The Stability Improvement of Aspergillus fumigatus α-Amylase by Immobilization onto Chitin-Bentonite Hybrid
Ezra Rheinsky Tiarsa, Yandri Yandri, Tati Suhartati, Heri Satria, Bambang Irawan, Sutopo Hadi
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 <a:math xmlns:a="http://www.w3.org/1998/Math/MathML" id="M1"> <a:mfenced open="(" close=")" separators="|"> <a:mrow> <a:msub> <a:mrow> <a:mi>V</a:mi> </a:mrow> <a:mrow> <a:mi mathvariant="normal">max</a:mi> </a:mrow> </a:msub> </a:mrow> </a:mfenced> </a:math> , 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, <g:math xmlns:g="http://www.w3.org/1998/Math/MathML" id="M2"> <g:msub> <g:mrow> <g:mi>V</g:mi> </g:mrow> <g:mrow> <g:mi mathvariant="normal">max</g:mi> </g:mrow> </g:msub> <g:mo>=</g:mo> <g:mn>10.90</g:mn> <g:mtext> </g:mtext> <g:msup> <g:mrow> <g:mi>μ</g:mi> <g:mtext>molemL</g:mtext> </g:mrow> <g:mrow> <g:mo>−</g:mo> <g:mn>1</g:mn> </g:mrow> </g:msup> <g:msup> <g:mrow> <g:mi mathvariant="normal">min</g:mi> </g:mrow> <g:mrow> <g:mo>−</g:mo> <g:mn>1</g:mn> </g:mrow> </g:msup> </g:math> , 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, <k:math xmlns:k="http://www.w3.org/1998/Math/MathML" id="M3"> <k:msub> <k:mrow> <k:mi>V</k:mi> </k:mrow> <k:mrow> <k:mi mathvariant="normal">max</k:mi> </k:mrow> </k:msub> <k:mo>=</k:mo> <k:mn>3.37</k:mn> <k:mtext> </k:mtext> <k:msup> <k:mrow> <k:mi>μ</k:mi> <k:mtext>molemL</k:mtext> </k:mrow> <k:mrow> <k:mo>−</k:mo> <k:mn>1</k:mn> </k:mrow> </k:msup> <k:msup> <k:mrow> <k:mi mathvariant="normal">min</k:mi> </k:mrow> <k:mrow> <k:mo>−</k:mo> <k:mn>1</k:mn> </k:mrow> </k:msup> </k:math> , 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.