Browsing by Author "Higgins, I. J."
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Item Open Access Biosensor and bioelectrocatalysis studies of enzymes immobilized on graphite electrode materials(1987-05) Schneider, B. H.; Higgins, I. J.; Turner, Anthony P. F.The immobilization of glucose oxidase and lipoamide dehydrogenase on graphite electrodes was studied for their application in mediated bioelectrochemical systems. These are techniques where the electrons involved in an enzymatic redox reaction are either supplied by, or given to an electrode from the enzyme. In the biosensor mode, where a current is measured due to product oxidation, the immobilized enzyme may be used to monitor its substrate, for example a biosensor for glucose based on the enzyme glucose oxidase. In the bioelectrosynthesis mode, when a cathodic potential is applied to the electrode, electrons may be transferred to the enzyme via a mediator thereby driving a thermodynamically difficult reaction, such as the reduction of the coenzyme NAD+ . A glucose sensor based on glucose oxidase immobilized onto graphite foil electrodes which had been doped with the mediator dimethylferrocene was found to produce both a linear and rapid response to changes in glucose concentration in solution. The kinetics of this particular system were studied in depth in order to explain its behaviour. Results showed that the porous nature of the electrode material resulted in immobilization of the enzyme throughout a number of layers• Consequently, diffusion of glucose into the electrode became the rate limiting process at low glucose concentrations, which resulted in linear calibration curves. This was characterized by a substrate modulus, which was found to be sufficiently large to predict diffusional limitations. From this the effective rate of glucose diffusion within the electrode was estimated, and found to be up to 5 times smaller than that in free solution. The rapid response of the sensor, on the other hand, was due to the absence of any mass transfer limitations external to the electrode surface. When lipoamide dehydrogenase was employed in the same system it was found that complications arose due to coupling of NADH oxidation with the adsorbed mediator. By employing a less anodic potential of +150 mV vs. Ag/AgCl at NADH concentrations below 1 mM the detection of enzymatic oxidation of the coenzyme was achieved. Enzymatic reduction of NAD+ was achieved using lipoamide dehydrogenase immobilized onto graphite felt electrodes. This reaction was mediated by methyl viologen, with the enzyme-modified electrode used to reduce the mediator at a potential of -0.7 V ( vs. SCE ). In this fashion the reduced form of methyl viologen was recycled, thereby driving the reduction of NAD . By coupling this system to lactate dehydrogenase it was possible to effect the reduction of pyruvate.Item Open Access The genetic improvement of brewing yeast(Cranfield University, 1989-08) Kirkham, Jennifer. Ann.; Higgins, I. J.Brewing yeast strains are usually aneuploid or polyploid with no true mating type. Thus many of the techniques which can be used to genetically modify laboratory yeast strains cannot be applied to them. This study was aimed at developing the technology for the genetic modification of brewing yeasts towards producing genetically improved brewing strains, suitable for use in large scale beer production. A system has been developed which can produce brewing yeast transformants with high genetic stability and unimpaired brewing performance. Such transformants contain only the required extra genetic material and no superfluous vector DNA. This system has also been demonstrated to have potential for the genetic labelling of yeast strains as a means of accurate identification which could also be used towards the patent protection of commercially important yeast strains. _ A method for the stabilisation of heterologous proteins produced in yeast has been investigated. The hypothesis that the glycosylation of heterologous proteins can improve their stability in yeast was tested by mutating the Escherichia coli ß-lactamase gene to produce N-linked glycosylation substrates. Two of the modified proteins were glycosylated when expressed in yeast, one of which had improved thermal stability. A wheat 11-amylase gene yeast expression cassette has been constructed using the wheat secretion signal and yeast gene control sequences. Expression of the cassette in yeast results in efficient starch-degrading activity both in liquid and on solid media. The enzyme is efficíently secreted and approximately 90% of the activity is found in yeast culture supernatants.Item Open Access Methanol dehydrogenase biofuel cells and enzyme-based electrodes(Cranfield University, 1984-12) Aston, W. J.; Higgins, I. J.; Turner, Anthony P. F.This thesis describes the linking of enzymes to electrodes and their application in biofuel cells and as analytical devices. Methanol dehydrogenase, an NAD independent enzyme was purified by two phase aqueous partition. The enzyme incorporated into a biofuel cell was capable of producing a current in the presence of either a soluble or insoluble mediator. Optimisation of the current was carried out and a variety of alternative membranes, mediators and electrodes were investigated for possible use in the biofuel cell. Although laboratory studies involved the use of platinum electrodes and the soluble mediator N,N,N',N' tetramethyl-p-phenylenediamine, other configurations were investigated, including modified membranes and electrodes. The ability to detect methanol coulometrically in solution led to the construction of a homogeneous poised potential sensor. The device was capable of detecting methanol at concentrations in solution as low as 0.02uM. the sensor was unaffected by many potential interfering compounds present in water supplies and was used in the analysis of water samples. The retention of the enzyme in the presence of the insoluble mediator 1,1'-dimethylferrocene allowed the construction of a probe. The probe was unaffected by fluctuations in the oxygen tension and was capable of detecting methanol in the range 0.9 uM to 0.1 mM. Covalent immobilisation of glucose oxidase in the presence of 1,1'-dimethylferrocene enabled the amperometric determination of glucose in the range 0.01 - 30 mM. The device was independent of pH in the normal physiological range with a temperature coefficient of 4.0% degrees-1. The electrode was used to assay both buffered glucose samples and preliminary work was carried out on untreated samples from diabetic and non-diabetic patients.