Characterization of Potential Plastic-Degradation Enzymes from Marine Bacteria.

Polyethylene terephthalate (PET) and polyethylene (PE) are prominent polymer materials that comprise a significant portion of commercial plastic waste. Their durability and slow degradation rate have resulted in significant accumulation of plastic on Earth. In a recent study, macrotranscriptomic profiling of a reconstituted marine bacterial community identified 10 putative enzymes capable of directly acting on PE or PET (PEases or PETases). Among these enzymes, three recombinant proteins were reported to possess PE degradation activity. To select potential plastic degrading enzyme candidates for protein engineering efforts, we expressed and purified eight out of the 10 candidates, excluding two due to poor expression and/or solubility. Notably, several candidate proteins displayed significant esterase activity on p- nitrophenyl butyrate and exhibited unexpected thermostability despite their marine origin. Additionally, we observed dose- and time-dependent hydrolytic activity on the PET trimer substrate. Structural analysis and mutagenesis of a candidate protein confirmed the presence of catalytic triad residues, classifying it as an esterase. Furthermore, we elucidated the structural importance of the two disulfide bonds. Through point mutation experiments, we observed an enhanced hydrolytic activity of a selected enzyme candidate on PET nanoparticles. Our findings challenge the classification of the enzymes directly acting on PE and highlight the significance and complexity of validating PE degradation enzymes identified through metagenomic analysis.