Understanding immobilised enzymes by NMR spectroscopy

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.

Biomimetic topography in orthopaedic ceramic

The primary objective of this research was to perform an in vitro assessment of the ability of microscale topography to alter cell behaviour, with specific regard to producing favourable topography in an orthopaedic ceramic material suitable for implantation in the treatment of arthritis. Topography at microscale and nanoscale alters the bioactivity of the material. This has been used in orthopaedics for some time as seen with optimal pore size in uncemented hip and knee implants. This level of topography involves scale in hundreds of micrometres and allows for the ingrowth of tissue. Topography at smaller scale is possible thanks to progressive miniaturisation of technology. A topographic feature was created in a readily available clinically licensed polymer, Polycaprolcatone (PCL). The effect of this topography was assessed in vitro. The same topography was transferred to the latest generation composite orthopaedic ceramic, zirconia toughened alumina (ZTA). The fidelity of reproduction of the topography was examined using scanning electron microscopy (SEM) and atomic force microscopy (AFM). These investigations showed more accurate reproduction of the topography in PCL than ZTA with some material artefacts in the ZTA. Cell culture in vitro was performed on the patterned substrates. The response of osteoprogenitor cells was assessed using immunohistochemistry, real-time polymerase chain reaction and alizarin staining. These results showed a small effect on cell behaviour. Finally metabolic comparison was made of the effects created by the two different materials and the topography in each. The results have shown a reproducible topography in orthopaedic ceramics. This topography has demonstrated a positive osteogenic effect in both polycaprolactone and zirconia toughened alumina across multiple assessment modalities.