspergillus niger; Aspergillus terreus; Michaelis–Menten parameters; enzymatic stability; optimization process; β-glucosidase activity.