Analysis of Poly(ethylene terephthalate) degradation kinetics of evolved IsPETase variants using a surface crowding model
Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved..
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.
| Errataetall: |
ErratumIn: J Biol Chem. 2024 Apr 4;300(4):107240. - PMID 38579375 |
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| Medienart: |
E-Artikel |
| Erscheinungsjahr: |
2024 |
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| Erschienen: |
2024 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:300 |
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| Enthalten in: |
The Journal of biological chemistry - 300(2024), 3 vom: 22. März, Seite 105783 |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Zhong-Johnson, En Ze Linda [VerfasserIn] |
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| Links: |
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| Themen: |
Biochemical model |
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| Anmerkungen: |
Date Completed 03.04.2024 Date Revised 10.04.2024 published: Print-Electronic ErratumIn: J Biol Chem. 2024 Apr 4;300(4):107240. - PMID 38579375 Citation Status MEDLINE |
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| doi: |
10.1016/j.jbc.2024.105783 |
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| 520 | |a Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved. | ||
| 520 | |a 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 | ||
| 650 | 4 | |a Journal Article | |
| 650 | 4 | |a IsPETase | |
| 650 | 4 | |a PET biodegradation | |
| 650 | 4 | |a PETase | |
| 650 | 4 | |a biochemical model | |
| 650 | 4 | |a kinetics | |
| 650 | 4 | |a surface crowding | |
| 650 | 7 | |a Hydrolases |2 NLM | |
| 650 | 7 | |a EC 3.- |2 NLM | |
| 650 | 7 | |a Polyethylene Terephthalates |2 NLM | |
| 700 | 1 | |a Dong, Ziyue |e verfasserin |4 aut | |
| 700 | 1 | |a Canova, Christopher T |e verfasserin |4 aut | |
| 700 | 1 | |a Destro, Francesco |e verfasserin |4 aut | |
| 700 | 1 | |a Cañellas, Marina |e verfasserin |4 aut | |
| 700 | 1 | |a Hoffman, Mikaila C |e verfasserin |4 aut | |
| 700 | 1 | |a Maréchal, Jeanne |e verfasserin |4 aut | |
| 700 | 1 | |a Johnson, Timothy M |e verfasserin |4 aut | |
| 700 | 1 | |a Zheng, Maya |e verfasserin |4 aut | |
| 700 | 1 | |a Schlau-Cohen, Gabriela S |e verfasserin |4 aut | |
| 700 | 1 | |a Lucas, Maria Fátima |e verfasserin |4 aut | |
| 700 | 1 | |a Braatz, Richard D |e verfasserin |4 aut | |
| 700 | 1 | |a Sprenger, Kayla G |e verfasserin |4 aut | |
| 700 | 1 | |a Voigt, Christopher A |e verfasserin |4 aut | |
| 700 | 1 | |a Sinskey, Anthony J |e verfasserin |4 aut | |
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