MPNN-Guided Redesign of PET Hydrolases with Enhanced Catalytic Activity below the PET Glass Transition Temperature

Abstract

Abstract Enzymatic depolymerization of polyethylene terephthalate (PET) presents a sustainable route for plastic circularity, but its industrial viability is disadvantaged by the need for thermostable enzymes active under mild, energy-efficient conditions. While polyester hydrolase Leipzig 7 (PHL7, also known as PES-H1) rapidly degrades amorphous PET near the glass transition temperature of this polymer (∼65 °C), its poor protein expression, inactivation above 60 °C, and slow depolymerization below 60 °C limit its practical application. Here, we employ ProteinMPNN and LigandMPNN, structural and evolutionary information, to redesign the sequence of PHL7 and improve protein expression, thermostability, and activity. We identified 2/36 experimentally tested variants (D5, D11) with enhanced PET depolymerization at 50 °C, achieving the same efficiency as PHL7 at 70 °C but with a shifted product profile, favoring mono-(2-hydroxyethyl) terephthalate (MHET) over terephthalate. Molecular dynamics revealed that these redesigns exhibit enhanced flexibility in active site regions, providing a mechanistic understanding of their low-temperature catalysis. These variants enable a potential route to resynthesize virgin PET via MHET polycondensation, offering an efficient circular economy pathway.