Diseño de la variante mutante S214H de la enzima PETasa de Ideonella sakaiensis

Abstract

The global accumulation of plastic waste, particularly polyethylene terephthalate (PET), constitutes a critical environmental problem due to its high resistance to natural degradation. Although mechanical and chemical recycling methods exist, they present limitations associated with the loss of material quality, the use of aggressive reagents, and high operational costs. In this context, enzymatic biodegradation emerges as a sustainable alternative to recover PET monomers under moderate conditions. The PETase enzyme from Ideonella sakaiensis has stood out for its ability to hydrolyze PET; however, its low thermostability and catalytic efficiency restrict its application on an industrial scale. This project focused on the design, purification, and evaluation of the S214H mutant variant, with the aim of improving its performance against PET. The mutation was incorporated through gene synthesis, cloned into the pET30a(+) vector, and expressed in Escherichia coli Rosetta (DE3). The recombinant protein was purified by affinity chromatography and analyzed by SDS-PAGE. Catalytic assays revealed that the S214H variant increased biodegradation efficiency, achieving 24.78 percent PET degradation under controlled conditions, exceeding the values obtained for the wild-type enzyme. This behavior suggests that the substitution at position 214 favors the stabilization of the catalytic microenvironment and improves interaction with the substrate. Taken together, these results position the S214H variant as an efficient alternative for the biological recycling of PET.