Fabrication of 1,4-alpha-D-glucan glucanohydrolase holding Gel-Scaffolds using Agar-Agar, a natural polysaccharide and Polyacrylamide, a synthetic organic polymer for continuous liquefaction of starch

Abstract

1,4-alpha-D-glucan glucanohydrolase is among the most widely used commercial hydrolytic enzymes acting randomly on the glycosidic linkages of starch resulting in its saccharification and liquefaction. Its applicability in different industries can be improved by enhancing its stability and reusability. Therefore, in the present study attempts have been made to enhance the industrial applicability of 1,4-alpha-D-glucan glucanohydrolase from Bacillus subtilis KIBGE-HAR by adapting immobilization technology. The study developed mechanically stable, enzyme containing gel-frameworks using two support matrices including agar-agar, a natural polysaccharide and polyacrylamide gel, a synthetic organic polymer. These catalytic gel-scaffolds were compared with each other in terms of kinetics and stability of entrapped 1,4-α-D-glucan glucanohydrolase. In case of polyacrylamide gel, K_m value for immobilized enzyme increased to 7.95 mg/mL, while immobilization in agar-agar resulted in decreased K_m value i.e 0.277 mg/mL as compared to free enzyme. It was found that immobilized enzyme showed maximum activity at 70 °C in both the supports as compared to free enzyme having maximum activity at 60 °C. Immobilized 1,4-α-D-glucan glucanohydrolase exhibited no change in optimal pH 7.0 before and after entrapment in polyacrylamide gel and agar-agar. The enzyme containing gel-scaffold was found suitable for repeated batches of starch liquefaction in industrial processes. Agar-agar entrapped 1,4-α-D-glucanglucanohydrolase was capable to degrade starch up to seven repeated operational cycles whereas polyacrylamide entrapped enzyme conserved its activity up to sixth operational cycle.