3. Electronic Theses and Dissertations (ETDs) - All submissions
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Item Ellagic acid-mediated inhibition of schistosoma japanicum glutathione transferase: enzyme kinetics and structural perspectives(2018) Oyiogu, Blessing OluebubeSchistosoma japonicum glutathione S-transferase (SjGST) is a homodimeric enzyme central to detoxification of electrophilic compounds in the parasite due to its lack of cytochrome P-450. Hence, SjGST is a potential therapeutic target for schistosomiasis. Akin to most cytosolic GSTs, SjGST is composed of two active sites per monomer, which are the glutathione binding site (G-site) and the hydrophobic binding site (H-site). Ellagic acid (EA), a polyphenolic compound ubiquitous in fruits and other plant-based products has been identified as a therapeutic agent against cancer, heart and liver diseases. The interaction between SjGST and EA was characterised in this study. Using 1-chloro-2,4 dinitrobenzene (CDNB) and glutathione (GSH) as SjGST substrates, EA was shown to inhibit SjGST activity, with an IC50 of 2.4 μM. The observed mode of inhibition with respect to the G-site and H-site was non-competitive and uncompetitive, respectively. The turnover rate of SjGST was reduced from 0.002 ± 0.0001 sec1 to 0.0006 ± 0.00001 sec-1 in the presence of 9 μM EA, while the catalytic efficiency (kcat/KM) was reduced from 0.02 ± 0.0002 M-1s-1 to 0.009 ± 0.0001 M-1s-1. Using ANS as an extrinsic fluorescence probe, SjGST bound to ANS in a concentration-dependent manner in the absence (Kd = 40.6 ± 2.6 μM) or presence of EA (Kd = 30.3 ± 1.4 μM). This observation was further probed by the displacement of ANS by EA, which suggest that about 64.5% of ANS was displaced by EA. The ANS studies indicated that EA binds to the H-site of the enzyme. Secondary and quaternary structure analyses indicate that EA does not alter the secondary and tertiary structures of SjGST. Thermodynamic parameters obtained by isothermal titration calorimetry (ITC) indicate that the interaction between SjGST and EA is enthalpically-driven and compensated by a negative entropy change (-T∆S = 20.40 ± 0.08 kJ/mol) because of enthalpy-entropy compensation. The free energy change (∆G = -29.88 ± 0.07 kJ/mol) is also favoured by entropic contributions; therefore, indicating a strong hydrophobic interaction. A stoichiometry of 4 molecule of EA per mole of the dimeric enzyme was obtained. Molecular modelling studies suggests that EA binds more to the dimer interface compared with the Hsite. Therefore, it is possible that EA may bind to two different sites in the enzyme, the H-site and the dimer interface.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 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 Development of lymphocyte specific internalising aptamers(2014-04-23) Millroy, Laura AnnAptamers are synthetic nucleic acid molecules designed to bind with high specificity and affinity to a selected target. The aptamer selection method, called the systematic evolution of ligands by exponential enrichment (SELEX), was first described in 1990 and has been adapted for the selection of aptamers for a number of applications. One such application is the selective targeting of cells for therapeutic delivery. This thesis explores this application with the selection and characterisation of internalising aptamers specific to the T lymphocyte specific receptor, CD7. The CD7 receptor is expressed on thymus derived progenitor lymphocytes and remains after T cell activation and expression of the CD4 receptor. As such, the CD7 receptor is a noteworthy target for lymphocyte cancers, HIV-1 and other T lymphocyte tropic viruses. A heterogeneous pool of internalising CD7-aptamers was enriched through six rounds of positive selection in a stably transduced CD7-HeLa cell line. Aptamers were selected using a modified whole cell SELEX method that selected specifically for internalising aptamers. Aptamer specificity for CD7-HeLa cells over HeLa cells was screened by flow cytometry. CD7 specific aptamers were screened for binding after blocking CD7-HeLa cells with an anti-CD7 antibody. Eight CD7 specific aptamer clones were selected from CD7-HeLa screening for evaluation in Jurkat cells (T lymphocyte cell line endogenously expressing the CD7 receptor). Three aptamer clones showed high level binding to Jurkat cells by flow cytometry (CSIR 3.14, CSIR 3.37 and CSIR 3.42). Kinetic analysis of aptamer internalisation was analysed using flow cytometry and determined to be within the femtomolar range. Aptamer CSIR 3.14 had a dissociation constant of 2.1 fM and an association rate of 4.7 ± 2.4 × 105 Molar-1minute-1, aptamer CSIR 3.37 had a dissociation constant of 0.23 fM and an association rate of 4.3 ± 3.3 × 106 Molar-1minute-1 and aptamer CSIR 3.42 had a dissociation constant of 1.1 fM with an association rate of 7.9 ± 5.1 × 105 Molar-1minute-1. Aptamer CSIR 3.14 internalisation was tracked by confocal microscopy and the kinetics calculated with an association rate of 6.3 × 104 Molar-1minute-1 and Kd of 13 fM. Deletions within the CSIR 3.14 sequence that altered the predicted structures significantly reduced the aptamer binding. Combined, the data presented in this thesis identifies aptamer CSIR 3.14 as a lymphocyte specific internalising aptamer with potential for therapeutic delivery.