Department of Biochemistry
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- ItemThe age-old problem of pollution, its role in endocrine disruption and the current analytical technologies that can be employed to monitor and assess waste water treatment plants(Stellenbosch : Stellenbosch University, 2017-03) Olivier, Daniel Wilhelm; Wolfaardt, Gideon M.; Swart, Pieter; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.ENGLISH ABSTRACT: Water scarcity is a global problem and pollution of this valuable resource is a growing concern. South Africa is no exception. As part of an on-going study aimed at developing decentralized water treatment systems based on biomimicry design, this project aimed to evaluate and optimize analytical methods that can be used to evaluate the efficiency of these systems in removing compounds with endocrine disrupting properties. In addition, this project also aimed to show the possible consequences if pollutants are not removed by investigating the effects of a number of endocrine disrupting chemicals in combination with one another and in combination with natural human hormones. This should provide a more realistic view of how a combination of pollutants that typically end up in the environment due to pollution can adversely affect organisms, and by extension, the ecosystem. It should also contribute to our understanding of how common pollutants that gets applied on the skin as personal care products (PCPs) can possibly influence human health and be linked to diseases such as breast cancer. The first aim resulted in a method that can be used to isolate, identify and quantify 11 endocrine disrupting chemicals (3 hormones, 1 synthetic hormone analog, 4 PCPs, 2 plasticizers and 1 anticonvulsant) and one human indicator. The method uses solid phase extraction to isolate compounds, dansyl chloride derivatization of compounds to enhance mass spectrometry detection and a novel super-critical fluid chromatography system, called an ultra-performance convergence chromatography (UPC2) system, coupled to tandem mass spectrometry for detection and quantification of each compound. This method can be used to evaluate the removal efficiency of waste water treatment systems. However, method validation revealed additional optimization and simplification should be considered. The second aim yielded data that showed the combined effect four common PCP pollutants as either being additive, antagonistic or synergistic. The data highlights how these PCPs can possibly interfere with the endocrine system of humans and animals if used as PCPs or are found in environment as pollutants. Finally, the data also suggest how common PCPs can influence diseases such as breast cancer.
- ItemAltered lipid metabolism as a possible mechanism in fumonisin-induced hepatocarcinogenesis in rats and investigations into risk assessment in humans(Stellenbosch : Stellenbosch University, 2013-12) Burger, Hester Maria; Gelderblom, W. C. A.; Swart, P.; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.ENGLISH ABSTRACT: Exposure to food contaminates such as mycotoxins have been associated with a variety of animal and human diseases worldwide. In South Africa, maize is the most To further refine risk assessment in the socio-demographic heterogeneous population of South Africa, the development and evaluation of a sensitive and interactive model the Mycotoxin Risk Assessment Model (MYCORAM) proofed to be more sensitive compared to the classical probable daily intake (PDI). The development of the MYCORAM was based on mycotoxin distribution during dry milling of maize in milling fractions intended for human consumption which was superimposed on the maize intake profiles of the South African population. Although dry milling, including a degerming step, is an effective way to reduce mycotoxins, risk and exposure assessment are influenced by maize dietary intakes, gender and ethnicity. This became evident when considering FB dietary exposure in rural maize subsistence farming communities in the Eastern Cape Province, South Africa confirmed the vulnerability of this subpopulation to risk of fumonisin exposure. Specific maximum tolerated maximum levels (MTL) to safeguard these communities fall outside the international regulatory processes and need to be urgently addressed. With the complex nature of cancer development in mind, integration of basic science and nutritional epidemiology will be important to contribute to our understanding of the adverse effects of FB and to define relevant risk assessment parameters. important commercial grain crop not just economically but also as a local food commodity both commercially and in subsistence rural farming communities. In order to control and manage mycotoxin contamination in food, evidence-based risk assessment is needed that includes mechanistic and human exposure studies. From this perspective the current study was conducted and aimed in further unravelling fumonisin B1 (FB1) mycotoxin induced hepatocarcinogenesis via the disruption of the lipid metabolism. The study also critically evaluates aspects of human risk assessment due to its relevance and importance to food safety known to impact on food security. This entails mycotoxin distribution during maize dry milling and the assessment of mycotoxin exposure in the South African population and vulnerable rural communities at risk. Fumonisin B1 affects the integrity of biological membranes by altering key lipid and fatty acid parameter in plasma, microsomal, mitochondrial and nuclear subcellular membrane fractions in rat liver. Changes in the major lipid constituents entailing an increase in cholesterol (CHOL) and phosphatidylethanolamine (PE) whilst sphingomyelin (SM) and phosphatidylcholine (PC) tended to decrease. Isolated plasma membrane lipid rafts, from rat primary hepatocytes exposed to FB1 augments the intricate effects exerted on the lipid metabolism regarding CHOL, SM and PE. The disruption of lipid and fatty acid constituents, such as arachidonic acid and ceramide, are likely to be key determinants affecting growth regulatory signaling pathways relevant to the critical balance between cell proliferation and apoptosis during cancer promotion. These changes provide further evidence that FB1 induce cancer promotion by differential inhibition and/or stimulation process whereby a few resistant “initiated” hepatocytes proliferate in an environment where the growth of normal cells is inhibited. A specific lipogenic phenotype is effected by FB1 which is closely associated with cancer development and considered to occur via an epigenetic-type of mechanism. These effects are not adequately addressed in defining risk assessment parameters.
