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.

Evaluating consumer price perception: a mental accounting and frame dependent perspective

In this thesis, we evaluate consumer purchase behaviour from the perspective of heuristic decision making. Heuristic decision processes are quick and easy mental shortcuts, adopted by individuals to reduce the amount of time spent in decision making. In particular, we examine those heuristics which are caused by framing – prospect theory and mental accounting, and examine these within price related decision scenarios. The impact of price framing on consumer behaviour has been studied under the broad umbrella of reference price, which suggests that decision makers use reference points as standards of comparison when making a purchase decision. We investigate four reference points - a retailer's past prices, a competitor's current prices, a competitor's past prices, and consumers' expectation of immediate future price changes, to further our understanding of the impact of price framing on mental accounting, and in turn, contribute to the growing body of reference price literature in Marketing research. We carry out experiments in which levels of price frame and monetary outcomes are manipulated in repeated measures analysis of variance (ANOVA). Our results show that where these reference points are clearly specified in decision problems, price framing significantly affects consumers' perceptions of monetary gains derived through discounts, and leads to reversals in consumer preferences. We also found that monetary losses were not sensitive to price frame manipulations.