The development of new and improved methods for downstream purification of proteins and enzymes from crude mixtures
Isgrove, Frances Helen
Cardiff Metropolitan University
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New economically viable methods for the industrial scale production and processing of proteins are constantly being sought. This project concentrates on two techniques which may be utilised in the purification of non-recombinant proteins. First, aqueous two-phase systems were investigated as a potential tool for the separation of individual proteose peptones from a total proteose peptone fraction of bovine milk. The optimum pH for the separation of β-CN-5P from the total proteose peptone components was pH7. Aqueous two-phase systems containing PEG with a mean molecular weight of less than 8000 were not found to separate the total proteose peptone components. The addition of sodium chloride into the aqueous two-phase systems encouraged a more effective extraction of β-CN-5P, although this effect was found to plateau at 5% NaCl. Second, a model enzyme was immobilised onto nylon film and the optimisation of binding conditions was studied using spectrophotometric procedures. Results suggested that slight variations in the concentration of the incubating solutions had a profound effect on the efficacy of the procedure. Additionally, a considerable quantity of observed binding appeared to be due to non-specific interactions between the ligand and the matrix. This binding was investigated and was found to be a result of both ionic and hydrophobic interactions.