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.

The regulation of AMP-activated protein kinase by vascular endothelial growth factor

The function of the vascular endothelium is to maintain vascular homeostasis, by providing an anti-thrombotic, anti-inflammatory and vasodilatory interface between circulating blood and the vessel wall, meanwhile facilitating the selective passage of blood components such as signaling molecules and immune cells. Dysfunction of the vascular endothelium is implicated in a number of pathological states including atherosclerosis and hypertension, and is thought to precede atherogenesis by a number of years. Vascular endothelial growth factor A (VEGF) is a crucial mitogenic signaling molecule, not only essential for embryonic development, but also in the adult for regulating both physiological and pathological angiogenesis. Previous studies by our laboratory have demonstrated that VEGF-A activates AMP-activated protein kinase (AMPK), the downstream component of a signaling cascade important in the regulation of whole body and cellular energy status. Furthermore, studies in our laboratory have indicated that AMPK is essential for VEGF-A-stimulated vascular endothelial cell proliferation. AMPK activation typically stimulates anabolic processes and inhibits catabolic processes including cell proliferation, with the ultimate aim of redressing energy imbalance, and as such is an attractive therapeutic target for the treatment of obesity, metabolic syndromes, and type 2 diabetes. Metabolic diseases are associated with adverse cardiovascular outcomes and AMPK activation is reported to have beneficial effects on the vascular endothelium. The mechanism by which VEGF-A stimulates AMPK, and the functional consequences of VEGF-A-stimulated AMPK activation remain uncertain. The present study therefore aimed to identify the specific mechanism(s) by which VEGF-A regulates the activity of AMPK in endothelial cells, and how this might differ from the activation of AMPK by other agents. Furthermore, the role of AMPK in the pro-proliferative actions of VEGF-A was further examined. Human aortic and umbilical vein endothelial cells were therefore used as a model system to characterise the specific effect(s) of VEGF-A stimulation on AMPK activation. The present study reports that AMPK α1 containing AMPK complexes account for the vast majority of both basal and VEGF-A-stimulated AMPK activity. Furthermore, AMPK α1 is localized to the endoplasmic reticulum when sub-confluent, but translocated to the Golgi apparatus when cells are cultured to confluence. AMPK α2 appears to be associated with a structural cellular component, but neither α1 nor α2 complexes appear to translocate in response to VEGF-A stimulation. The present study confirms previous reports that when measured using the MTS cell proliferation assay, AMPK is required for VEGF-A-stimulated endothelial cell proliferation. However, parallel experiments measuring cell proliferation using the Real-Time Cell Analyzer xCELLigence system, do not agree with these previous reports, suggesting that AMPK may in fact be required for an aspect of mitochondrial metabolism which is enhanced by VEGF-A. Studies into the mitochondrial activity of endothelial cells have proved inconclusive at this time, but further studies into this are warranted. During previous studies in our laboratory, it was suggested that VEGF-A-stimulated AMPK activation may be mediated via the diacylglycerol (DAG)-sensitive transient receptor potential cation channel (TRPCs -3, -6 or -7) family of ion channels. The present study can neither confirm, nor exclude the expression of TRPCs in vascular endothelial cells, nor rule out their involvement in VEGF-A-stimulated AMPK activation; more specific investigative tools are required in order to characterise their involvement. Furthermore, nicotinic acid adenine dinucleotide phosphate (NAADP)-stimulated Ca2+ release from acidic intracellular organelles is not required for AMPK activation by VEGF-A. Despite what is known about the mechanisms by which AMPK is activated, far less is known concerning the downregulation of AMPK activity, as observed in human and animal models of metabolic disease. Phosphorylation of AMPK α1 Ser485 (α2 Ser491) has recently been characterised as a mechanism by which the activity of AMPK is negatively regulated. We report here for the first time that VEGF-A stimulates AMPK α1 Ser485 phosphorylation independently of the previously reported AMPK α1 Ser485 kinases Akt (protein kinase B) and ERK1/2 (extracellular signal-regulated kinase 1/2). Furthermore, inhibition of protein kinase C (PKC), the activity of which is reported to be elevated in metabolic disease, attenuates VEGF-A- and phorbol 12-myristate 13-acetate (PMA)-stimulated AMPK α1 Ser485 phosphorylation, and increases basal AMPK activity. In contrast to this, PKC activation reduces AMPK activity in human vascular endothelial cells. Attempts to identify the PKC isoform responsible for inhibiting AMPK activity suggest that it is one (or more) of the Ca2+-regulated DAG-sensitive isoforms of PKC, however cross regulation of PKC isoform expression has limited the present study. Furthermore, AMPK α1 Ser485 phosphorylation was inversely correlated with human muscle insulin sensitivity. As such, enhanced AMPK α1 Ser485 phosphorylation, potentially mediated by increased PKC activation may help explain some of the reduced AMPK activity observed in metabolic disease.

