Covalent immobilization and thermodynamic characterization of pullulanase for the hydrolysis of pullulan in batch system

Pullulanase showed significant improvement in thermal stability after covalent immobilization on Duolite XAD761. Optimum temperature of immobilized enzyme was 60 °C, which was 10 °C higher than the free enzyme. Apparent Km values for pullulan, soluble starch and dextran were 4.4, 20 and 50 mg mL−1, respectively. Mn2+ and Ca2+ showed 2.0- and 2.2-fold increase in enzyme activity. Activation energy (Ea) of immobilized biocatalyst was 22.38 kJ mol−1. Thermodynamic parameters (ΔH*, ΔG*, ΔS*) for irreversible inactivation of immobilized pullulanase at different temperatures (60–70 °C) were also determined. D-value was maximum (95.95 h) at 60 °C and temperature quotient (Q10) was 1.29. Immobilized biocatalyst was effectively used for pullulan hydrolysis in a batch system. Stationary phase in hydrolysis (95.70 ± 1.36%) was reached after 300 min at 125 rpm. Pullulan hydrolysis yielded 4.21 ± 0.06% reducing sugars under optimal conditions. Immobilized biocatalyst was successfully recycled for 33 batches, but the enzyme activity was reduced to half after 25th cycle.