Caracterización cinética de las variantes I208V/N212A, I208V/S238Y y N212A/S238Y de la enzima Petasa de Ideonella sakaiensis. Articulado al proyecto de investigación aprobado mediante Resolución Nro. UTA-CONIN-2024-0075-R y reformado mediante la Resolución Nro. UTACONIN-2025-0003-R

Abstract

The accumulation of plastic waste, especially polyethylene terephthalate (PET), represents a serious global environmental problem that demands sustainable alternatives. One promising solution is enzymatic biodegradation, in which the PETase enzyme from Ideonella sakaiensis stands out for its ability to degrade PET under moderate conditions. However, its limited thermostability restricts its industrial use. In this context, the present study aimed to kinetically characterize three mutant variants of the PETase enzyme (I208V-N212A, I208V-S238Y, and N212A-S238Y), previously designed to improve catalytic efficiency and thermal stability. The methodology involved the production of the variants through expression in E. coli Rosetta (DE3), purification by affinity chromatography using a Ni-NTA column, and subsequent evaluation of kinetic parameters using p(NP)-acetate as a model substrate. Additionally, the effect of temperature on enzymatic activity was analyzed. The results indicated that all variants adequately followed the Michaelis-Menten model, but the I208V-S238Y variant showed the best overall performance, with the lowest Km value (2.77 mM), the highest Vmax (41.86 U), and the greatest specificity constant (CE = 10.08 1 sachet mM per s), indicating higher substrate affinity and superior catalytic efficiency. Moreover, this variant exhibited good protein expression and thermal stability, meeting the three established objectives. The I208V/S238Y variant therefore emerges as the most promising candidate for biotechnological applications focused on efficient PET degradation, offering a viable alternative for sustainably mitigating plastic pollution.