Enzyme Engineering to Enhance Polyethylene Terephthalate Degradation

Poly(ethylene terephthalate) (PET) is a petroleum-derived polymer widely used in the manufacture of packaging materials and textile products. However, it is estimated that PET requires hundreds of years to degrade naturally, leading to significant environmental concerns. Initially, recycling was adopted as the primary strategy to reduce plastic waste. Nevertheless, only 9% of the plastic produced worldwide is recycled with the actual technologies due to the high energy costs associated with conventional recycling methods, limiting their large-scale implementation. Furthermore, recycled PET often exhibits physicochemical properties inferior to those of the virgin polymer. These limitations highlight the need for the development of alternative approaches. 

In 2016, Yoshida et al. discovered the bacterium Ideonella sakaiensis 201-F6, which produces a group of enzymes known as PETases that can hydrolyze the ester bonds present in the PET polymer. However, these enzymes exhibit relatively low catalytic efficiency. The predominant interactions identified were hydrophobic interactions and hydrogen bonds. The main objective of this research is to enhance the catalytic efficiency of the PETase from Ideonella sakaiensis through the rational redesign of its active site by introducing targeted mutations. A computational approach was employed for this purpose. The mutations were introduced in silico, and the resulting structures were subjected to energy minimization. The effects of the amino acid substitutions were evaluated using molecular docking and enzyme–substrate interaction analyses.  To investigate the effects of the mutations on ligand stability within the active site, molecular dynamics simulations were performed. The resulting trajectories were analyzed by calculating the root mean square fluctuation (RMSF), allowing the evaluation of ligand mobility and its retention mechanism within the active site. The first proposed mutation consists of replacing the isoleucine residue at position 208 in the wild-type enzyme with alanine. This substitution increases the volume of the enzyme's active site, aiming to facilitate the degradation  of larger PET fragments.