Engineering and evaluation of thermostable <i>Is</i>PETase variants for PET degradation

Abstract

Polyethylene terephthalate (PET) is a mass-produced petroleum-based synthetic polymer. Enzymatic PET degradation using, for example, <i>Ideonella sakaiensis</i> PETase (<i>Is</i>PETase) can be a more environmentally friendly and energy-saving alternative to the chemical recycling of PET. However, <i>Is</i>PETase is a mesophilic enzyme with an optimal reaction temperature lower than the glass transition temperature (<i>T</i> _g) of PET, where the amorphous polymers can be readily accessed for enzymatic breakdown. In this study, we used error-prone PCR to generate a mutant library based on a thermostable triple mutant (TM) of <i>Is</i>PETase. The library was screened against the commercially available polyester-polyurethane Impranil DLN W 50 for more thermostable <i>Is</i>PETase variants, yielding four variants with higher melting points. The most promising <i>Is</i>PETaseTM^K95N/F201I variant had a 5.0°C higher melting point than <i>Is</i>PETaseTM. Although this variant showed a slightly lower activity on PET at lower incubation temperatures, its increased thermostability makes it a more active PET hydrolase at higher reaction temperatures up to 60°C. Several other variants were compared and combined with selected previously published <i>Is</i>PETase mutants in terms of thermostability and hydrolytic activity against PET nanoparticles and amorphous PET films. Our findings indicate that thermostability is one of the most important characteristics of an effective PET hydrolase.