Browsing by Author "Burton, Stephanie Gail"

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    Biocatalytic studies of phenol oxidases producing antioxidants
    (2007) Ncanana, Sandile Welcome; Burton, Stephanie Gail
    In recent years there has been increasing interest in the production of oligomers and polymers of economic importance using biocatalysts; the application of enzymes in dimerisation or polymerisation processes can lead to the synthesis of unique compounds, with novel properties, that could not be easily achieved by conventional methods. Further, the application of biocatalysts in polymerization processes can be exploited in development of bioremediation systems, and there is a demand for new technologies that can be utilized in the removal of organic pollutants such as phenolics from contaminated environment. This study reports on the potential application of laccase, obtained from the white rot fungi Trametes pubescens, in the synthesis of organic compounds which are dimers or polymers, and in the development of bioprocesses of potential economic importance. The focus of this study is, particularly, on the effect of organic solvents and the structure of the substrates on the nature of products formed. The thesis also gives some insight into the relationship between the structure of laccase products and their biological (antioxidant and antimalarial) activity. The compounds tyrosol, hydroxytyrosol, 8-hydroxyquinoline, and totarol were selected as the model compounds for laccase reactions. Tyrosol was oxidised by laccase, yielding dimeric and polymeric compounds which were identified by LC-MS and IH-NMR. As a comparative study, hydroxytyrosol was also oxidised by laccase yielding dimeric, trimeric and polymeric compounds which were identified by LC-MS. Manipulation of this biocatalytic system resulted in development of an efficient process that allows for selectivity with respect to the products. A system was then developed whereby oxidation of hydroxytyrosol by laccase would selectively yield either dimers or oligomeric products. Thus, use of 50 % acetone in the reaction medium favoured the synthesis of dimeric products, and 20 % methanol resulted in the formation of a polymeric product. These results showed that hydroxytyrosol-laccase reactions were more readily controlled than tyrosol-laccase reactions, and this difference was attributed to structural configuration of these substrates.
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    Characterization of a thermostable amidase and development of a bioreactor process for lactic acid production
    (2005) Makhongela, Happy Steven; Burton, Stephanie Gail; Cowan, DA
    This thesis reports studies of the biochemical properties of a newly isolated thermostable amidase from Bacillus sp. RAPc8, and development of a continuous reactor process for the production of a target product. The amidase was cloned and over-expressed in E. coli BL21 strain pNH 223 pLySs by Cameron (2001). Earlier work by Cameron (2001) on the production and purification of the recombinant amidase showed that the enzyme could be produced with high levels of expression in shake-flask culture. Furthermore, preliminary studies have also shown that the molecular weight of the amidase was approximately 35kDa, and that it acts optimally at a temperature and pH of 50°C and 7.2 respectively.
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    An investigation into the biocatalytic application of the thermostable nitrile hydratase from the thermophilic strain Geobacillus pallidus RAPc8
    (2008) Chiyanzu, Idan; Burton, Stephanie Gail
    Nitrile hydratases (NHases) are bacterial metalloenzymes that catalyze the hydration of nitriles to their corresponding amides. The enzymes have been found in several microorganisms and participate in the metabolism of nitrile compounds as source of carbon and nitrogen. The commercial use of NHases is well recognized and has prompted research interest as well as the application of the enzymes in the manufacture of commodity amide chemicals. This has largely been due to the versatile nature of the enzymes, associated with their physiochemical properties and broad substrate specificity. However, the widespread application of nitrile-converting enzymes in the industrial processes has been restricted in part by the thermal instability of the mesophilic-derived enzymes, and thus there is an increased focus on NHases from thermophilic microorganisms. A novel moderately thermophilic microorganism, Geobacillus pallidus RAPc8, was isolated by our collaborators (Pereira and co-workers, 1998). The strain has an optimal growth temperature of 65oC and constutitively expresses a thermostable nitrile hydratase. The gene cluster containing the nitrile hydratase were cloned, sequenced, and inducibly expressed in E. coli BL21 (DE3) to levels of approximately 49 U/mg. The NHase was purified by four steps including heat treatment, ammonium sulfate precipitation, hydrophobic interaction chromatography and ion exchange chromatography.
