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 (KM), maximum velocity (Vmax), thermal inactivation rate constant (ki), half-life (t1/2), and the change of energy due to denaturation (ΔGi). The results showed that the soluble enzyme has an optimum temperature of 55°C, KM of 3.04 mg mL−1 substrate, Vmax of 10.90 μmole mL−1 min−1, ki of 0.0171 min−1, t1/2 of 40.53 min, and ΔGi of 104.47 kJ mole−1, while the immobilized enzyme has an optimum temperature of 70°C, KM of 8.31 mg mL−1 substrate, Vmax of 1.44 μmole mL−1 min−1, ki of 0.0060 min−1, t1/2 of 115.50 min, and ΔGi 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.
Highlights
Hitherto, microbial α-amylase is used commercially as biocatalysts in various industries, such as detergent, syrup, bread and cake, dairy products, starch processing, animal feed, textile and leather, pulp and paper, candy, sugar, bioethanol, pharmaceuticals, and waste treatment [1, 2]
Despite its wide usage on the industrial scale, mostly enzymes are soluble in water when applied in batch processes, and this weakens the hydrogen bonds which contribute to stability [4]. e decrease in the stability of enzymes causes denaturation due to the loss of equilibrium in the noncovalent bonds [5], resulting in the once use of nonimmobilized enzymes
0.75 ml of the starch substrate was added to the immobilized enzyme, which was incubated at its optimum temperature for 30 min, and centrifuged for about 15 min. e supernatant was assayed for α-amylase enzyme activity by Mandel’s method
Summary
Microbial α-amylase is used commercially as biocatalysts in various industries, such as detergent, syrup, bread and cake, dairy products, starch processing, animal feed, textile and leather, pulp and paper, candy, sugar, bioethanol, pharmaceuticals, and waste treatment [1, 2]. E soluble enzyme was immobilized on a bentonite matrix using 0.1 M phosphate buffer with variations in pH which includes 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, and 8.0. 0.5 ml of the soluble enzyme and 0.5 mL of initial buffer were added to the matrix Afterward, it was incubated at 4°C for 30 min and centrifuged for about 15 min. 0.75 ml of the starch substrate was added to the immobilized enzyme, which was incubated at its optimum temperature for 30 min, and centrifuged for about 15 min. E enzyme activity is proportional to the enzyme-catalyzed reaction rate [17] Both were analyzed by Mandel’s method at each optimum temperature, and the KM values of soluble and immobilized enzymes were used as each optimum substrate concentration for subsequent procedures. Determination of t1/2, ki, and ΔGi. e thermal inactivation rate constant (ki) and half-life (t1/2) were calculated using the following first-order enzyme inactivation rate equation
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