Kinetics and thermodynamics of catalysis and thermal inactivation of a novel α-amylase (Tp-AmyS) from Thermotoga petrophila

Abstract

This research is focussed on kinetic, thermodynamic and thermal inactivation of a novel thermostable recombinant α-amylase (Tp-AmyS) from Thermotoga petrophila. The amylase gene was cloned in pHIS-parallel1 expression vector and overexpressed in Escherichia coli. The steady-state kinetic parameters (Vmax, Km, kcat and kcat/Km) for the hydrolysis of amylose (1.39 mg/min, 0.57 mg, 148.6 s−1, 260.7), amylopectin (2.3 mg/min, 1.09 mg, 247.1 s−1, 226.7), soluble starch (2.67 mg/min, 2.98 mg, 284.2 s−1, 95.4) and raw starch (2.1 mg/min, 3.6 mg, 224.7 s−1, 61.9) were determined. The activation energy (Ea), free energy (ΔG), enthalpy (ΔH) and entropy of activation (ΔS) at 98 °C were 42.9 kJ mol−1, 74 kJ mol−1, 39.9 kJ mol−1 and −92.3 J mol−1 K−1, respectively, for soluble starch hydrolysis. While ΔG of substrate binding (ΔGE-S) and ΔG of transition state binding (ΔGE-T) were 3.38 and −14.1 kJ mol−1, respectively. Whereas, EaD, Gibbs free energy (ΔG*), increase in the enthalpy (ΔH*) and activation entropy (ΔS*) for activation of the unfolding of transition state were 108, 107, 105 kJ mol−1 and −4.1 J mol−1 K−1. The thermodynamics of irreversible thermal inactivation of Tp-AmyS revealed that at high temperature the process involves the aggregation of the protein.