3. Electronic Theses and Dissertations (ETDs) - All submissions
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Item Comparison of activities, electrophoretic patterns and kinetic properties of some enzymes in normal and cancerous liver(1976-11) Hammond, Kathryn D.Glucose present in tissues is metabolised either by means of glycolysis, gluconeogenesis, glycogenesis or glycogenolysis, as shown in Figs. 1 and 2. Glycolysis occurs in almost all tissues, whereas gluconeogenesis is restricted mainly to liver and kidney cortex; the synthesis and degradation of glycogen takes place mainly in liver, skeletal muscle and heart. The patterns of carbohydrate metabolism characteristic of normal tissue are often altered in various diseased states; this is particularly evident in cancer. Tumour tissues, such as hepatoma, appear to lose the functional properties of adult differentiated tissue and their metabolism often seems to resemble that of rapidly-dividing, undifferentiated foetal tissue.Item Application of laccase enzymes in organic synthesis(2018) Maphupha, Mudzuli MaxwellThe use of enzymes as catalysts in various synthetic procedures appears to be an economical and profound way of providing selective processes in synthetic organic chemistry. Enzymes provide alternative and sustainable processes and have helped to avoid limitations encountered when using traditional heterogeneous and homogeneous catalysts; this includes the use of toxic substances, use of expensive heavy metals, extensive use of harmful organic solvents, harsh reaction conditions, and also poor selectivity of many catalysts. Laccases are oxidoreductase enzymes capable of catalysing oxidation reactions of several low molecular weight organic compounds such as polyphenols, aminophenols, methoxyphenols, polyamines, and lignin-related molecules. The catalytic process of these enzymes occurs though a one-electron oxidation and water is released as the only by-product. In this project we investigated the range and limitations of applications of laccase enzymes in organic synthesis. The project focus was on method development for cross-coupling reactions of Carbon, Nitrogen, Oxygen, and Sulphur substituted aromatic compounds. The laccase facilitated synthesis of five classes of compounds; biaryl compounds, benzoxazoles, benzimidazoles, benzothiazoles, and aminobenzoquinones, was investigated. The research explored the synthesis of biaryl compounds from simple substituted phenol substrates. The optimal reaction conditions for the synthesis of biaryl compounds from simple phenols were investigated. A condensation reaction between 2-aminophenol and aryl aldehyde derivatives was performed with the aim of synthesising 2-arylbenzoxazole derivatives; however various aminophenol derivatives were formed as the phenolic Schiff base failed to cyclise. Alternatively, when including the laccase-mediator ABTS, dimerization of 2-aminophenol to 2-amino-3H-phenoxazin-3-one (4) occurred. A chemo-selective method for the synthesis of 2-aryl-1H-benzimidazoles from condensation of 2-phenelynediamine and aryl aldehydes was developed using laccase as an oxidising catalyst. Optimal conditions for synthesising 2-aryl-1H-benzimidazoles were identified while using acetate buffer (0.1 M, pH 4.5), acetonitrile as a co-solvent and the commercial laccase preparation Novoprime base 268. A modern and practical laccase-catalysed route suitable for the synthesis of 2-arylbenzothiazoles was developed. To the best of our knowledge, the laccase catalysed method for preparation of 2-arylbenzothiazole derivatives derived from condensation–dehydration reaction of 2-aminothiophenol with aryl-aldehydes has not been reported before. The method described is green, effective and simply requires a facile work-up routine, utilising solvents such as acetonitrile and DMF as co-solvents. Finally, factors limiting yields for the synthesis of aminobenzoquinones were investigated by varying the reaction conditions. The laccase catalysed nuclear diamination of aromatic hydrobenzoquinones with aliphatic and aromatic amine molecules was investigated under mild reaction conditions using commercial laccases from Novozymes (Suberase®, Denilite® II Base, and Novoprime Base 268). Conducting the reactions under dilute conditions, sequential addition of enzyme and substrate over time and using Novoprime Base 268 as our laccase increased the yields to up to 100%.Item The characterization of the phosphatidyl-inositol-3-kinase in plasmodium falciparum and the effect of selective inhibitors of this enzyme on the parasite(2004-05-04) Mtombeni, NokuhleMalaria is the most prevalent parasitic disease in the world and the emergence of drug resistant strains of Plasmodium falciparum has made the search for new antimalarial drugs important. Protein kinases play an important role in cellular function and the phosphatidylinositol 