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.

Re-imagining the theology of human sexuality in the Methodist Church: the use of narrative in theological methodology

This thesis proposes the development of a narrative methodology in the British Methodist Church. Such a methodology embraces and communicates both felt experience and critical theological thinking, thus producing and presenting a theology that might have a constructive transformative impact on wider society. In chapter one I explore the ways in which the Church speaks in public, identify some of the challenges it faces, and consider four models of engagement. If the Church is to engage in public discourses then I argue that its words need to be relevant and connect with people’s experiences. To ground the thinking I focus on the context of the British Methodist Church and explore how the Church engages in theological reflection through the lens of its thinking on issues of human sexuality. Chapter two reviews how theological reflection is undertaken in the British Methodist Church. I describe how the Methodist Quadrilateral of Scripture, tradition, reason and experience remains a foundational framework for theological reflection within the Methodist Church and consider the impact of institutional processes and the ways in which the Methodist people actually engage with theological thinking. The third and fourth chapters focus on how the British Methodist Church has produced its theology of human sexuality, giving particular attention to the use of personal and sexual stories in this process. I find that whilst there has been a desire to listen to the stories of the Methodist people, there has not been a corresponding interrogation or analysis of their stories so as to enable robust and constructive theological reflection on these experiences. Using resources from Foucauldian approaches to discourse analysis, I critique key statements and the processes involved in their production, offering an analysis of this body of theological thinking and indicating where possibilities for alternative ways of thinking and acting arise. The proposed methodology draws upon resources from social science methodologies, and in chapter five I look at the use of personal experience and relevant strategies of inquiry that prompt reflection on the hermeneutical process and employ narrative approaches in undertaking, analysing and presenting research. The exploration shows that qualitative research methodologies offer resources and methods of inquiry that could help the Church to engage with personal stories in its theological thinking in a robust, interrogative and imaginative way. In chapter six an examination of story and narrative is undertaken, to show how they have been understood as ways of knowing and how they relate to theological inquiry. Whilst acknowledging some of the limitations of narrative, I indicate how it offers constructive possibilities for theological reflection and could be a means for the British Methodist Church to engage in public discourse. This is explored further in chapter seven, which looks in more detail at how the British Methodist Church has used narrative in its theological thinking, and outlines areas requiring further attention in order for a narrative theological methodology to be developed, namely: attention to the question ‘whose experience?’; investigation of issues of power and the dynamics involved in the process of the production of theological thought; how personal stories and experiences are interrogated and how narrative is constructed; and how narrative might be employed within the Methodist Quadrilateral. The final chapter considers the advantages and limitations of such an approach, whether the development of such a method is possible in the Methodist Church today and its potential for helping the Church to engage in public discourse more effectively. I argue that this methodology can provoke new theological insights and enable new ways of being in the world