- ItemAntibody production against Staphylococcus aureus CoA biosynthesis enzymes and their application in protein level quantification(Stellenbosch : Stellenbosch University, 2021-04) Bothma, Karli; De Villiers, Marianne; Bellstedt, D. U.; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.ENGLISH ABSTRACT: Antimicrobial resistance has become an increased burden worldwide as more and more human pathogens are becoming resistant to current antimicrobials. Therefore, the identification of novel drug targets and development of new antimicrobial drugs are currently of high priority. A drug target that has gained increased attention is the coenzyme A (CoA) biosynthesis pathway. CoA is an essential cofactor that is necessary for life in all organisms, including human pathogens, making it an attractive target for the development of new antimicrobial drugs. The CoA biosynthesis pathway of Staphylococcus aureus, which is the leading cause of hospital-associated infections, was the focus of this study. Although various studies have investigated this pathway in S. aureus as a possible drug target, there is still a lot that needs to be elucidated in this regard. One gap in our knowledge is that the levels of the CoA biosynthesis enzymes (PanK, CoaBC, PPAT and DPCK) under physiological conditions are currently unknown. This study therefore aimed to develop immunological techniques which could be implemented as tools to quantify the levels of these enzymes at different growth phases of S. aureus cultivated under physiological growth conditions. To achieve this aim, the four CoA biosynthesis enzymes of S. aureus were recombinantly expressed and purified using established methods. Polyclonal antibodies were raised in rabbits by immunising the animals with the respective enzymes adsorbed to acid-treated, naked Salmonella minnesota R595. This method has been used to successfully produce antibodies to a wide variety of antigens, especially in cases where only small amounts of the antigen were available. With these antibodies, indirect competition enzyme-linked immunosorbent assays (ELISAs) with excellent standard curves were obtained for the quantification of each of the respective enzymes. Furthermore, cross-reactivity studies performed with ELISA and western blot revealed that the anti-SaPanK, anti-SaPPAT and anti-SaDPCK antibodies showed limited cross-reactivity. In an attempt to quantify the amount of cross-reactivity of each antibody-antigen pair, however, it was found that only the cross-reactivity of the anti-SaPPAT antibodies had an effect on the final optimised assay. In this study an original, highly sensitive ELISA method for the detection and quantification of each of the enzymes of the CoA biosynthesis pathway of S. aureus was developed. These assays provide a cost-effective method for enzyme level quantification that have the potential to provide better insight on the levels of the different enzymes under physiological conditions and ultimately aid in the development of new antimicrobial drugs.
