Caracterización cinética y estructural de la enzima ancestral N1 PETasa

Abstract

The contamination caused by polyethylene terephthalate (PET) represents one of the most critical environmental challenges today, due to its high global production, persistence, and the limited efficiency of conventional recycling methods. In this context, enzyme-based degradation strategies have emerged as sustainable and highly specific alternatives. The methodology included structural modeling using AlphaFold 3.0, multiple sequence alignments, comparative analysis with modern PETases, and the experimental evaluation of enzymatic activity on pNP-acetate, pNP-butyrate, and post-consumer PET. This research aligns with the development of biotechnologies applied to the enzymatic recycling of polymers. Structural analyses revealed that N1 PETase retains the α/β-hydrolase fold, the Ser–Asp–His catalytic triad, and functional motifs associated with polyester hydrolysis. The ancestral N1 PETase enzyme emerges as a promising candidate for designing optimized biocatalysts. It also provides evidence on the relationship between structural architecture, loop dynamics, and catalytic efficiency, contributing to the advancement of biotechnological solutions for the circular economy. In this context, the present study carried out the structural, functional, and kinetic characterization of the ancestral N1 PETase enzyme, reconstructed through phylogenetic methods, to determine its potential as a biocatalyst for PET depolymerization.