Characterization of Immobilized β-Amylase Enzyme Isolated from Sweet Potato and prepared by Entrapment Method

Abstract

Aim: This study attempted to isolate β-amylase from sweet potato and enzyme immobilized by encapsulation method, and characterized with various parameters. 
 Methods: The enzyme β-amylase was isolated with phosphate-buffered saline and purified by centrifugation with ammonium sulfate. The purified enzyme was immobilized on chitosan (0.25 g) and sodium alginate (0.25 g) polymers by entrapment method in the presence of calcium chloride (0.5 M). The immobilized enzyme was characterized by a starch hydrolysis test, the optimal pH and temperature were studied and the stability of the immobilized enzyme was also determined. SEM analysis was performed and Vm and Km were also found. 
 Results: The starch hydrolysis test showed positive results on the starch agar plates for immobilized enzymes. The thermal inactivation showed a severe loss in the activity of the free enzymes (49.3 %) while the temperature profile of the immobilized enzymes was much broader (84.55 %) at higher temperatures (80° C). The optimal pH and stability indicated that the immobilized enzyme has higher stability in the pH range of 5-8. The Km and Vmax value of free and immobilized enzyme was 7.67 mmol, 21.15 µmol (R2 0.8880), and 4.72 mmol,16.79 µmol (R2 0.8446) respectively. The storage of free and immobilized enzymes for one month showed that 83.5 % and 40 % of free enzymes and 11.6 % and 8.6 % of immobilized enzymes lost activity at 25° C and 4° C, respectively. SEM analysis shows the smooth, porous surface. 
 Conclusion: Immobilized enzymes (natural polymers) exhibit higher thermal stability the optimal pH and stability indicate immobilized enzyme has higher stability in the pH range of 5-8, and achieves a relative activity of 69.7 %. After 6 uses, the reuse efficiency of the immobilized enzyme decreased from 99.8 % to 52.3 %. The storage of the immobilized enzyme showed much higher stability than the found-free enzyme.