The irreversible thermal inactivation and the thermodynamics of calcium ion binding of Bacillus amyloliquefaciens α-amylase in the absence of substrates were studied. The enzyme inactivation on heating was apparently followed by first-order kinetics. The enzyme was stabilized with an increased concentration of calcium ion and thus the inactivation was highly dependent on the state of calcium binding. The activation parameter for the inactivation suggests an unfolding of the enzyme protein upon heating. Values of both the activation enthalpy and entropy were increased with a higher calcium ion concentration. An inactivation kinetic model is based on the assumption of a two-stage unfolding transition in which the bivalent ion dissociation occurs in the first step followed by the secondary structural unfolding. This simple kinetic model provides both a qualitative and quantitative interpretation of calcium ion binding to the enzyme and its effect on the inactivation properties. The specific approximations of the kinetic model were strictly followed in the analysis to calculate the apparent inactivation rate at each calcium ion concentration in terms of the calcium-binding parameters. The enthalpy and entropy changes for the calcium ion binding were calculated to be −149kJ/mol and −360J·mol−1·K−1 respectively and these values suggest a strong enthalpic affinity for the bivalent ion binding to the enzyme protein. The thermodynamical interpretation attempts to provide clear relations between the terms of an apparent inactivation rate and the calcium binding.
Abbreviations used: BAA, α-amylase from Bacillus amyloliquefaciens; PPA, α-amylase from porcine pancreas; TAA, α-amylase from Aspergillus oryzae.