Open AccessEngineering and evaluation of thermostable Is PETase variants for PET degradation
Author(s) -
Brott Stefan,
Pfaff Lara,
Schuricht Josephine,
Schwarz JanNiklas,
Böttcher Dominique,
Badenhorst Christoffel P. S.,
Wei Ren,
Bornscheuer Uwe T.
Publication year - 2022
Publication title -
engineering in life sciences
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.547
H-Index - 57
eISSN - 1618-2863
pISSN - 1618-0240
DOI - 10.1002/elsc.202100105
Subject(s) - thermostability , polyethylene terephthalate , polyester , glass transition , polymer , amorphous solid , directed evolution , hydrolysis , materials science , mutant , melting point , hydrolase , chemistry , enzyme , chemical engineering , organic chemistry , biochemistry , composite material , gene , engineering
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.