Faculty of Applied Sciences
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Item Laccase-mediated biotransformation of phenolic compounds for the synthesis of new antioxidants(2020) Mazibuko, Bodine; Kudanga, TukayiThe increased incidences, mortality rate and economic impact of noncommunicable diseases (e.g. high blood pressure and diabetes) associated with oxidative stress, have led to the higher demand for antioxidant supplements for their prevention. The use of naturally occurring antioxidants is becoming a more attractive option due to the health risks associated with synthetic antioxidants. Phenolic compounds from plants have been shown to have antioxidant properties with the potential to be used as substitutes to synthetic antioxidants. However, monomeric phenolic compounds have several short comings such as low bioavailability, poor solubility, and low antioxidant capacity while some have pro-oxidant properties at high concentrations. Hence there has been increasing research focused on the biotransformation of these phenolic antioxidants through enzymatic oligomerisation to higher molecular weight compounds with improved antioxidant capacity and stability. Of the investigated enzymes, laccases have shown the most promise owing to their green catalytic properties. Their reaction mechanism involves the use of molecular oxygen as a co- substrate in oxidising phenolic compounds to corresponding radicals, with water as the only by- product. This study focused on the synthesis of antioxidants with enhanced antioxidant capacity using a laccase from Trametes pubescens as biocatalyst. To establish the potential of the phenolic compounds for use as substrates for the coupling reactions, a preliminary screening process was done. Guaiacol, caffeic acid, vanillic acid, eugenol, catechol, gallic acid, ferulic acid and quercetin hydrate were identified as suitable substrates for the laccase enzyme. However, only products from eugenol, coumaric acid and quercetin could be isolated, hence coupling reactions were carried out using these substrates in monophasic systems. Reaction products were monitored using thin layer chromatography (TLC) and high-performance liquid chromatography (HPLC). Purification was carried out using preparative TLC and characterisation using liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR). The antioxidant capacities of reaction products were determined using ABTS (2,2’-Azinobis 3- ethylbenzthiazoline-6-sulfonic acid), DPPH (2,2-diphenyl-1-picrylhydrazyl) and FRAP (ferric- reducing antioxidant power) assays. Quercetin hydrate oxidation produced one product which was purified and characterised. The product had an Rf of 0.68, tR 13.567 and m/z 601 in negative mode, indicating that it was a dimeric form of quercetin. Oxidation of ρ-coumaric acid resulted in the production of two products designated P1 (Rf 0.47) and P2 (Rf 0.42). Further characterisation was done on product P2 since product P1 could not be successfully purified. P2 had a retention time of 11.295 and m/z 325, indicating that it was a dimer of ρ-coumaric. The ρ-coumaric dimer had an enhanced antioxidant capacity, approximately 2-fold, 3-fold and 6-fold higher compared to the substrate, as demonstrated by the ABTS, DPPH and FRAP assays, respectively. A symmetrical 5-5 eugenol dimer (m/z 325, [M] =326), bis-eugenol, was produced from eugenol oxidation. Maximum product formation (50% yield) was obtained in a monophasic system with 40% v/v dioxane as co-solvent after incubation for 18 h. The bis- eugenol dimer had an improved antioxidant capacity of up to three and four times that of eugenol as demonstrated by the ABTS and DPPH assays, respectively. In conclusion, two dimers with high antioxidant capacity were successfully produced, purified and characterised. The study has demonstrated the potential of the T. pubescens laccase as a catalyst for the synthesis of phenolic compounds with enhanced antioxidant capacity.Item Fabrication of electrochemical biosensors for the determination of phenolic compounds by experimental and computational methods(2018) Kunene, Kwanele Winterose; Bisetty, Krishna; Kanchi, SuvardhanThe polyphenolic compounds of interest, bisphenol A (BPA) and its analogue bisphenol S (BPS) used in the plastic industry to manufacture baby bottles and beverage containers, were used in this study. They are generally used in the manufacture of polycarbonates, epoxy resins and unsaturated polystyrene resins. There is a growing concern in the public and scientific community about these organic compounds due to their endocrine disrupting activity and negative toxic impact on the wildlife. This has encouraged scientists to embark on research to find a sensitive and selective technique that will adequately determine these compounds even in trace amounts. The experimental research strategy adopted in this work was supported by computational methods. This work was conducted in two stages; Firstly, a sensitive EC biosensor was developed using a carbon screen printed electrode fabricated with the combination of silver doped zinc oxide nanoparticles with multiwalled carbon nanotubes (MWCNTs) and laccase enzyme. The EC behaviour of BPA towards the fabricated biosensor was investigated using cyclic voltammetry and differential pulse voltammetry under optimum experimental conditions. Secondly, a novel and selective PEC sensor was developed for the first time to detect BPS based on the vertically aligned ZnO nanorods (ZnO NRs) with a molecularly imprinted polypyrrole (PPy). Amperometric, cyclic voltammetry and impedance spectroscopy were used for the investigation of the photo induced electrochemical behaviour of BPS. Different characterisation techniques such as ultra-violet visible spectroscopy, fourier transform infrared spectroscopy, scanning electron microscopy, transmission electron microscopy, photoluminescence, Raman spectroscopy, grazing incidence X-ray diffraction and diffuse reflectance were used to characterize the synthesized nanostructures. Results revealed that the fabricated EC and PEC sensors exhibited good catalytic activity towards the determination of BPA and BPS respectively, in samples extracted from plastic water bottles. For the EC method, a low detection limit of 0.08 μM for BPA in a linear range 0.5 to 2.99 µM was detected. However, in the case of BPS, a highly selective PEC method was attained linearly ranging from 2.5 to 12.5 µM with a much higher limit of detection of 0.7 μM. Experimental results were further supported computationally for a better understanding of the optical properties of ZnO NRs-polypyrrole complex. Computational results were in good agreement with experimental results.