Stability enhancement attempted in this study demonstrated that significant improvement in the stability of the α-amylase isolated from Aspergillus fumigatus was achieved by immobilizing the enzyme on a bentonite/chitosan hybrid matrix using the adsorption method. Centrifugation was used to isolate the α-amylase, which was then refined using (NH4)2SO4 salt precipitation and dialysis. The purity of the α-amylase improved 19.40 times when compared to that of the crude extract. The optimal temperature for free α-amylase is 50˚C, while the optimum temperature for α-amylase/bentonite/chitosan is 60˚C. The KM value of α-amylase/bentonite/chitosan was 1.69 ± 0.08 mg mL-1 substrate and the Vmax value was 52.32 ± 3.29 µmol mL-1 min-1, whereas for free α-amylase, the KM value of 2.56 ± 0.09 mg mL-1 substrate and the Vmax value of 3.78 ± 0.09 µmol mL-1 min-1 were obtained. The ΔGi value of free α-amylase is 102.68 ± 0.30 kJ mol-1 and the t½ is 21.23 ± 0.23 min, whereas the ΔGi value of α-amylase/bentonite/chitosan is 104.43 ± 0.00 kJ mol-1 and the t½ is 94.29 ± 0.91 min. The higher values of ΔGi and t½demonstrated that α-amylase/bentonite/chitosan has better stability than that of free α-amylase. Another important finding is that α-amylase /bentonite/chitosan was able to retain their activity as high as 47.61 ± 0.53% after six recycles, indicating that the enzyme has the potential to be used in industry. Doi: 10.28991/ESJ-2023-07-05-023 Full Text: PDF

In this paper, the A. fumigatus α-amylase had been immobilized onto zeolite/chitosan hybrid to improve its thermal-stabilization for industrial needs. The methods applied enzyme production, isolation, partial purification, immobilization, and characterization. The optimum temperatures of the native and immobilized enzymes were 50 and 55˚C, respectively. The native enzyme has KM of 3.478 ± 0.271 mg mL-1 substrate and Vmax of 2.211± 0.096 µmole mL-1 min-1, while the immobilized enzyme has KM value of 12.051 ± 4.949 mg mL-1 substrate and Vmax of 1.602 ± 0.576 µmole mL-1 min-1. The residual activity of the immobilized enzyme retained up 10.97% after fifth reuse cycles. The native enzyme has ΔGi of 104.35 ± 1.09 kJ mole-1 and t½ of 38.75 ± 1.53 min, while the immobilized enzyme has ΔGi of 108.03 ± 0.05 kJ mole-1 and t½ of 180.03 ± 3.31 min. According to the increase in half-life (t½), stability improvement of the A. fumigatusα-amylase was 4.65 times greater than the native enzyme. Thus, the zeolite/chitosan hybrid is used as a new supporting matrix for further enzyme immobilization to stabilize the enzymes. Doi: 10.28991/ESJ-2022-06-03-06 Full Text: PDF

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 , 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, , 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, , 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.

In this research, immobilization of A. fumigatus α-amylase on chitin was studied with the main purpose to improve the characteristics of the enzyme. A series of experiments were carried out to study stability improvement, thermodynamic parameters, include ki, ΔGi, and t½, and reusability of the immobilized enzyme. The experimental results indicate that significant thermal stability was achieved, as indicates by the ability of the enzyme to retain its relative activity above 39% after 80 min of incubation at 60oC. Thermodynamic parameters, include ki, ΔGi, and t½, indicate that the immobilized enzyme is more rigid, stable, and less flexible in the water, resulting in increased stability up to 1.5 times compared to that of the native enzyme. Furthermore, the immobilized enzyme was able to retain over 46% of its initial activity after six consecutive applications for starch hydrolysis, confirming the potential of chitin for the production of immobilized enzymes on an industrial scale. Doi: 10.28991/ESJ-2023-07-01-06 Full Text: PDF

In this research, the immobilization of α-amylase from Bacillus subtilis ITBCCB148 by crosslinking method on chitosan matrix has been performed. This research aims to know the effect of immobilization on the thermal stability of α-amylase. The results showed that the native α-amylase has an optimum temperature of 65oC, KM = 1.6 mg mL-1 substrate, and Vmax = 39.7 µmol mL-1 min-1. The immobilized α-amylase has optimum temperature of 75oC, KM = 3.5 mg mL-1 substrate, and Vmax = 7.05 µmol mL-1 min-1. The residual activity of the native and immobilized enzyme on thermal stability test at 65oC for 80 minutes was 58% and 86.15%, respectively. The immobilized enzyme can be reused up to six repeated cycles.The thermodynamic data of native enzyme was t½ = 113.6 min, ki = 6.1x10-3 min-1, and ΔGi = 107.3 kJ mol-1, while the immobilized enzyme was t½ = 433.1 min, ki= 1.6x10-3 min-1, and ΔGi 111.1 kJ mol-1. Based on the decrease of ki, and the increase of ΔGi and half-life(t½) values, the immobilization of α-amylase with chitosan can increase the thermal stability of this enzyme.

The stability of enzymes which play an important role as biocatalysts in many industrial processes is a persistent challenge with significant impact on production costs. In this study, improvement of the stability of α-amylase obtained from Aspergillus fumigatus was attempted by immobilizing the enzyme onto zeolite using adsorption method. For purification, the isolated enzyme was subsequently subjected to centrifugation, fractionation, and dialysis. The native enzyme was found to have an optimum temperature of 50 °C, while the immobilized enzyme, the optimum temperature of 60 °C was found. The immobilized enzyme was found to have the K _M value of 11.685 ± 0.183 mg mL⁻¹ substrate and V _max of 1.406 ± 0.049 μmol mL⁻¹ min⁻¹, while for the native enzyme, the K _M value of 3.478 ± 0.271 mg mL⁻¹ substrate and the V _max of 2.211 ± 0.096 μmol mL⁻¹ min⁻¹ were obtained. Furthermore, the immobilized enzyme displays the ΔG_i of 106.76 ± 0.00 kJ mol⁻¹ and t _½ of 90.40 ± 0.00 min, while the native enzyme, the values obtained are ΔG_i of 104.35 ± 1.09 kJ mol⁻¹ and t_½ of 38.75 ± 1.53 min. As can be seen, the t _½ of immobilized enzyme is 2.38 times longer than that of native enzyme, justifying a very significant stability enhancement of the enzyme as a result of. Another important finding is that the immobilized α-amylase enzyme was able to retain its activity as high as 13.80 ± 1.19% activity after five cycles, indicating its potential for industrial use.