Ecology, physiology and performance in high-rate anaerobic digestion

The design demands on water and sanitation engineers are rapidly changing. The global population is set to rise from 7 billion to 10 billion by 2083. Urbanisation in developing regions is increasing at such a rate that a predicted 56% of the global population will live in an urban setting by 2025. Compounding these problems, the global water and energy crises are impacting the Global North and South alike. High-rate anaerobic digestion offers a low-cost, low-energy treatment alternative to the energy intensive aerobic technologies used today. Widespread implementation however is hindered by the lack of capacity to engineer high-rate anaerobic digestion for the treatment of complex wastes such as sewage. This thesis utilises the Expanded Granular Sludge Bed bioreactor (EGSB) as a model system in which to study the ecology, physiology and performance of high-rate anaerobic digestion of complex wastes. The impacts of a range of engineered parameters including reactor geometry, wastewater type, operating temperature and organic loading rate are systematically investigated using lab-scale EGSB bioreactors. Next generation sequencing of 16S amplicons is utilised as a means of monitoring microbial ecology. Microbial community physiology is monitored by means of specific methanogenic activity testing and a range of physical and chemical methods are applied to assess reactor performance. Finally, the limit state approach is trialled as a method for testing the EGSB and is proposed as a standard method for biotechnology testing enabling improved process control at full-scale. The arising data is assessed both qualitatively and quantitatively. Lab-scale reactor design is demonstrated to significantly influence the spatial distribution of the underlying ecology and community physiology in lab-scale reactors, a vital finding for both researchers and full-scale plant operators responsible for monitoring EGSB reactors. Recurrent trends in the data indicate that hydrogenotrophic methanogenesis dominates in high-rate anaerobic digestion at both full- and lab-scale when subject to engineered or operational stresses including low-temperature and variable feeding regimes. This is of relevance for those seeking to define new directions in fundamental understanding of syntrophic and competitive relations in methanogenic communities and also to design engineers in determining operating parameters for full-scale digesters. The adoption of the limit state approach enabled identification of biological indicators providing early warning of failure under high-solids loading, a vital insight for those currently working empirically towards the development of new biotechnologies at lab-scale.

A contribution towards the performance and structural understanding of copper and nickel salts as ammonia stores

The ongoing depletion of fossil fuels and the severe consequences of the greenhouse effect make the development of alternative energy systems crucially important. While hydrogen is, in principle, a promising alternative, releasing nothing but energy and pure water. Hydrogen storage is complicated and no completely viable technique has been proposed so far. This work is concerned with the study of one potential alternative to pure hydrogen: ammonia, and more specifically its storage in solids. Ammonia, NH3, can be regarded as a chemical hydrogen carrier with the advantages of strongly reduced flammability and explosiveness as compared to hydrogen. Furthermore, ammine metal salts presented here as promising ammonia stores easily store up to 50 wt.-% ammonia, giving them a volumetric energy density comparable to natural gas. The model system NiX2–NH3 ( X = Cl, Br, I) is studied thoroughly with respect to ammine salt formation, thermal decomposition, air stability and structural effects. The system CuX2–NH3 ( X = Cl, Br) has an adverse thermal decomposition behaviour, making it impractical for use as an ammonia store. This system is, however, most interesting from a structural point of view and some work concerning the study of the structural behaviour of this system is presented. Finally, close chemical relatives to the metal ammine halides, the metal ammine nitrates are studied. They exhibit interesting anion arrangements, which is an impressive showcase for the combination of diffraction and spectroscopic information. The characterisation techniques in this thesis range from powder diffraction over single crystal diffraction, spectroscopy, computational modelling, thermal analyses to gravimetric uptake experiments. Further highlights are the structure solutions and refinements from powder data of (NH4)2[NiCl4(H2O)(NH3)] and Ni(NH3)2(NO3)2, the combination of crystallographic and chemical information for the elucidation of the (NH4)2[NiCl4(H2O)(NH3)] formation reaction and the growth of single crystals under ammonia flow, a technique allowing the first documented successful growth and single crystal diffraction measurement for [Cu(NH3)6]Cl2.