ANCUT1, a Fungal Cutinase MgCl2-Activated by a Non-Essential Activation Mechanism for Poly(ethylene terephthalate) Hydrolysis

Plastic waste, particularly poly(ethylene terephthalate) (PET), negatively impacts the environment and human health. Biotechnology could become an alternative to managing PET waste if enzymes ensure the recovery of terephthalic acid with efficiencies comparable to those of chemical treatments. Recent research has highlighted the potential of fungal cutinases, such as wild-type ANCUT1 (ANCUT1wt) from Aspergillus nidulans, in achieving PET depolymerization. Fungal cutinases’ structures differ from those of bacterial cutinases, while their PET depolymerization mechanism has not been well studied. Here, a reliable model of the ANCUT1wt was obtained using AlphaFold 2.0. Computational chemistry revealed potential cation-binding sites, which had not been described regarding enzymatic activation in fungal cutinases. Moreover, it allowed the prediction of residues with the ability to interact with a PET trimer that were mutation candidates to engineer the substrate binding cleft, seeking enhancements of PET hydrolysis. Enzyme kinetics revealed that both ANCUT1wt and ANCUT1N73V/L171Q (DM) were activated by MgCl2, increasing the dissociation constant of the substrate and maximal reaction rate. We found that in the presence of MgCl2, DM hydrolyzed different PET samples and released 9.1-fold more products than ANCUT1wt. Scanning Electron Microscopy revealed a different hydrolysis mode of these enzymes, influenced by the polymer’s crystallinity and structure.