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    An investigation into the bioconversion of South African sub-bituminous coal by Trametes species and Trichoderma atroviride
    (2008) Vengadajellum, Caryn J; Burton, Stephanie Gail
    The work presented in this thesis describes the preliminary investigation of the bioconversion of a South African (SA) sub-bituminous coal to produce low molecular mass intermediates which may be used in further processes to yield value-added compounds. Low rank coals (LRC), which are less desirable for combustion processes than high rank coals, are more amenable to biodegradation due to their high moisture content and oxygen-containing functionalities. LRC are therefore ideal for application to biological processing. Microorganisms that are capable of modifying the coal structure by different mechanisms have been identified. Among these are the well-characterised lignin-degrading fungal systems, which have been commonly investigated for the bioconversion of LRC due to the ligninocellulosic origin of LRC, and thus its structural similarities to lignin. The bioconversion of coal to yield value added products is still at the fundamental research stage and has focused mainly on the biosolubilisation of low rank coals such as lignite. There is still a need to discover the ideal combination of coal type and microbial strain.
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    Investigation into the bioremediation and benefication of olive-derived wastewaters from the Western Cape
    (2005) Garcin, C J; Burton, Stephanie Gail
    Since this was the first research project into olive wastewaters produced in South Africa, the scope of the project was broad, and recommendations were made for further research in several directions. Most importantly, the development of smallscale treatment systems that could be used on site would be of great benefit to olive and olive oil producers, as they are often remote and do not have access to appropriate treatment facilities. In addition, the combination of unit operations, such as an extraction system and a biological degradation system, would allow for the recovery of a valuable product that would offset the cost of producing a treated effluent.
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    An investigation into the bioremediation of black olive brine wastewater
    (2005) Werner, Craig Michael; Burton, Stephanie Gail
    In South Africa, the table olive industry is burgeoning and as a consequence, the large volume of fermentation wastewaters that are generated have created environmental concerns, as these wastewaters have the potential to pollute rivers and ground waters. Currently, these wastewaters are disposed of in large evaporation ponds, but this is not considered to be the optimal treatment solution, due to the potential for pollution and environmental damage. This thesis describes an investigation into the bioremediation of black olive fermentation wastewaters. Wastewaters, from both the table olive and olive oil industries, are toxic and this toxicity can be attributed to the phenolic compounds present. These compounds are known to have antimicrobial and phytotoxic effects. Aerobic biological treatments have been extensively investigated in order to reduce the phenolic fraction of olive mill wastewaters (OMW) with relative success. Biological treatment methods are also cheaper than chemical or physical treatment methods. Therefore, it was decided to investigate aerobic biodegradation of black olive fermentation wastewater (olive wastewater) from the production of black (Kalamata) olives.
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    An investigation into the production of polyphenol antioxidants from grape marc
    (2006) Germanis, Jason; Burton, Stephanie Gail
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    Investigation of the antioxidant activity of phenolic compounds in cell culture systems
    (2009) Zwane, Eunice Nonhlanhla; Burton, Stephanie Gail
    This study focuses on newly synthesized polyphenolic compounds which were expected to be antioxidants in vivo. Antioxidants are compounds which at low concentrations can prevent or reduce the deleterious effects caused by reactive oxygen species (ROS) induced by UVA exposure of the skin. This study focuses on developing methods required to measure the effects of these synthesized compounds in ultraviolet radiation-exposed cultured skin cells. Biocatalytic synthesis of polyphenolic compounds was carried out using isolated enzymes, tyrosinase from Agaricus biosporus, and laccase from Trametes pubescens to produce oxidized oligomeric products.
