Abstract
Plastic pollution represents a critical global challenge, necessitating the development of sustainable end-of-life solutions for ubiquitous materials such as poly(ethylene terephthalate) (PET). While traditional mechanical recycling of PET is limited by property degradation and a finite number of recycling cycles, and chemical recycling typically requires high temperatures, pressures, and harsh reagents, enzymatic depolymerisation offers a promising alternative that enables complete breakdown of PET into its constituent monomers for re-synthesis or up-cycling under milder conditions. However, achieving industrial viability for this biocatalytic approach requires overcoming key challenges, notably the inherent recalcitrance of PET arising from its high crystallinity and morphological diversity, as well as the need for robust and efficient enzymes capable of operating under industrially relevant conditions.This PhD thesis investigates enzymatic PET deconstruction with a focus on overcoming these limitations through a combined approach of enzyme engineering and substrate modification.
A comprehensive laboratory-scale workflow was developed for full biocatalytic recycling of PET, encompassing recombinant expression and purification of PET-hydrolysing enzymes, bioreactor-scale depolymerisation, and subsequent monomer recovery and upcycling. Using this system, the recovered monomers were successfully repolymerised, achieving over 40 % PET hydrolysis following substrate pretreatment, with the recovered terephthalic acid (TPA) further upcycled into adipic acid and vanillin, demonstrating closed-loop recycling and chemical valorisation.
Protein engineering efforts focused on improving enzyme stability and catalytic efficiency. The introduction of disulphide bridges into Saccharopolyspora genus cutinase variants did not significantly enhance thermostability, whereas a targeted charge substitution distal to the active site played a key role in modulating PET film substrate binding. Among three novel PET hydrolases characterised, a single distal charge mutation in the SfCut enzyme increased PET film degradation from 5 % to 25 %, while in a faster variant the substitution reduced activity from 44 % to 14 %, supporting the influence of surface charge in rational enzyme design and substrate selectivity.
To enhance substrate affinity, carbohydrate-binding modules (CBMs) were explored through fusion of a CBM64 domain to a PET-degrading enzyme, with the aim of increasing enzyme binding affinity, particularly towards highly crystalline materials, including challenging post-consumer textile waste samples. Notably, the CBM fusion enhanced activity on amorphous substrates, with a marked improvement observed on amorphised textile samples, where hydrolytic activity increased from 35 % to 80 %, demonstrating the potential of CBM-assisted enzymes to improve depolymerisation efficiency, solids loading dependent.
Alongside enzyme engineering, this thesis also investigates polymer pre-treatment strategies, specifically dissolution and re-precipitation, to effectively amorphise highly crystalline and laboratory-dyed textile PET, thereby rendering it more accessible to enzymatic hydrolysis. It was shown that solvent concentrations as low as 4 % dramatically decrease enzyme activity through protein denaturation, while substrates with crystallinity above 30 % can still support substantial PET-degrading activity. These findings indicate that substrate bioavailability, rather than crystallinity alone, is a key determinant of enzymatic PET degradation efficiency.
By integrating enzyme engineering with substrate conditioning, this work provides a detailed understanding of the interdependent factors controlling enzymatic PET depolymerisation. The findings offer a mechanistic and practical framework to accelerate the development of industrially viable biocatalytic recycling processes, contributing to the broader transition towards a circular plastics economy.
Keywords: biocatalytic recycling; carbohydrate-binding modules; circular economy; enzymatic depolymerisation; poly(ethylene terephthalate); protein engineering; substrate crystallinity; textile waste; thermostability; bioreactor hydrolysis.
| Date of Award | 6 Jan 2026 |
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| Original language | English |
| Awarding Institution |
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| Supervisor | Andrew Pickford (Supervisor), Victoria Bemmer (Supervisor) & Bruce Lichtenstein (Supervisor) |