Characterization of Beta-Galactosidase Enzyme Kinetics and Thermodynamics Produced from Aspergillus Oryzae
Sami Kadhim Hassan
This study investigates the properties of β-galactosidase enzyme derived from Aspergillus oryzae, a mold fungus. The Michaelis-Menten constants (Km and Vmax) were determined using o-nitrophenyl-β-galactoside (ONPG) as the substrate. The Km value, calculated from the Lineweaver-Burk plot, was found to be 0.800 mM, with a Vmax of 0.0864 (A/min). Additional Km and Vmax values were obtained from different plots: Michaelis-Menten (Km = 0.840 mM, Vmax = 0.0838 A/min), Direct Linear (Km = 0.800 mM, Vmax = 0.0850 A/min), Hanes (Km = 0.633 mM, Vmax = 0.1216 A/min), and Eadie-Hofstee (Km = 0.043 mM, Vmax = 0.0867 A/min). The enzymatic activity was explored across a pH range of 5.0 to 8.0, revealing an optimum activity at pH 7.5. The enzyme exhibited a preference for slightly alkaline conditions, as its activity decreased with decreasing pH, reaching zero at pH 4.0. Thermal stability was assessed by determining the activation energy (Ea) and denaturation kinetics. The rate constant of denaturation increased with temperature, ranging from 0.2366 to 0.4768 min⁻¹ as the temperature rose from 60 to 62°C. Denaturation times at 60, 61, and 62°C were 20.172, 19.821, and 10.028 min, respectively, indicating enhanced thermal stability at 61 and 61.5°C for 1 and 2 minutes. However, the enzyme showed reduced stability at 62°C after 4 minutes. The activation energy for β-galactosidase inactivation (Ea) was determined as 32,430.63 J/mol.
Keywords: β-galactosidase enzyme, O-nitrophenyl-β-galactoside, Substrate, Km, Vmax, PH sensitivity, Thermal stability, Activation energy, Aspergillus oryzae
