Biodegradation of bio-based polyesters by PET hydrolytic enzymes

In everyday life the consumption of plastic is steadily increasing, arising some concerns not only regarding the origin of these materials, which is generally fossil-based, but also for their end-of-life treatment. If a solution for the first aspect may be found in the utilization of biobased plastics, the other matter is still under investigation. Recycling plastic is a complex process, due to the broad range of polymers used, but also for the presence of contaminants and additives; for these reasons, these substrates are sent, in almost their totality, to landfills or burnt in incinerations, increasing the carbon dioxide content in the atmosphere and producing microplastics. Nowadays some innovative treatments, aimed in improving a circular economy, have been proposed. Among those, employing plastic-growing microorganisms and their hydrolysing enzymes seems a useful tool and it is the object of this study. This research focuses on a possible degradation via enzymatic hydrolysis of four crosslinked bio-polyesters, two which are semi-aromatic and the remaining aliphatic, performing a theorical and an experimental analysis. Two esterases, PETase and cutinase, are responsible of the degradation: the theorical investigation brought attention on them, which, based on some previous studies, could hydrolase the bonds of polyethylene terephthalate (PET). Results of former research were used to describe their proposed structures and the followed mechanisms. Then, comparing the chemical structure of the substrates and of PET, a possible reaction mechanism was here suggested. This result was tested through the experimental analysis, after having expressed and purified the enzymes. The washed bio-polyesters were then incubated with those proteins in two concentrations for 48 hours and then the degradation products were analysed with liquid and, some in cases, gas chromatography techniques. Samples where PETase was involved were also investigated using scanning electron microscopy (SEM). PETase could degrade only semi-aromatic polyesters and the products were mainly dimers, as the gas chromatography mass spectrometer confirmed. This was expected since the same result was obtained in other studies. Furthermore, this behaviour occurred only when the samples were washed before the reaction, since their surface was easier to access for the active site. On the other hand, cutinase continued the degradation until reaching the formation of one monomer, the diol, and, generally, the reaction was successful for every substrate and released a higher quantity of dimers. This was explainable not only thanks to a lower selectivity of the enzyme, but also for the higher operational temperature applied, which came from a major thermostability of cutinase compared to PETase. Unfortunately, the SEM images did not give any concluding result, not showing high differences between polymers surfaces treated with the enzymes and the ones that were just incubated without the proteins. In conclusion, the type and the quantity of the released products confirmed that PETase and cutinase can be applied in degrading not only PET, but also other polyesters. That considered, some future work, mainly aimed in applying a more thermostable PETase and in treating the crosslinks before the incubation, can still be done, before scaling-up the process and substitute the already followed chemical methods applying non sustainable chemicals.