Data for: Measuring PETase enzyme kinetics by single-molecule microscopy

Polyethylene terephthalate (PET) is one of the most widely produced man-made polymers and is a significant contributor to microplastics pollution. The environmental and human health impacts of microplastics pollution have motivated a concerted effort to develop microbe- and enzyme-based strategies to degrade PET and similar plastics. A PETase derived from the bacteria Ideonella sakaiensis was previously shown to enzymatically degrade PET, triggering multidisciplinary efforts to improve the robustness and activity of this and other PETases. However, because these enzymes only erode the surface of the insoluble PET substrate, it is difficult to measure standard kinetic parameters, such as kon, koff and kcat, complicating interpretation of the activity of mutants using traditional enzyme kinetics frameworks. To address this challenge, we developed a single-molecule microscopy assay that quantifies the landing rate and binding duration of quantum dot-labeled PETase enzymes interacting with a surface-immobilized PET film. Wild-type PETase binding durations were well fit by a biexponential with a fast population having a 2.7 s time constant, interpreted as active binding events, and a slow population interpreted as non-specific binding interactions that last tens of seconds. A previously described hyperactive mutant, S238F/W159H had both a faster apparent on-rate and a slower off-rate than wild-type PETase, potentially explaining its enhanced activity. Because this single-molecule approach provides a more detailed mechanistic picture of PETase enzymatic activity than standard bulk assays, it should aid future efforts to engineer more robust and active PETases to combat global microplastics pollution.

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Work Title Data for: Measuring PETase enzyme kinetics by single-molecule microscopy
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Open Access
Creators
  1. Yuwei Zhang
  2. William O. Hancock
Keyword
  1. Polyethylene terephthalate
  2. PETase
  3. Single-molecule
  4. Microscopy
License CC BY-NC-ND 4.0 (Attribution-NonCommercial-NoDerivatives)
Work Type Other
Acknowledgments
  1. The authors thank members of the Hancock lab for helpful suggestions and Dr. Justin Brown for advice in PET preparation
Publication Date 2024
Related URLs
Deposited August 09, 2024

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Version 1
published

  • Created
  • Updated
  • Updated Description, Publication Date Show Changes
    Description
    • Polyethylene terephthalate (PET) is one of the most widely produced man-made polymers and is a significant contributor to microplastics pollution. The environmental and human health impacts of microplastics pollution have motivated a concerted effort to develop microbe- and enzyme-based strategies to degrade PET and similar plastics. A PETase derived from the bacteria Ideonella sakaiensis was previously shown to enzymatically degrade PET, triggering multidisciplinary efforts to improve the robustness and activity of this and other PETases. However, because these enzymes only erode the surface of the insoluble PET substrate, it is difficult to measure standard kinetic parameters, such as kon, koff and kcat, complicating interpretation of the activity of mutants using traditional enzyme kinetics frameworks. To address this challenge, we developed a single-molecule microscopy assay that quantifies the landing rate and binding duration of quantum dot-labeled PETase enzymes interacting with a surface-immobilized PET film. Wild-type PETase binding durations were well fit by a biexponential with a fast population having a 2.7 s time constant, interpreted as active binding events, and a slow population interpreted as non-specific binding interactions that last tens of seconds. A previously described hyperactive mutant, S238F/W159H had both a faster apparent on-rate and a slower off-rate than wild-type PETase, potentially explaining its enhanced activity. Because this single-molecule approach provides a more detailed mechanistic picture of PETase enzymatic activity than standard bulk assays, it should aid future efforts to engineer more robust and active PETases to combat global microplastics pollution.
    Publication Date
    • 2024-11
  • Updated Acknowledgments Show Changes
    Acknowledgments
    • The authors thank members of the Hancock lab for helpful suggestions and Dr. Justin Brown for advice in PET preparation
  • Added Creator Yuwei Zhang
  • Added Creator WILLIAM O HANCOCK
  • Added 20230328_125059.txt
  • Added 20230510_164107.txt
  • Added 20230613_170443.txt
  • Added 20230815_151208_8nMPWT.txt
  • Added 20230823_163112_8nMPWT.txt
  • Added 20240327_104113_8nMPWT.txt
  • Added 20240327_111502_8nMPWT.txt
  • Added 20240327_125639_8nMPWT.txt
  • Added 20240327_144709_8nMPWT.txt
  • Updated Keyword, License Show Changes
    Keyword
    • Polyethylene terephthalate, PETase, Single-molecule, Microscopy
    License
    • https://creativecommons.org/licenses/by-nc-nd/4.0/
  • Published
  • Updated

Version 2
published

  • Created
  • Added hyperactive mutant.zip
  • Added Inactive mutant.zip
  • Added Laser70mW_150mW.zip
  • Added Qdot.zip
  • Added wild type PETase.zip
  • Deleted 20240327_144709_8nMPWT.txt
  • Deleted 20240327_125639_8nMPWT.txt
  • Deleted 20230328_125059.txt
  • Deleted 20230510_164107.txt
  • Deleted 20230613_170443.txt
  • Deleted 20230815_151208_8nMPWT.txt
  • Deleted 20230823_163112_8nMPWT.txt
  • Deleted 20240327_104113_8nMPWT.txt
  • Deleted 20240327_111502_8nMPWT.txt
  • Published
  • Updated
  • Updated Work Title Show Changes
    Work Title
    • Measuring PETase enzyme kinetics by single-molecule microscopy
    • Data for: Measuring PETase enzyme kinetics by single-molecule microscopy
  • Updated Related URLs, Publication Date Show Changes
    Related URLs
    • https://doi.org/10.1016/j.bpj.2024.09.016
    Publication Date
    • 2024-11
    • 2024
  • Renamed Creator William O. Hancock Show Changes
    • WILLIAM O HANCOCK
    • William O. Hancock