3-kinase (PI3K) signal transduction pathway is implicated in diverse cellular processes such as glucose transport, cell survival and proliferation. A homology based approach identified an open reading frame (ORF) coding for the catalytic region of part of the 6.4 Kb ORF of PFE0765w gene sequence found at plasmoDB. The ORF consisted of 1 758 base pairs which coded for a 586 amino acid protein with a molecular weight of 68.5 KDa. The PfPI3K ORF was amplified from P.falciparum DNA, subcloned into an expression vector and the sequence verified. Analysis of the expressed protein obtained by Western blotting and probing with anti-His monoclonal antibody showed a protein of 68.5 KDa as well as some smaller products.Item Synthesis of biodiesel from rubber seed oil for internal compression ignition engine(2017) Onoji, Samuel ErhigareABSTRACT Biodiesel has been identified as a good complement and plausible replacement of fossil diesel because of the overwhelming characteristic properties similar to fossil diesel in addition to its good lubricity, biodegradability, non-toxicity and eco-friendliness when used in diesel engines. The production of biodiesel from edible vegetable oils competes with food sources, thereby resulting in high cost of food and biodiesel. Studies have shown that rubber seed contains 35 45 % oil, which portrays a better competitor to other non-edible oil bearing plants in biodiesel production. In this study, non-edible vegetable oils from underutilized Nigerian NIG800 clonal rubber seeds were extracted from 0.5 mm kernel particle size using n-hexane as solvent to obtain a yield of 43 wt.% over an extraction time of 1 h. The oil was characterized for fatty acids by using gas chromatography-mass spectrometry (GC-MS), and for structural properties by Fourier transform-infrared (FT-IR) and nuclear magnetic resonance (NMR) analyses. The optimization of the process conditions of the vegetable oil extraction was evaluated using response surface methodology (RSM) and artificial neural network (ANN) techniques both of which, were based on a statistically designed experimentation via the Box-Behnken design (BBD). A three-level, three-factor BBD was employed using rubber seed powder (X1), volume of n-hexane (X2) and extraction time (X3) as process variables. The RSM model predicted optimal oil yield of 42.98 wt. % at conditions of X1 (60 g), X2 (250 mL) and X3 (45 min) and experimentally validated as 42.64 wt. %. The ANN model predicted optimal oil yield of 43 wt. % at conditions of X1 (40 g), X2 (202 mL) and X3 (49.99 min) and validated as 42.96 wt. %. Both models were effective in describing the parametric effect of the considered operating variables on the extraction of oil from the rubber seeds. On further examinations of the potentials of the vegetable oil, the kinetics of thermo-oxidative degradation of the oil was investigated. The kinetics produced a first-order reaction, with activation energy of 13.07 kJ/mol within the temperature range of 100 250 oC. In a bid to attain enhanced yield of biodiesel produced via heterogeneous catalysis, coupled with the carbonaceous potentials of the pericarp and mesocarp of rubber seed shell casing as a suitable catalytic material, the rubber seed shells (RSS) were used to develop a heterogeneous catalyst. RSS was washed 3 4 times with hot distilled water, dried at 110 oC for 5 h, ground to powder, and calcined at 800 oC at a heating rate of 10 oC/min as a catalyst and analyzed for thermal, structural, and textural properties using thermogravimetric analyzer, x-ray diffractometer, and nitrogen adsorption/desorption analyzer, respectively. The catalyst was further analyzed for elemental compositions and surface morphology by x-ray fluorescence and scanning electron microscopy, respectively. The catalyst was then applied in biodiesel production from rubber seed oil. A central composite design (CCD) was employed together with RSM and ANN to obtain optimal conditions of the process variables consisting of reaction time, methanol/oil ratio, and catalyst loading on biodiesel yield. The optimum conditions obtained using RSM were as follows: reaction time (60 min), methanol/oil ratio (0.20 vol/vol), and catalyst loading (2.5 g) with biodiesel yield of 83.11% which was validated experimentally as 83.06 0.013%. Whereas, those obtained via ANN were reaction time (56.7 min), methanol/oil ratio (0.21 vol/vol), and catalyst loading (2.2 g) with a biodiesel yield of 85.07%, which was validated experimentally as 85.03 0.013%. The characterized biodiesel complied with ASTM D 6751 and EN 14214 biodiesel standards and was used in modern diesel test engine without technical modifications. Though the produced biodiesel has a lower energy content compared with conventional diesel fuel, in all the cases of blends considered, the optimal engine speed for higher performance and