Purification and characterization of two intracellular β-glucosidases from the Neurospora crassa mutant cell-1

Abstract

Two intracellular β-glucosidases (E.C. 3.2.1.21) were purified from the filamentous fungus Neurospora crassa, mutant cell-1 (FGSC no. 4335) and characterized. The extent of purification were 2.55- and 28.89-fold for β-glucosidase A and β-glucosidase B, respectively. β-Glucosidase A was a dimeric protein, and B a monomeric protein, with molecular masses of 178 and 106 kDa, respectively. Both isoenzymes were glycoproteins with relatively high carbohydrate contents (β-glucosidase A, 29.2%; β-glucosidase B, 34.2%). The isoelectric points determined by IEF were 6.27 and 4.72, respectively. pH optima for activity were determined to be 5.0 and 5.5, and temperature optima to be 55 and 60 °C, for β-glucosidases A and B, respectively. Both purified β-glucosidases. especially β-glucosidase B, showed relatively high stability against pH and temperature. Both enzymes were stable in the pH range of 5.0–9.0. The activities were completely retained up to 48 h at temperatures below 40 °C. At higher temperatures, enzymes were relatively unstable and lost their activities at 60 °C after 24 h. Both β-glucosidases were highly activated by CuCl₂, and inhibited by SnCl₂ and KMnO₄. Hg²⁺ and Ag⁺ also inhibited severely β-glucosidase B. The K _m and V _max values of the isoenzymes against cellobiose as substrate were 1.50 mM and 12.2μmol min⁻¹ mg⁻¹ for β-glucosidase A and 2.76 mM and 143.5 μmol min⁻¹ mg⁻¹ for β-glucosidase B.