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

Author(s)
Zhong-Johnson, En Ze Linda; Dong, Ziyue; Canova, Christopher T; Destro, Francesco; Cañellas, Marina; Hoffman, Mikaila C; Maréchal, Jeanne; Johnson, Timothy M; Zheng, Maya; Schlau-Cohen, Gabriela S; Lucas, Maria Fátima; Braatz, Richard D; Sprenger, Kayla G; Voigt, Christopher A; Sinskey, Anthony J; ... Show more Show less
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Abstract
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. IsPETase 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 IsPETase productivity, we develop a directed evolution assay to identify mutations beneficial to PET film degradation at 30 °C. IsPETase also displays enzyme concentration-dependent inhibition effects, and surface crowding has been proposed as a causal phenomenon. Based on total internal reflectance fluorescence microscopy and adsorption experiments, IsPETase is likely experiencing crowded conditions on PET films. Molecular dynamics simulations of IsPETase variants reveal a decrease in active site flexibility 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 find 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.
Date issued
2024-03
URI
https://hdl.handle.net/1721.1/157674
Department
Massachusetts Institute of Technology. Department of Biology; Massachusetts Institute of Technology. Department of Chemical Engineering; Massachusetts Institute of Technology. Department of Chemistry; Massachusetts Institute of Technology. Plasma Science and Fusion Center; Massachusetts Institute of Technology. Department of Biological Engineering
Journal
Journal of Biological Chemistry
Publisher
Elsevier BV
Citation
Zhong-Johnson, En Ze Linda, Dong, Ziyue, Canova, Christopher T, Destro, Francesco, Cañellas, Marina et al. 2024. "Analysis of Poly(ethylene terephthalate) degradation kinetics of evolved IsPETase variants using a surface crowding model." Journal of Biological Chemistry, 300 (3).
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