Caracterización cinética de las variantes silvestre y mutantes I208V, N212A y S238Y de la enzima PETasa de Ideonella sakaiensis

Abstract

The accumulation of plastic waste has reached critical levels, with 8 million tons polluting the oceans annually. The resulting microplastics enter ecosystems and organisms, posing a serious threat to environmental health. In response, bioremediation has emerged as a sustainable alternative, employing microorganisms or enzymes to degrade polymers. The enzyme IsPETase from Ideonella sakaiensis is particularly more effective than other PET-degrading enzymes. Thus, mutations close to the active site (S238Y, N212A and I208V) have been recently designed to improve its activity and thermostability, evaluated in qualitative analyses. Therefore, in this research the kinetics of these mutants was characterized by evaluating the kinetic parameters of the reactions catalyzed by these enzymes, using p(NP)-acetate as substrate and varying the temperature (25, 35 and 45 degrees Celsius). The data were analyzed with GraphPad Prism. A reduction in KM was observed in the mutants, indicating that the mutations increased the affinity for the substrate, although the decrease in kcat, reflected a lower catalytic capacity, especially for the I208V variant. Overall, all three mutations are less efficient than the WT variant in hydrolyzing phenyl esters under saturated conditions. Regarding the effect of temperature, the N212A variant showed the highest activity at 45 degrees Celsius, standing out as the most thermostable. This study aims to deepen the catalytic efficiency of IsPETase mutants, contributing to the development of biotechnological recycling for plastic wastes.