Biochemical studies on immobilized fungal β-glucosidase

Abstract

β-Glucosidase from Aspergillus niger was immobilized on sponge by covalent binding through a spacer group (glutaraldehyde). Sponge-immobilized enzyme had the highest immobilization yield (95.67%) and retained 63.66% of the original activity exhibited by the free enzyme. The optimum pH of the immobilized enzyme remains almost the same as for the free enzyme (pH 4.0). The optimum temperature for β-glucosidase activity was increased by 10 °C after immobilization. The activation energy (Ea) of the immobilized β-glucosidase was lower than the free enzyme (3.34 and 4.55 kcal/mol), respectively. Immobilized β-glucosidase exhibited great thermal stability and retained all the initial activity after incubation at 55 °C for 2 h; however, the free enzyme retained 89.25% under the same condition. The calculated half-life (t1/2) value of heat inactivation of immobilized enzyme at 60, 65 and 70 °C was 213.62, 72.95 and 56.80 min, respectively, whereas at these temperatures the free enzyme was less stable (half-life of 200.0, 55.31 and 49.5 min, respectively). The deactivation rate constant at 65 °C for the immobilized β-glucosidase is 9.5x10-3/ min, which was lower than that of the free form (12.53x10-3/ min). The immobilization process improved the pH stability of the enzyme (immobilized and free enzyme retained 69.35 and 39.86%, respectively, of their initial activity after 45 min at pH 7.5). The effect of some chemical substances on the activity of the immobilized and free β-glucosidase has been investigated. In the presence of sodium dodecyl sulfate (SDS) and p-chloromercuri benzoate (p-CMB) the immobilized enzyme retained 36.13 and 45.34%, respectively, of the initial activity, which is higher than that of free enzyme (13.71 and 1.61%, respectively). The Michaelis constant (Km) value of the free enzyme was 40.0 mM, while the apparent Km value for the immobilized enzyme was 46.51 mM. The maximum reaction rate (vmax) of immobilized β-glucosidase was smaller than that of the free enzyme by 7.69%. Sponge-immobilized β-glucosidase was repeatedly used to hydrolyze cellobiose (5 and 8 cycles with retained activity of 67.32 and 51.04%, respectively).