Purification and characterization of two forms of extracellular β-glucosidase from jute pathogenic fungus Macrophomina phaseolina

Two forms of β-glucosidase (β-d-glucoside glucohydrolase, EC 3.2.1.21) from the culture filtrate of Macrophomina phaseolina were separated and partially purified by (NH₄)₂SO₄ precipitation, ion-exchange chromatography (DE-52) and gel filtration. The final preparation was purified 103-fold and 88-fold for β-glucosidase-I and β-glucosidase-II, respectively. Polyacrylamide gel electrophoresis of the purified enzymes imparted a single band at pH 8.3. The two forms differ from each other with respect to molecular weight (323 600 for β-glucosidase I and 220 000 for β-glucosidase II)pH optima, temperature optima, electrophoretic mobility and substrate specificity. The two forms of β-glucosidase may also be differentiated by inhibition experiments using inhibitors like glucono-δ-lactone and nojirimycin. Of the two inhibitors tested nojirimycin is more potent for β-glucosidase-I than that for β-glucosidase-II. The energy of activation for the two enzymes is also different (12.02 kcal/mol for glucosidase I and 10.0 kcal/mol for glucosidase II).