lower emissions was observed at 2500 rpm. In this study, the B20 blend has best engine performance with a lower emission profile, and was closely followed by B50 blend.Item Application of biocatalysts in the resolution of peptidomimetic compounds from multi-component reactions(2017) Kola, FatimaA library of potentially bioactive peptidomimetic compounds has been created using the Ugi and Passerini multi-component reactions. The products were isolated as racemic mixtures which we aimed to separate by enzymatic means. The Ugi enantiomers were obtained in exceedingly low yields due to considerable byproduct formation encountered with the use of ammonia as reactant and methanol as solvent. A threefold increase in the yields was achieved when employing the less nucleophilic 2,2,2-trifluoroethanol as solvent. An endeavour to resolve the racemates using proteases and penicillin G acylase proved unsuccessful, possibly attributable to the poor solubility of the Ugi products in a broad range of organic solvents. Enantiomers obtained from the Passerini reaction were efficaciously hydrolysed by lipase enzymes. Hence, the enantioselectivity of the hydrolysis reactions was explored by the employment of chiral HPLC and Mosher ester analysis. The biocatalysts displayed adequate selectivity for one of the adducts, making prospective separation of the racemic mixture viable. An investigation into the diastereoselectivity of the Passerini reaction was also performed. The application of phenylalanine-derived chiral starting materials in the reaction was seen to have a slight effect on reaction diastereoselectivity, giving on average a 2:1 diastereomeric ratio. Slightly greater d.r. values were obtained when bulkier isocyanides were employed. The diastereomeric mixtures were effectively separated via preparative HPLC to obtain pure compounds. 13C NMR spectroscopy indicated a trend in the chemical shift values for the newly formed stereogenic carbon centres for most of the two separated diastereomers. The C-2 values of most of the minor diastereomers appeared more downfield in comparison to the major diastereomers. Distinguishing the stereochemistry of the major and minor diastereomers through X-ray crystallography was not possible as the compounds were non-crystalline. Thus the configurations of the newly formed stereogenic centres of the major compounds were assigned by analogy with similar compounds previously identified in our laboratory. The major compounds were described as R at the newly formed stereogenic centre when L-phenylalaninal was used, and the minor products S. When D-phenylalaninal was used, the major diastereomer was assigned an S configuration at the newly formed stereocentre and the minor one was assigned R. The formation of the major diastereomer could be explained using a Felkin-Anh chelation controlled model, which has been observed in other reactions of mono protected amino aldehydes.Item Class pi glutathione S-transferase: unfolding and conformational stability in the absence and presence of G-site ligands(1996) Erhardt, JulijaThe glutathione S-transferases (GST) are a supergene family of h0111o-or heterodimeric Phase II detoxification enzymes which catalyse the S-conjugation between glutathione and an electrophilic substrate. The active site can be divided into two adjacent functional regions; a highly specific Gssite for binding the physiological substrate glutathione and a nonspecific If-site for binding nonpolar electrophilic substrates. Unfolding of porcine class Pi isoenzyme (pGSTPl~l) was monitored under equilibrium conditions using different physicochemical parameters. The coincidence of unfolding curves obtained with functional and structural probes, the absence of thermodynamically stable intermediates such as a folded monomer, and the dependence of pGSTPl··l stability upon protein concentration, indicate a cooperative and concerted two-state unfolding transition between native dimeric pGSTPl-l and unfolded monomeric enzyme. Equilibrium and kinetic unfolding experiments employing tryptophan fluorescence and enzyme activity measurements were preformed to study the effect of ligand binding to the G-site on the unfolding and stability of the porcine class pi glutathione S-transferase against urea. The presence of glutathione caused a shift in the equilibrium-unfolding curves towards lower urea concentrations and enhanced the first-order rate constant for unfolding suggesting a destabilisation of the pGSTPl-l structure against urea. The presence of either glutathione sulphonate or S-hexylglutathione, however, produced the opposite effect in that their binding to the G-site appeared to exert a stabilising effect against urea. The binding of these glutathione analogues also reduced significantly the degree of cooperativity of unfolding indicating a possible change in the protein's unfolding pathway.