Plastics are ubiquitous in daily life as a useful material across many widespread sectors including transport, medicine, packaging and textiles. However, lack of end-of-life management and limitations in current recycling technologies have led to a global plastic pollution crisis. Polyethylene terephthalate (PET) is the most commonly used single use plastic for food packaging, water bottles and polyester textiles. Naturally occurring cutinase enzymes have been shown to hydrolyse PET. Increasing levels of PET pollution worldwide coupled with challenges faced by current recycling technologies require a solution. Enzymes as biocatalysts could offer a viable methodology but require fine-tuning in order to withstand industrial conditions, whilst still remaining an economically competitive and environmentally desirable approach. The studies described herein aim to understand PET degrading enzyme function and application at scale. This is achieved by biophysical characterisation, analysis of optimal conditions and product inhibition sensitivity of PETase and a PETase(W159H/S258F) variant. Following this, 74 putative PETases were discovered using a bioinformatics approach constrained to identify thermophilic proteins. As a result many new candidate enzymes were discovered showing desirable properties including thermostability and crystalline PET tolerance. Additionally, utilising synergistic enzymes systems to understand optimal ratios of PETase and a secondary enzyme MHETase to eliminate the intermediate product mono(2-hydroxyethyl) terephthalate (MHET) for a homogenous monomer generation; further engineering of a PETase:MHETase dual enzyme fusion improved PET hydrolysis by 6 fold. Finally, characterisation of the performance of accessory domains for industrial scale PET bio-recycling at high solids loadings of >15wt%, a key condition for industrial viability. We show that carbohydrate binding modules (CBMs) selected in this study do not enhance hydrolysis of PET film when fused to novel leaf compost cutinase variant (LCCYCCG) at high solids loading. Expanding this research to include different PET morphologies and %- crystallinities with an alternative catalytic domain from Saccharopolyspora flava (SfCut) and CBM64 from Spirochaete thermophila shows despite enhanced binding to crystalline PET moieties, there is no enhancement to PET hydrolysis. The insights from these studies enhance our understanding of PET degrading enzymes and the fundamentals for process design, such as product inhibition profiles, thermostability, crystalline PET tolerance and performance at industrial solids loadings which is directly linked to a more feasible PET biorecycling approach.
|Date of Award||7 Feb 2023|
|Supervisor||Andrew Pickford (Supervisor), John Edward McGeehan (Supervisor) & Gregg T. Beckham (Supervisor)|
Development of plastic degrading enzymes for future industrial PET biorecycling
Graham, R. (Author). 7 Feb 2023
Student thesis: Doctoral Thesis