Analysis of Poly(ethylene terephthalate) degradation kinetics of evolved IsPETase variants using a surface crowding model

Poly(ethylene terephthalate) (PET) is a major plastic polymer utilized in the single-use and textile industries. The discovery of PET-degrading enzymes (PETases) has led to an increased interest in the biological recycling of PET in addition to mechanical recycling. Is PETase from Ideonella sakaiensis is a candidate catalyst, but little is understood about its structure- function relationships with regards to PET degradation. To understand the effects of mutations on Is PETase productivity, we develop a directed evolution assay to identify mutations beneficial to PET fi lm degradation at 30 degrees C. Is PETase also displays enzyme concentration-dependent inhibition effects, and surface crowding has been proposed as a causal phenomenon. Based on total internal reflectance fl uorescence microscopy and adsorption experiments, Is PETase is likely experiencing crowded conditions on PET fi lms. Molecular dynamics simulations of Is PETase variants reveal a decrease in active site fl exibility in free enzymes and reduced probability of productive active site formation in substrate-bound enzymes under crowding. Hence, we develop a surface crowding model to analyze the biochemical effects of three hit mutations (T116P, S238N, S290P) that enhanced ambient temperature activity and/or thermostability. We fi nd that T116P decreases susceptibility to crowding, resulting in higher PET degradation product accumulation despite no change in intrinsic catalytic rate. In conclusion, we show that a macromolecular crowding- based biochemical model can be used to analyze the effects of mutations on properties of PETases and that crowding behavior is a major property to be targeted for enzyme engineering for improved PET degradation.