Kinetic study of the thermal denaturation of a hyperthermostable extracellular α-amylase from Pyrococcus furiosus.
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.
School of Chemical Engineering, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK.
I
I
Brown
T R
TR
Dafforn
P J
PJ
Fryer
P W
PW
Cox
D019843:Archaeal Proteins / Q000737:chemistry*; D004795:Enzyme Stability; D006358:Hot Temperature; D007700:Kinetics; D008956:Models, Chemical; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D020027:Pyrococcus furiosus / Q000201:enzymology*; D000516:alpha-Amylases / Q000737:chemistry*
D019843:Archaeal Proteins; D000516:alpha-Amylases
Denaturation; Pyrococcus furiosus; Sterilisation; TTI; α-Amylase