Biochemical Analysis of Poly(ethylene terephthalate) Film Degradation Kinetics of Engineered IsPETase Variants

Abstract

Plastic production and pollution have become a global crisis, with 79% of waste plastics landfilled in 2015 and only 12% recycled, demonstrating the need for rapid improvements in waste management and recycling technologies. Poly(ethylene terephthalate) (PET) is a major plastic polymer that is heavily investigated for enzymatic recycling. The presence of the ester bond in the polymer allows hydrolysis via serine esterases, such as cutinases and lipases. However, little is known about the surface reaction and how biochemical behavior might differ on a 2D solid surface compared to solution phase. Consequently, traditional solution-phase biochemical models, such as Michaelis-Menten, may not be directly applicable to kinetics of these enzymes, as the catalysis is occurring under a heterogeneous phase. To improve the fundamental understanding of the enzymatic reaction on the surface and derive an appropriate biochemical model for kinetic analysis, this thesis aims to develop a simple kinetic assay of PET biodegradation, identify mutations that positively impact product formation rates, and develop a novel biochemical model to analyze these mutations that fully describe the kinetic profiles observed for these enzymes. I developed a kinetic assay based on spectrophotometric measurements of UVabsorbance of the products in the reaction supernatant, as degradation products harboring the benzene ring will absorb between 240-280 nm. The method was found to be reliable to obtain relative measurements of initial reaction rates but cannot be used to determine the absolute concentration of products in the supernatant. I also developed a directed evolution assay of IsPETase using solid PET film substrates and found that mutation T116P improved maximum product accumulation by 30% based on kinetic studies and thermostability, while mutations S238N and S290P improved purification yield and thermostability. Finally, my collaborators and I found that the activity of IsPETase is impacted by surface crowding and developed a biochemical model to analyze the kinetic data of mutants. Based on the kinetic model, T116P reduced crowding susceptibility with no impact on activity, resulting in improved macroscopic degradation rates. In conclusion, crowding tendency may become a major property to be targeted for enzyme engineering to improve solid-substrate depolymerases for industrial applications.