Chemistry

Materials science

Hydrolase

Engineering

Composite material

Biochemistry

Enzyme

Polyester

Melting point

Organic chemistry

Gene

Mutant

Amorphous solid

Polyethylene terephthalate

Chemical engineering

Directed evolution

Hydrolysis

Glass transition

Thermostability

Polymer

Engineering in Life Sciences

Polyethylene terephthalate (PET) is a mass‐produced petroleum‐based synthetic polymer. Enzymatic PET degradation using, for example, Ideonella sakaiensis PETase ( Is PETase) can be a more environmentally friendly and energy‐saving alternative to the chemical recycling of PET. However, Is PETase 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 Is PETase. The library was screened against the commercially available polyester‐polyurethane Impranil DLN W 50 for more thermostable Is PETase variants, yielding four variants with higher melting points. The most promising Is PETaseTM K95N/F201I variant had a 5.0°C higher melting point than Is 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 Is 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.

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

Engineering and evaluation of thermostable IsPETase variants for PET degradation