Carrier free co-immobilization of glucoamylase and pullulanase as combi-cross linked enzyme aggregates (combi-CLEAs)

Herein we report the successful preparation of carrier free co-immobilized form of glucoamylase and pullulanase using combi-CLEAs method from commercial multi-enzyme preparation OPTIMAX® 7525 HP. Glucoamylase and pullulanase were precipitated using ammonium sulfate, followed by cross-linking for 8 h with 2% (v/v) glutaraldehyde to produce insoluble catalytically active co-immobilized form of enzymes. The effects of precipitant type and cross-linking were studied and the biocatalyst was characterized. Scanning electron microscopy analysis showed that co-immobilized form of enzymes was of spherical structure. For starch hydrolytic activity, shift in optimum pH from 5 to 7 and temperature from 60 to 70 °C were observed after co-immobilization of enzymes. After starch hydrolysis reaction in batch mode, 100, 80 and 30% conversions were obtained with co-immobilized enzymes, mixture of separate CLEAs and free enzymes, respectively. Co-immobilization also enhanced the thermal stability and storage stability. After eight reuses with 30 min reaction time, co-immobilized glucoamylase and pullulanase retained 90 and 85% of its initial activity, respectively.