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Updated: May 7, 2026

A New Screening Method for the Directed Evolution of Thermostable Bacteriolytic Enzymes
Published on: November 7, 2012
Kinetic study of the thermal denaturation of a hyperthermostable extracellular α-amylase from Pyrococcus furiosus
I Brown1, T R Dafforn, P J Fryer
1School of Chemical Engineering, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK.
Abstract:
Hyperthermophilic enzymes are of industrial importance and interest, especially due to their denaturation kinetics at commercial sterilisation temperatures inside safety indicating time-temperature integrators (TTIs). The thermal stability and irreversible thermal inactivation of native extracellular Pyrococcus furiosus α-amylase were investigated using differential scanning calorimetry, circular dichroism and Fourier transform infrared spectroscopy. Denaturation of the amylase was irreversible above a Tm of approximately 106°C and could be described by a one-step irreversible model. The activation energy at 121°C was found to be 316kJ/mol. Using CD and FT-IR spectroscopy it was shown that folding and stability greatly increase with temperature. Under an isothermal holding temperature of 121°C, the structure of the PFA changes during denaturation from an α-helical structure, through a β-sheet structure to an aggregated protein. Such data reinforces the use of P. furiosus α-amylase as a labile species in TTIs.
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