Heat-induced structural changes of Aspergillus oryzae pectin methylesterase (PME) were studied by means of fluorescence spectroscopy and molecular modeling, whereas the functional enzyme stability was monitored by inactivation studies. The fluorescence spectroscopy experiments were performed at two pH value (4.5 and 7.0). At both pH values, the phase diagrams were linear, indicating the presence of two molecular species induced by thermal treatment. A red shift of 7 nm was observed at neutral pH by increasing temperature up to 60°C, followed by a blue shift of 4 nm at 70°C, suggesting significant conformational rearrangements. The quenching experiments using acrylamide and iodide demonstrate a more flexible conformation of enzyme with increasing temperature, especially at neutral pH. The experimental results were complemented with atomic level observations on PME model behavior after performing molecular dynamics simulations at different temperatures. The inactivation kinetics of PME in buffer solutions was fitted using a first-order kinetics model, resulting in activation energy of 241.4±7.51 kJ mol(-1).
Keywords: Conformation; Fluorescence spectroscopy; Kinetics; Molecular dynamics; Pectin methylesterase.
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