Unveiling PET Hydrolase Surface Dynamics through Fluorescence Microscopy

A. P. Rennison, A. Nousi, P. Westh*, R. Marie*, M. S. Møller

*Corresponding author for this work

Research output: Contribution to journalJournal articleResearchpeer-review

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Abstract

PET hydrolases are an emerging class of enzymes that are being heavily researched for their use in bioprocessing polyethylene terephthalate (PET). While work has been done in studying the binding of PET oligomers to the active site of these enzymes, the dynamics of PET hydrolases binding to a bulk PET surface is an unexplored area. Here, methods were developed for total internal reflection fluorescence (TIRF) microscopy and fluorescence recovery after photobleaching (FRAP) microscopy to study the adsorption and desorption dynamics of these proteins onto a PET surface. TIRF microscopy was employed to measure both on and off rates of two of the most commonly studied PET hydrolases, PHL7 and LCC, on a PET surface. It was found that these proteins have a much slower off rates on the order of 10-3 s-1, comparable to non-productive binding in enzymes such as cellulose. In combination with FRAP microscopy, a dynamic model is proposed in which adsorption and desorption dominates over lateral diffusion over the surface. The results of this study could have implications for the future engineering of PET hydrolases, either to target them to a PET surface or to modulate interaction with their substrate.
Original languageEnglish
Article numbere202300661
JournalChemBioChem
Volume25
Issue number5
Number of pages13
ISSN1439-4227
DOIs
Publication statusPublished - 2024

Keywords

  • PET hydrolases
  • Interfacial catalysis
  • Fluorescence microscopy
  • Surface dynamics
  • Enzyme catalysis
  • Green chemistry

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