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Abu Hassan, Aziana (2024) Experimental and computational study of latex clearing protein LcpK30 for rubber degradation. PhD thesis, University of Nottingham.

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Abstract

The demand for rubber products grows exponentially. This condition results in significant amounts of rubber waste in the environment. Most of the rubber waste would come from synthetic rubber. Rubber waste management without a sustainable strategy will harm the environment and the general public's health. This work aims to introduce a sustainable approach to degrading rubber using enzymes. Latex-clearing protein K30 (LcpK30) catalyses the oxidative cleavage of the C=C bond in cis-1,4-polyisoprene natural rubber (NR). This enzyme can degrade synthetic polyisoprene, but the degradation rate is still low, and its potential to degrade other diene rubbers remains poorly understood. The ability of LcpK30 to interact with a sizeable hydrophobic substrate is interesting. It thus raises the question of how the substrates can access the catalytic site that is buried inside the protein cavity.



The work presented in this thesis explores ways to increase the applicability of LcpK30 to rubber degradation and provides a further understanding of how LcpK30 interacts with the polyisoprene substrate. Optimum production of the oligomers is essential to materialise the utilisation of the products from enzymatic degradation of NR for high-value applications. The optimum yield of oligomer at 65 % was obtained when the NR degradation was performed with 20 mg mL-1 NR emulsion at 30 °C and 6 µM LcpK30 added in three batches over 24 hours.

Degradation of other diene rubbers with varied molecular weights, chemical structure and morphology catalysed by LcpK30 was assisted by pre-treating the rubber into film, particles and co-solvent emulsion. The degradability was evaluated based on the production of oligomers with carbonyl end-group. The oligomers were analysed using high performance liquid chromatography (HPLC), 1H Nuclear Magnetic Resonance (NMR) and gel permeation chromatography (GPC). It was found that the catalytic ability of LcpK30 to degrade other rubbers depended on the chemical structure and physical morphology of the rubber during the degradation. LcpK30 could degrade epoxidised NR with a low epoxide level (25 %), and the pretreatment improved LcpK30's contact with synthetic isoprene rubber (IR) molecules. The presence of other structural units (e.g. vinyl) in polybutadiene rubber or stereostructural changes (trans- polyisoprene) inhibits its catalytic activity.

Protein tunnel identification using CAVER-Pymol plugin 3.0.3 revealed two dominant tunnels that could offer access to the substrate from the surface to the buried active site. Computational modelling of protein (LcpK30) and ligand (cis-1,4-polyisoprene with ten C=C bonds) interaction using GOLD molecular docking showed two potential binding modes for the substrate, which are in extended and folded conformation that mostly interacted with LcpK30 through hydrophobic contact. Intriguingly, this study provided insight into interactions taking place further away from the active site, which a short substrate model cannot fully explain. The potential site for introducing mutation that no studies have yet reported was identified, which were Ala159, Ile396, and Leu171. In this study, the Leu171Phe mutant was prepared and showed an improved heme occupancy with similar activity to the wild type.

Findings from this study have enhanced the fundamental understanding needed to advance the research of LcpK30 to increase its potential as a biocatalyst for the environmentally friendly treatment of rubber waste. The application of LcpK30 can also be expanded as a tool for NR modification to develop new speciality rubber.

Item Type:	Thesis (University of Nottingham only) (PhD)
Supervisors:	Pordea, Anca
Keywords:	Rubber waste treatment; Rubber degradation; Polyisoprene substrate; Oligomers; Biocatalysts
Subjects:	T Technology > TD Environmental technology. Sanitary engineering
Faculties/Schools:	UK Campuses > Faculty of Engineering > Department of Chemical and Environmental Engineering
Item ID:	77126
Depositing User:	Abu Hassan, Aziana
Date Deposited:	18 Jul 2024 04:40
Last Modified:	18 Jul 2024 04:40
URI:	https://eprints.nottingham.ac.uk/id/eprint/77126

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