Energetic Constraints in the Enzymatic Depolymerization of Crystalline PET

Polyethylene terephthalate (PET) is a widely used thermoplastic whose high crystallinity poses a major barrier to upscaling enzymatic recycling. While PETases with high activity and stability have been reported, no enzyme capable of directly depolymerizing crystalline PET (cPET) has been discovered, and the molecular determinants limiting their efficacy remain difficult to characterize. Here, we integrate experimental conformational ratios of crystalline and amorphous PET chains with enhanced-sampling molecular dynamics simulations to map the free-energy landscape of a prototypical PETase bound to PET oligomers, revealing how structural equilibria translate to catalytic function. Surprisingly, productive enzyme-substrate catalytic configurations can be reached for both crystalline and amorphous PET chains. However, forming catalytic ensembles with cPET requires ~25 kJ/mol more than with aPET, with an additional ~17 kJ/mol per monomer needed for chain separation, which further limits enzymatic activity on crystalline substrates. The model highlights limitations of current alpha/beta-hydrolase scaffolds used for PET depolymerization and indicates directions for their redesign to enable cPET depolymerization. Our approach showcases a general strategy to explore substrate-enzyme catalytic ensembles in plastic depolymerization and guide enzyme design with built-in sustainability.