Browsing by Author "Ncanana, Sandile Welcome"

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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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    Biolistic-mediated transformation of Eragrostis Curvula with the HSP 12 gene
    (2003) Ncanana, Sandile Welcome; Farrant, Jill M; Lindsey, George G; Brandt, Wolf F
    Eragrostis curvula is a desiccation sensitive monocotyledonous plant and an economically important forage grass in southern Africa. This species has a potential to be improved for drought and salt resistance among other important agronomic traits. In this study, E. curvula was used as a model system to explore the feasibility of producing pasture grasses with increased tolerance to water deficit. To date, no reports have been published on transformation of this species. This study reports the transformation of E. curvula with Saccharomyces cerevisiae Hsp 12 gene using biolistic-mediated transformation. Firstly, a tissue culture protocol was established for E. curvula that was suitable for transformation studies. Although this species has been previously regenerated in vitro using inflorescence tissues, this study established new protocols that utilize leaf and seed as source of material. The aim of which was to find the best regenerable tissue that could be used for transformation studies. Plant regeneration was achieved from shoot explants cultured on MS medium supplemented with either 0.5 mg/I thidiazuron (TDZ) or 0.5 mg/I 6-benzylaminopurine (BAP) through the process of direct organogenesis. It was found that TDZ was the most effective cytokinin. Plant regeneration was also achieved from callus induced from immature leaves on MS medium supplemented with 2 mg/I 2,4- 0,0.01 mg/I BAP and 6 % (w/v) sucrose. Histological experiments performed gave clear evidence that plant regeneration from callus was through a process of indirect organogenesis. The regeneration protocol was combined with an optimized biolistic mediated transformation protocol using the PDS-I00/He apparatus of which both shoot explants and callus were used as target tissues. In the molecular aspect of the study, the Hsp 12 gene was ligated in the Sac I restriction site of pCAMBIAUbeeQ vector. The successful cloning of the Hsp 12 gene was confirmed by PCR and restriction endonuclease digestion. The resultant vector pCAMBIAUbeeQ Hsp 12 was purified and subsequently used for biolistic transformation of E. curvula. The regenerable shoot explants and callus tissue were bombarded with DNA (pCAMBIAUBeeQHsp12) coated on gold particles. As a comparative study, Agrobacterium tumefaciens was transformed with pCAMBIAUBeeQHsp12 vector for subsequent transformation of Nicotiana tabacum. Transient expression of GUS gene in transformed E. curvula shoot explants was visualized 72 h after bombardment. Optimized conditions for expression of GUS gene were gold micro projectiles, 7 cm travel distance and helium pressure of 9100 kPa. Transformed tissues were cultured on the regeneration medium without antibiotic selection. Putative transformants were generated and the presence of Hsp 12 gene was verified by PCR and its expression at RNA level was confirmed by RT-PCR. The presence of Hsp 12 protein in the transformed plants was analyzed using SDS-PAGE and MALDI-TOF peptide mass spectrometry.