Construcción de variantes mutantes de la enzima PETasa de Ideonella sakaiensis

Abstract

Indiscriminate plastic production has become a serious environmental problem due to the lack of an efficient treatment. Polyethylene terephthalate (PET) is one of the most used polymers, however chemical and physical options for its degradation are not efficient, neither environmentally friendly. During the last decade, PET biologic treatment has aroused great interest, since it allows the complete degradation of this polymer, and its degradation products work as raw material to produce new PET. Consequently, biological degradation makes it possible to establish a circular system for the use of PET. This investigation built mutant variants of the Ideonella sakaiensis PETase, based on a structural and computational analysis of the enzyme to rationally design variants that potentially increase the enzymatic activity on PET. I208V, N212A and S238Y mutations presented a greater difference in binding energies, suggesting high affinity for the substrate and a low one for the reaction products. Mutant variants were built by site-directed mutagenesis using QuickChange II kit, and through Sanger sequencing it was confirmed that at least two clones of each mutant variant got the desired sequence. Obtained mutations were expressed in E. coli Rosetta (DE3) under expression assay established conditions, 0.1 mM IPTG and 22°C overnight (approx. 15 h). Finally, expressed mutants were purified through affinity chromatography (FPLC) using a Ni-NTA column coupled to an FPLC system. The presence of the enzyme of interest was verified by SDS-PAGE electrophoresis.