ABSTRACT
Enzyme immobilization is a powerful method to improve the stability, reuse, and enzymatic properties of enzymes. The immobilization of the α-amylase enzyme from Aspergillus fumigatus on a chitin-bentonite (CB) hybrid has been studied to improve its stability. Therefore, this study aims to obtain the higher stability of α-amylase enzyme to reduce industrial costs. The procedures were performed as follows: production, isolation, partial purification, immobilization, and characterization of the free and immobilized enzymes. The CB hybrid was synthesized by bentonite, chitin, and glutaraldehyde as a cross-linker. The free enzyme was immobilized onto CB hybrid using 0.1 M phosphate buffer pH 7.5. The free and immobilized enzymes were characterized by optimum temperature, Michaelis constant (K M), maximum velocity (V max), thermal inactivation rate constant (k i ), half-life (t 1/2), and transformation of free energy because of denaturation (ΔG i ). The free enzyme has optimum temperature of 55°C, K M = 3.04 mg mL-1 substrate, V max=10.90 µmolemL-1min-1, k i = 0.0171 min-1, t 1/2 = 40.53 min, and ΔG i = 104.47 kJ mole-1. Meanwhile, the immobilized enzyme has optimum temperature of 60°C, K M = 11.57 mg mL-1 substrate, V max=3.37 µmolemL-1min-1, k i = 0.0045 min-1, t 1/2 = 154.00 min, and ΔG i = 108.17 kJ mole-1. After sixth cycle of reuse, the residual activity of the immobilized enzyme was 38%. The improvement in the stability of α-amylase immobilized on the CB hybrid based on the increase in half-life was four times of the free enzyme.
ABSTRACT
The stability of the α-amylase enzyme has been improved from Aspergillus fumigatus using the immobilization method on a bentonite matrix. Therefore, this study aims to obtain the higher stability of α-amylase enzyme from A. fumigatus; hence, it is used repeatedly to reduce industrial costs. The procedures involved enzyme production, isolation, partial purification, immobilization, and characterization. Furthermore, the soluble enzyme was immobilized using 0.1 M phosphate buffer of pH 7.5 on a bentonite matrix, after which it was characterized with the following parameters such as optimum temperature, Michaelis constant (K M ), maximum velocity (V max), thermal inactivation rate constant (k i), half-life (t 1/2), and the change of energy due to denaturation (ΔG i ). The results showed that the soluble enzyme has an optimum temperature of 55°C, K M of 3.04 mg mL-1 substrate, V max of 10.90 µmole mL-1 min-1, k i of 0.0171 min-1, t1/2 of 40.53 min, and ΔG i of 104.47 kJ mole-1, while the immobilized enzyme has an optimum temperature of 70°C, K M of 8.31 mg mL-1 substrate, V max of 1.44 µmole mL-1 min-1, k i of 0.0060 min-1, t 1/2 of 115.50 min, and ΔG i of 107.37 kJ mole-1. Considering the results, the immobilized enzyme retained 42% of its residual activity after six reuse cycles. Additionally, the stability improvement of the α-amylase enzyme by immobilization on a bentonite matrix, based on the increase in half-life, was three times greater than the soluble enzyme.
