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April 2nd, 2025
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Norwegian University of Science and Technology
biochemistry
biorxiv

Development of a Vibrio natriegens-based plate-clearing assay for rapid screening of PET-hydrolyzing enzymes

Bolstad, I. M.Open in Google Scholar•Trooyen, S. H.Open in Google Scholar•Luciano, D.Open in Google Scholar•Petersen, E.Open in Google Scholar•Courtade, G.Open in Google Scholar

Polyethylene terephthalate (PET) is a major contributor to plastic waste. Enzymatic PET degradation offers a sustainable recycling approach, but screening for effective enzymes remains challenging. Herein, we established an experimental plate-clearing assay with Vibrio natriegens, exploiting its protein-secretion ability to rapidly identify catalytic activity without time-consuming downstream processing. To validate the assay\'s robustness, we tested mutants of Fusarium solani pisi cutinase (FsC) and discovered a highly active FsC mutant, T45P, with three-fold higher activity and TPA-to-MHET ratio compared to the wildtype. As a further test case, we designed novel protein structures by scaffolding functional sites from the PET-degrading enzyme LCC-ICCG using the machine-learning models RFdiffusion and ProteinMPNN. Although we successfully produced one de novo protein, we did not detect catalytic activity on PET or BHET, nor did we detect substrate binding ability. This work provides a framework for experimental screening of PET-hydrolytic activity through a robust V. natriegens-based assay system.

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