The production of plastic waste is increasing exponentially every year. In 2023, we produced more than 410 million metric tonnes of plastic worldwide, over 90% of which was fossil fuel based. In the search for new ways to recycle plastic waste, scientists have discovered microorganisms that secrete enzymes into their environment and utilise the degradation products of plastic polymers as a source of energy. Polyethylene terephthalate (PET) is a polymer consisting of terephthalic acid and ethylene glycol, which are linked by a hydrolysable ester bond that can be cleaved by PET hydrolases. The adsorption of enzymes on the surface of a hydrophobic polymer is required for subsequent degradation.
Our aim was to produce fusion and chimeric protein oligomers of CaPETaseM9 from the bacterium Cryptosporangium aurantiacum, TurboPETase from the bacterium HR29 and BHETase BsEstΔ5 from the bacterium Bacillus subtilis. BsEstΔ5 degrades intermediate products of PET degradation and thus prevents the inhibition of PET hydrolases. To achieve oligomerisation, we introduced parallel and antiparallel coiled-coil domains of GCN4 from the mesophilic yeast Saccharomyces cerevisiae and of Sso10a1 from the hyperthermophilic archaeon Sulfolobus solfataricus into monomeric enzymes. Our goal was to determine whether fusion and chimeric versions of the enzymes dimerise and whether this improves adsorption to the surface of PET and subsequent activity. Oligomerisation was analysed by size exclusion chromatography and mass photometry. Activity was measured by determining the concentration of PET film degradation products and by an enzymatic assay with the small-molecule substrate p-nitrophenyl butyrate (pNPB). We found that C-terminal fusions with the coiled-coil domain of GCN4 form not only dimers but also higher oligomeric states, regardless of the length of the linker sequence between the enzyme and the coiled-coil domain. These constructs retain activity on pNPB but exhibit reduced activity on the polymeric substrate PET, under our experimental conditions. Due to expression and stability difficulties of protein constructs containing TurboPETase, we were unable to detect dimer formation for either the fusion or chimeric enzymes with the coiled-coil domain of Sso10a1, within the tested concentration range. This could explain the reduced loss of enzyme activity on pNPB as well as on PET. Using dynamic light scattering measurements, we confirmed that the thermal stability of the studied enzymes is not affected by the introduction of the coiled-coil domain.
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