Abstract
Plastic waste is a global issue in marine and domestic environments. For thermoplastics like poly(ethylene terephthalate) (PET), a robust method for end-of-life management of these polymers is required to reduce the damage to the environment. PET is the most commonly used plastic in the food industry as packaging, often single-use bottles and food containers. Despite PET being mechanically recyclable, this process can only be repeated four to five time before some of the mechanical properties of the plastic are lost. Recently, a new method of small-scale recycling has been developed using enzymes to fully depolymerise PET to its monomeric units: terephthalic acid (TPA) and ethylene glycol (EG). To create a method of recycling using these enzymes, the properties must be improved for viability at industrial scale.In order to investigate upscale of enzymatic PET hydrolysis several studies were conducted. The analysis of denaturation kinetics of two cutinases, BTA-1 and BTA-2 (Thermobifida fusca), was investigated and compared to bioreactor experiments. Following this, a new directed evolution platform is employed to improve thermostability and activity of LCC-ICCG, the current benchmark enzyme for PET hydrolysis at scale. The same platform for directed evolution was utilised to improve another PET hydrolase (PHL7). These experiments generated LCC-ICCG and PHL7-Jemez which were subsequently tested in bioreactors. Further studies investigated glycosylation as a means to improve thermostability. The naturally non- glycosylated Fusarium solani cutinase (FsC) was engineered to produce three glycosylated variants. Analysis of denaturation kinetics of these mutants was carried out to predict the temperature at which the most insightful comparison of durability could be made, followed by high solids loading poly(butylene succinate) (PBS) depolymerisations in bioreactors at said temperature. Furthermore, to gain a deeper understanding of glycosylation and its effect on thermostability, modular cloning was employed to generate multi-glycan variants of LCC. These variants were then analysed to determine that glycosylation may have a cumulative effect on thermostability.
The results from these studies provide insights into how enzymes can be optimized for industrial biorecycling. Investigations into the denaturation kinetics of BTA-1 and BTA-2 have shown that small scale experiments can predict enzyme thermostability on a large scale. The directed evolution studies demonstrated the potential to tailor enzymes for improved PET hydrolysis at scale. Analysing the denaturation kinetics of glycosylated FsC variants allowed the prediction of the most thermostable variant highlighting this techniques utility for identifying enzymes suited for large-scale PET hydrolysis.
These studies demonstrate that enzymes can be optimized for improved activity in industrial-like conditions and that testing these enzymes with high solids loading is an important step in developing enzymes for large-scale biorecycling.
| Date of Award | 10 Sept 2024 |
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| Original language | English |
| Awarding Institution |
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| Supervisor | Andrew Pickford (Supervisor), Sam Robson (Supervisor) & Tobias von der Haar (Supervisor) |