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    The Laccase from Micromonospora sp.044 30-1 as a biocatalyst for synthesis of antioxidant compounds
    (2010) Goodwin, Candice Michelle; Burton, Stephanie Gail
    Laccases (EC 1.10.3.2) are blue multicopper oxidases that catalyse a single electron oxidation of various phenolic substrates with an associated four-electron reduction of dioxygen to water. The varied uses of laccase as a biocatalyst can be attributed to its ability to produce a free radical from a suitable substrate. Of importance to the pharmaceutical, chemical, and industrial sectors, are the laccase-catalysed reactions providing means for the synthesis of dimeric phenolics showing biological activity, including antioxidant activity. The objective of this research was to investigate the production of value-added compounds, with biological activity, via laccase-catalysed oxidation reactions. Our laboratory has access to several unique and previously unexploited culture collections obtained from extreme environments spanning the globe. Potentially novel strains were screened for the ability to produce laccases: 14 environmental isolates, of which 2 strains were fungi, 7 were streptomycetes, and 5 were non-streptomycetes, representing the rare actinomycete genera Gordonia, Rhodococcus, Mycobacterium, Amycolatopsis, and Micromonospora were screened. This is the first report of laccase production in these species. A screening protocol, using criteria specifically suited to bioprocess development, was developed to investigate variables affecting the production of laccase by the native strains. Variables investigated included different types of media (nutritional variables), pH, temperature, incubation times, aeration and agitation, salt concentrations, and the effect of inducers on laccase production by the native strains. Of the isolates investigated, actinomycete strain Micromonospora sp. 044 30-1 showed the greatest potential for the production of laccase. This strain may be novel and the role of laccase in this strain may be related to sporulation. Various growth requirements were investigated in order to optimise for maximal laccase production by strain 044 30-1. The optimal medium for laccase production was M172F medium, pH 5, supplemented with a high concentration of Cu2+ (8 mM), and 2.0% sodium chloride. Extracellular laccase production was higher than intracellular laccase production. The successful application of a Micromonospora strain in an airlift bioreactor specifically for the synthesis of laccase was demonstrated. The biocatalytic potential of the laccase from Micromonospora sp. 044 30-1 was investigated. Laccases are responsible for the formation of radicals that can react non-enzymatically with each other to form dimers or oligomers linked by C-C or C-O bonds. This study reports on laccasecatalysed oxidative reactions involving, primarily, tyrosol, monoacetyltyrosol, and to a lesser extent, reactions with totarol, 3-hydroxyanthranilic acid, and 8-hydroxyquinoline. This study reports for the first time the isolation and structure determination of novel biocatalysis reaction products, specifically the dimeric products obtained through biocatalytic reaction of monoacetyltyrosol with Trametes versicolor laccase, and Micromonospora sp. 044 30-1 laccase. The biocatalysis reaction products of the laccase-catalysed oxidation of tyrosol and monoacetylated tyrosol showed higher antioxidant activity than the parent compounds, as determined by the 2,2- diphenyl-1-picrylhydrazyl (DPPH) and low density lipoprotein (LDL) assays, showing that the dimeric derivatives of laccase-catalysed reactions with phenolic compounds have enhanced antioxidant capabilities. The biocatalysis products were also evaluated as antimicrobials and showed antimicrobial activity against E. coli, a vancomycin resistant clinical strain of Enterococcus faecium, a clinically relevant strain of Micrococcus, and Mycobacterium aurum, a strain that displays a similar antibiotic susceptibility profile to Mycobacterium tuberculosis, the causative agent of the prevalent disease tuberculosis.
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    The recovery of phenolic antioxidants from fruit processing wastewaters of South Africa by Chipo H. Mupure.
    (2009) Mupure, Chipo H; Burton, Stephanie Gail
    Thus, this study focused on the determination of antioxidant activity and the extraction of phenolic antioxidants from fruit processing wastewaters obtained from fruit processors in the Western Cape region of South Africa, using various extraction techniques. The chemical analysis of the fruit processing wastewaters showed that the silage water SW had the highest concentration of total phenols of 399.52 mgIL gallic acid equivalents (GAE) and apple wastewater AWl had the lowest concentration of 7.61 mg/L GAE. The fruit processing wastewaters also contained sugars and complex carbohydrates.