Enzymatic recycling of PET plastic requires enzymes with high stability and increased catalytic efficiency, at high temperatures. IsPETase degrades PET polymers, to its monomers, at ambient temperatures, through a Serine-Histidine-Aspartic catalytic triad. HotPETase, designed with directed evolution, is a thermotolerant variant of IsPETase able to degrade PET at 65 ^oC. In this work, experimental and computational approaches
were used in order to investigate the thermostability and catalytic activity of these two enzymes, as well as their intermediates. Protein unfolding was studied with differential scanning calorimetry. Activation energy and activation temperature showed an increase during the evolution process. HotPETase and one predecessor (M10*) displayed increased thermodynamic properties and resilience to denaturation. The PET degradation rates of all enzymes were studied with time course assays, revealing different kinetic stabilities. The experimentally observed optimum temperature for activity was increased from IsPETase to HotPETase. HotPETase was then tested in 200 mL bioreactor reactions in order to evaluate its potential for large scale applications . Binding of PET to the active site was investigated with molecular docking calculations. The best docked poses were further refined with molecular dynamics simulations to account for flexibility and temperature effects. Trajectory analysis of the molecular dynamics simulations of IsPETase, M10* and HotPETase at 30 ^oC indicated the probability of PET obtaining the optimum distances within the active site. Finally, for simulations at 60 ^oC, HotPETase and M10* were found to have a significantly large probability of obtaining the necessary distances, whereas IsPETase was found to be incapable of retaining the PET in the optimum distances.

Carmen Gomez Centurion Golmayo

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