Surface modification of biomolecules for catalysis and delivery applications
File(s)
Author(s)
Chen, Yiyan
Type
Thesis
Abstract
The research is motivated by the urgent need to overcome the logistical and stability limitations of biomolecular therapeutics which often require cold chain storage due to the inherent instability of proteins and nucleic acids at room temperatures. This dissertation explores the surface modification of biomacromolecules, including proteins and DNAs, to enhance their thermal stability, solubility, and functional utility for applications in biocatalysis and therapeutic delivery.
Surface engineering was performed via cationization to increase the thermal stability of β-glucosidase. A comparative analysis between a commercial glucosidase and a thermostable variant (A5IL97) revealed superior activity of the latter in ionic liquids (ILs), achieving high glucose yields in ionic liquids at elevated temperatures. However, attempts to further stabilise the enzyme via carbodiimide-mediated cationisation and surfactant conjugation led to significant loss in activity, attributed to structural disruption during the modification process.
A novel strategy was subsequently developed for the direct conjugation of myoglobin with amine-functionalised ionic liquids, forming protein–ionic liquid complexes (PILs). MALDI-TOF confirmed the attachment of 3-14 IL molecules per protein, drastically altering its solubility profile: while native myoglobin was soluble only in water and DMSO, PILs demonstrated remarkable solubility in a range of organic solvents. Circular dichroism confirmed the retention of modified myoglobin secondary structure up to 95°C, underscoring the potential of PILs to enable biocatalysis in non-aqueous environments.
The research then extended to nucleic acid stabilisation through the development of DNA polyplex–surfactant biofluids (DNA-P-S). These were formed by complexing plasmid DNA with a cationic polymer and an oxidised surfactant. Characterisation via dynamic light scattering and circular dichroism confirmed successful assembly and structural preservation. The DNA-P-S biofluids exhibited a significant increase in thermal stability. These formulations retained transfection efficacy after 9 months at room temperature and heat shock at 70°C for 4 days, while unmodified counterparts lost all activity.
Surface engineering was performed via cationization to increase the thermal stability of β-glucosidase. A comparative analysis between a commercial glucosidase and a thermostable variant (A5IL97) revealed superior activity of the latter in ionic liquids (ILs), achieving high glucose yields in ionic liquids at elevated temperatures. However, attempts to further stabilise the enzyme via carbodiimide-mediated cationisation and surfactant conjugation led to significant loss in activity, attributed to structural disruption during the modification process.
A novel strategy was subsequently developed for the direct conjugation of myoglobin with amine-functionalised ionic liquids, forming protein–ionic liquid complexes (PILs). MALDI-TOF confirmed the attachment of 3-14 IL molecules per protein, drastically altering its solubility profile: while native myoglobin was soluble only in water and DMSO, PILs demonstrated remarkable solubility in a range of organic solvents. Circular dichroism confirmed the retention of modified myoglobin secondary structure up to 95°C, underscoring the potential of PILs to enable biocatalysis in non-aqueous environments.
The research then extended to nucleic acid stabilisation through the development of DNA polyplex–surfactant biofluids (DNA-P-S). These were formed by complexing plasmid DNA with a cationic polymer and an oxidised surfactant. Characterisation via dynamic light scattering and circular dichroism confirmed successful assembly and structural preservation. The DNA-P-S biofluids exhibited a significant increase in thermal stability. These formulations retained transfection efficacy after 9 months at room temperature and heat shock at 70°C for 4 days, while unmodified counterparts lost all activity.
Version
Open Access
Date Issued
2025-09-21
Date Awarded
01/01/2026
License URL
Advisor
Hallett, Jason
Publisher Department
Department of Chemical Engineering
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)