Engineering and evaluation of thermostable IsPETase variants for PET degradation

Polyethylene terephthalate (PET) is a mass-produced petroleum-based synthetic polymer. Enzymatic PET degradation using, for example, Ideonella sakaiensis PETase (IsPETase) can be a more environmentally friendly and energy-saving alternative to the chemical recycling of PET. However, IsPETase is a mesophilic enzyme with an optimal reaction temperature lower than the glass transition temperature (T-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 IsPETase. The library was screened against the commercially available polyester-polyurethane Impranil DLN W 50 for more thermostable IsPETase variants, yielding four variants with higher melting points. The most promising IsPETaseTM(K95N/F201I) variant had a 5.0 degrees C higher melting point than IsPETaseTM. 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 degrees C. Several other variants were compared and combined with selected previously published IsPETase 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.

Automated summary

In this study, a mutant library of a thermostable triple mutant (TM) of IsPETase was generated using error-prone PCR, resulting in the discovery of four more thermostable IsPETase variants when screened against Impranil DLN W 50. The most promising variant IsPETaseTM(K95N/F201I) exhibited a 5.0 degrees C higher melting point than IsPETaseTM, making it a more active PET hydrolase at higher reaction temperatures up to 60 degrees C. Thermostability was highlighted as a crucial characteristic for an effective PET hydrolase based on the findings.