Development of a Vibrio natriegens-based plate-clearing assay for rapid screening of PET-hydrolyzing enzymes
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 using Vibrio natriegens, exploiting its protein secretion system to rapidly identify catalytic activity without time-consuming downstream processing. To validate the assays robustness, we tested mutants of Fusarium solani pisi cutinase (FsC) and found that one mutant, T45P, exhibited threefold higher activity and a TPA-to-MHET ratio than the wild-type. As a further test case, we screened an error-prone PCR library of FsC using the assay, obtaining a 25% hit rate after screening 150 colonies. This demonstrates the scalability of the method for screening PET-hydrolytic activity in a large number of enzymes.