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Research Articles

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

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Pages 227-233 | Received 20 Jul 2019, Accepted 22 Feb 2020, Published online: 11 Mar 2020
 

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.

Acknowledgements

We gratefully acknowledge the late Professor, Dr. David B. Wilson (Department of Molecular Biology and Genetics, Cornell University, NY, USA) for his guidance, provision of lab facilities, and preparation of this manuscript. His contribution to this work was of great significance.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work was supported by International Research Support Initiative Programme, awarded to Uzma Hameed for Ph.D research work, by the Higher Education Commission of Pakistan.

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