Abstract
Since their discovery in the early 1900’s, plastics have become ubiquitous in everyday life. Possessing some exceptional physical and chemical properties, they have become extensively used in almost all industrial sectors, with the packaging, textiles, and building and construction sectors being the biggest producers and consumers. However, due to poor end-of-life management and extreme variability in recycling technologies coupled with high consumer demand and the short shelf-life of plastic packaging, this material has accumulated rapidly in the environment and as a result has found its way into every ecosystem on earth. Polyethylene terephthalate (PET) is a petroleum-derived synthetic polyester and one of the most common single-use plastics. It is used extensively in food packaging, water and soft drink bottles and in textiles for items such as clothes and carpets. Certain plant polymers such as cellulose, lignin and cutin can be broken down by naturally occurring enzymes and a number of these enzymes, particularly cutinases, have been shown to degrade PET.In 2016, a bacterium Ideonella sakaiensis was observed using PET plastic as its sole carbon source by producing an enzyme called PETase. PETase, like other PET- degrading enzymes, breaks the polyester chain into its smaller chemical building blocks, which can then be reformed to produce new, recycled PET. The use of PET- degrading enzymes on an industrial scale could be an invaluable approach towards a closed-loop plastic recycling process, by reducing, and ultimately ending, the use of oil-based chemicals for PET production.
Firstly, this thesis presents a structural and functional comparison of wild-type PETase to a previously designed double-mutant variant, PETaseW159H/S238F. We further highlight the effect of chemical additives, reaction temperature, substrate crystallinity and product accumulation on the catalytic ability of both enzymes which have implications for large scale enzymatic recycling of PET waste.
Secondly, we present an exhaustive and collaborative approach to discovering and characterising new thermophilic PET hydrolases from natural diversity, expanding the current pool of PET hydrolases that exist in a narrow sequence space. Finally, we structurally and biophysically characterise a subset of hyper-thermophilic putative PET-hydrolases. We use a combination of X-ray crystallography and AlphaFold to assess the tertiary structure, assess the catalytic ability across three morphologies of PET substrate and engineer a ‘lidless’ variant, with the hope of improving catalytic turnover.
| Date of Award | 21 May 2025 |
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| Original language | English |
| Awarding Institution |
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| Supervisor | Andrew Pickford (Supervisor), John Edward McGeehan (Supervisor) & Gregg T. Beckham (Supervisor) |