Understanding immobilised enzymes by NMR spectroscopy

Fauré De la Barra, Nicole Eloísa (2016) Understanding immobilised enzymes by NMR spectroscopy. PhD thesis, University of Glasgow.

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Printed Thesis Information: https://eleanor.lib.gla.ac.uk/record=b3153678


Enzyme immobilisation is the conversion of a soluble enzyme molecule into a solid particle form. This allows the recovery of the enzyme catalyst for its re-use and avoids protein contamination of the product streams. A better understanding of immobilised enzymes is necessary for their rational development. A more rational design can help enormously in the applicability of these systems in different areas, from biosensors to chemical industry. Immobilised enzymes are challenging systems to study and very little information is given by conventional biochemical analysis such as catalytic activity and amount of protein. Here, solid-state NMR has been applied as the main technique to study these systems and evaluate them more precisely. Various approaches are presented for a better understanding of immobilised enzymes, which is the aim of this thesis. Firstly, the requirements of a model system of study will be discussed. The selected systems will be comprehensibly characterised by a variety of techniques but mainly by solid-state NMR. The chosen system will essentially be the enzyme α-chymotrypsin covalently immobilised on two functionalised inorganic supports – epoxide silica and epoxide alumina – and an organic support – Eupergit®. The study of interactions of immobilised enzymes with other species is vital for understanding the macromolecular function and for predicting and engineering protein behaviour. The study of water, ions and inhibitors interacting with various immobilised enzyme systems is covered here. The interactions of water and sodium ions were studied by 17O and 23Na multiple-quantum techniques, respectively. Various pore sizes of the supports were studied for the immobilised enzyme in the presence of labelled water and sodium cations. Finally, interactions between two fluorinated inhibitors and the active site of the enzyme will be explored using 19F NMR, offering a unique approach to evaluate catalytic behaviour. These interactions will be explored by solution-state NMR firstly, then by solid-state NMR. NMR has the potential to give information about the state of the protein in the solid support, but the precise molecular interpretation is a difficult task.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Solid-state NMR, immobilised enzyme, NMR, porous support, ligands interaction
Subjects: Q Science > Q Science (General)
Q Science > QD Chemistry
Colleges/Schools: College of Science and Engineering > School of Chemistry
Supervisor's Name: David, Professor Lennon
Date of Award: 2016
Depositing User: Miss Nicole Fauré
Unique ID: glathesis:2016-7319
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 24 May 2016 13:46
Last Modified: 16 Jun 2016 07:55
URI: https://theses.gla.ac.uk/id/eprint/7319

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