Current research deals with immobilization of amyloglucosidase through carrier-free approach using cross-linking strategy. Cross-linked amyloglucosidase aggregates (CLAAs) with aggregation yield of 94% were prepared in 04 h by incorporating 40% ammonium sulfate and 1.5% glutaraldehyde in enzyme solution. CLAAs were characterized by optimizing various conditions including reaction time, pH, temperature and substrate concentration. It was noticed that after cross-linking no change in optimum reaction time and substrate concentration was observed however, a 5-degree shift in optimum temperature from 60 °C to 65 °C was obtained as compared to soluble amyloglucosidase. Activation energy (Ea) of amyloglucosidase as calculated from Arrhenius plot was 5.5 kcal mol-1 and 5.2 kcal mol-1 for soluble and cross-linked aggregates, respectively. Stability studies revealed that CLAAs can be used at higher temperatures for longer time period than soluble amyloglucosidase. Furthermore, data of recycling studies showed that CLAAs can be efficiently reused for 20 cycles with the retention of 63% of its initial activity. Due to the continuous reusability of CLAAs, the product formation is also increased 8 times from 5.71 mg ml-1 (soluble enzyme) to 46.548 mg ml-1 (CLAAs). Findings of this research show that carrier-free strategy is more effective for continuous hydrolysis of starch and production of glucose.
Keywords: Carrier-free immobilization; Glucose production; Starch saccharification.
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