The Stability Improvement of Aspergillus fumigatus α-Amylase by Immobilization onto Chitin-Bentonite Hybrid

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 (KM), maximum velocity V max , thermal inactivation rate constant (ki), half-life (t1/2), and transformation of free energy because of denaturation (ΔGi). The free enzyme has optimum temperature of 55°C, KM = 3.04 mg mL−1 substrate, V max = 10.90 μ molemL − 1 min − 1 , ki = 0.0171 min−1, t1/2 = 40.53 min, and ΔGi = 104.47 kJ mole−1. Meanwhile, the immobilized enzyme has optimum temperature of 60°C, KM = 11.57 mg mL−1 substrate, V max = 3.37 μ molemL − 1 min − 1 , ki = 0.0045 min−1, t1/2 = 154.00 min, and ΔGi = 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.

Immobilization and Stabilization of Aspergillus Fumigatus α-Amylase by Adsorption on a Chitin

“…This procedure was based on previous researches [22,24]. The native enzyme (0.5 mL) was immobilized onto 0.20 g of chitin using 0.1 M acetate/phosphate buffer with a pH variation of 4.0; 4.5; 5.0; 5.5; and 6.0.…”

“…Immobilization was conducted following the method as described in pervious works [22,24,26]. The native enzyme (0.5 mL) was immobilized onto 0.20 g of chitin using 0.5 mL of the buffer with the predetermined initial pH.…”

“…This variable was determined since each enzyme has a temperature range, known as optimum temperature, for achieving the maximum rate of reaction [2]. According to the previous findings, the optimum temperature of the A. fumigatus α-amylase was in the range of 50-55 o C, but higher range was reported after immobilization [5,22,24]. In this present study, determination of optimum temperature was carried out by performing the experiments at different incubation temperatures as specified in Mandel's assay, which are 50, 55, 60, 65, 70, and 75 o C. The experiments were run for 30 min, and the incubation temperature at which the highest enzyme activity was achieved was taken as the optimum temperature.…”

“…The KM constant indicates the amount of substrate in order to become saturated by the enzyme and generally refer to KM as a measure of the affinity of the enzyme for its substrate (The KM is a constant that is equivalent to the concentration of substrate at which the reaction takes place at one half its maximum rate), while the Vmax measures the extent of the activity [20]. According to previous works, the optimum substrate concentration for the A. fumigatus α-amylase was in the range of 0.2-0.4% and become higher 2-4 times after immobilization [5,22,24]. In present study, the Lineweaver-Burk graph was constructed by plotting the enzyme activity against the concentrations of starch (0.2, 0.4, 0.6, 0.8, and 1.0%).…”

Novel Magnetic Polymeric Filters with Laccase-Based Nanoparticles for Improving Congo Red Decolorization in Bioreactors

“…Several functionalization and immobilization strategies have been proposed to improve enzyme stability and resistance to environmental changes, such as the use of surface fillers, and polymers, among others [13,14]. Pertaining to immobilization processes for surface coverage, protein-protein interactions (e.g., electrostatic forces, hydrogen bonding) are favored over surface-protein interactions (e.g., bonding, chemical or physical adsorption) and the appropriate immobilization method will be chosen depending on conditions, such as pH, temperature, reaction time, among others [14,15].…”