Pectin cross-linked enzyme aggregates (pectin-CLEAs) of glucoamylase

Abstract

Pectin cross-linked enzyme aggregates (pectin-CLEAs) of glucoamylase were prepared for the first time with pectin as cross-linking agent. Pectin as a biocompatible, biodegradable, non-toxic, renewable and macromolecular cross-linker was used instead of traditional micro-molecular glutaraldehyde cross-linker. The cross-linker was prepared by controlled sodium metaperiodate oxidation of native pectin. The effects of precipitant type, amount of precipitant and cross-linking on activity recovery of glucoamylase in pectin-CLEAs were studied. After aggregation of glucoamylase with ammonium sulphate, when formed aggregates were cross-linked by pectin, 83% activity recovery was achieved in pectin-CLEAs, whereas when cross-linked by traditional cross-linker glutaraldehyde, 64% activity recovery was achieved in glutaraldehyde-CLEAs. After formation of pectin-CLEAs and glutaraldehyde-CLEAs, the optimum temperature for glucoamylase activity was shifted from 50 to 55 °C. The free enzyme and pectin-CLEAs displayed an optimal pH of 5, whereas the optimal pH of glutaraldehyde-CLEAs was shifted to pH 6. Compared with the free enzyme and glutaraldehyde-CLEAs, lower inactivation rate constant of glucoamylase in pectin-CLEAs within the temperature range of 50–70 °C was observed. Moreover, the activation energy required for denaturation of glucoamylase in pectin-CLEAs was higher than glutaraldehyde-CLEAs and free enzyme. Kinetic studies show that the K_m and V_max of glucoamylase remained unchanged after pectin-CLEAs formation, whereas K_m was increased and V_max was decreased after glutaraldehyde-CLEAs formation. Finally upon 10 consecutive uses, pectin-CLEAs retained 55% initial activity and glutaraldehyde-CLEAs retained only 29% initial activity. These results suggest that this pectin-CLEA is potentially usable in industrial applications.