Efficient mild depolymerization of polyester plastics accomplished by engineered PETase via directed evolution of flexible loops

Due to inadequate disposal and recycling of polyethylene terephthalate (PET) plastic products, there has been growing concern about the severe environmental consequences. Biocatalytic degradation of PET has emerged as a promising solution. The PET hydrolase Is PETase from Ideonella sakaiensis exhibits distinctive substrate activity at mild temperatures, but its practical application is limited by restricted catalytic efficiency. In this study, the highly flexible loops of Is PETase are targeted for engineering after a detailed structural analysis. A customized dual-fluorescence high-throughput assay is then developed to facilitate the directed evolution of these selected loops in order to enhance Is PETase's catalytic performance, specifically at mild temperatures. Through implementation of this system, the evolved Is PETase mutant M4-Q demonstrates exceptional PET biodegradation activity at mild temperatures and advantages in degrading various aliphatic-aromatic copolyesters. These findings offer innovative insights into the enzymatic degradation of PET and other polyester plastic waste.