- ItemAntifungal activity of antimicrobial polymers created with peptides from the tyrothricin complex(Stellenbosch : Stellenbosch University, 2024-03) Mitha, Priyata; Rautenbach, Marina; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.ENGLISH ABSTRACT: The global burden posed by the lack of effective antifungal agents in addressing conditions like vulvovaginal candidiasis (VVC), diaper dermatitis, and nosocomial infections, caused by Candida species, has prompted an urgent need for innovative interventions. The increasing resistance to current antifungal agents further highlights the urgency of this issue. To address these challenges, the exploration of novel solutions or modifications to existing applications is paramount. In this context, antifungal peptides (AFPs) have emerged as promising candidates. This study specifically focuses on investigating anti-Candida activity, with a particular emphasis on the tyrocidines (Trcs), a natural cyclodecapeptide antibiotic complex. These peptides are considered good potential candidates for further development due to their broad-spectrum activity against bacteria, fungi, viruses, and parasites, as well as their inherent ability to adhere to various materials. Recent studies have indicated that the peptide adopts two major self-assembly modes depending on its surrounding environment, and the potent antibacterial activity of the peptide is relatively non- selective in terms of the type of material being functionalised. This study aimed to explore the effect of different solvent systems and Trc peptide complexes on anti-Candida activity of Trc-functionalised materials, as well as the biophysical tracking of peptide self-assembly and material binding. The investigated materials include polystyrene plastic and cellulose filter paper (used as controls), viscose and polypropylene sheets/wipes. The Trc mixture, Phe-rich and Trp-rich peptide complexes were formulated in four solvents: acetonitrile (ACN), methanol (MeOH), ethanol (EtOH) and isopropanol (IPA). Optimisation studies by assessing the target cell metabolism over time revealed that EtOH promotes good material adherence and effective antifungal activity for the Trc mixture and Phe-rich complex, while MeOH and IPA have similar effects on the Phe-rich and Trp-rich peptide complexes. The robustness and stability of the functionalised materials were assessed through a series of washing steps. These findings highlighted the significance of amino acid composition in the optimal peptide deposition onto the materials. Testing an industry-derived method for material treatment led to the conclusion that the average polarity of the peptide complex, solvent system, and the material each play a role. The Trc mixture and Phe-rich peptides associated more readily with the hydrophobic polystyrene, while the more polar Trp-rich peptide complex associated readily to the more polar cellulose and viscose. Furthermore, the viscose material allows for even distribution of the peptide, resulting in potent antifungal activity. Furthermore, the link between surface-derived antifungal activity and biophysical properties of the peptides in the different ethanolic solutions during deposition on materials was assessed by observing changes in Trp and Tyr fluorescence. This provided insight into the impact of peptide conformation in solution and its binding to a specific material on antifungal activity. This study indicated that peptide oligomers, driven by hydrophobic interactions and aromatic stacking, lead to the assembly of metastable oligomers that are crucial for material association and antifungal activity. In aqueous solvents, water propelled the formation of hydrophobic interaction-driven oligomers, represented by fluorescence quenching/loss. In these types of peptide structures, the polar amino acids decorate the outside of the oligomers with the Orn⁹/Lys⁹ residues, previously found to be essential for activity, interacting with the electronegative target cell wall. Active peptide moieties, such as amphipathic dimers, are then released from the peptide layers on the material to elicit antifungal activity. After the washing steps, only peptides strongly associated to the material were retained, and if in the correct conformation, could elicit the antifungal response. Organic solvents at higher concentrations resulted in a decreased hydrophobic effect on the peptides, represented by fluorescence dequenching/gain and poor antifungal activity. Here, peptides may either be in the incorrect conformation, stacked too tightly, or deposited as inactive stable oligomers. The potential of the tyrocidines in material functionalisation and the results in this study pave way towards the development of effective antifungal materials. With little to no anticipation of resistance emergence, and by prevention of chronic fungal infections, this study shows the potential of the Trc-functionalised materials for female and baby hygiene products, as well as surface sterilisation.
- ItemApplications of generalised supply-demand analysis(Stellenbosch : Stellenbosch University, 2013-03) Christensen, Carl David; Rohwer, J. M.; Hofmeyr, J-H. S.; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.ENGLISH ABSTRACT: Supply-demand analysis (SDA) is a tool that allows for the control, regulation and behaviour of metabolic pathways to be understood. In this framework, reactions are grouped into reaction blocks that represent the supply and demand of a metabolic product. The elasticities of these supply and demand blocks can be used to determine the degree of control either block has over the flux in the pathway and the degree of homoeostasis of the metabolic product that links the blocks. Rate characteristic plots, on which the rates of supply and demand blocks are plotted as functions of the concentration of the linking metabolite, represent a powerful visual tool in this framework. Generalised supply-demand analysis (GSDA) allows for the analysis of metabolic models of arbitrary size and complexity without prior knowledge of the regulatory structure of the pathway. This is achieved by performing SDA on each variable metabolite in a pathway instead of choosing a single linking metabolite. GSDA also provides other benefits over SDA as it allows for potential sites of regulation and regulatory metabolites to be identified. Additionally it allows for the identification and quantification of the relative contribution of di erent routes of regulation from an intermediate to a reaction block. Moiety-conserved cycles present a challenge in performing in silico SDA or GSDA, as the total concentration of a moiety must remain constant, thereby limiting the range of possible concentrations of the metabolites between which it cycles. The first goal of this thesis was to develop methods to perform GSDA on two-membered and interlinked moiety-conserved cycles. We showed that by expressing the members of a moiety-conserved cycle as a ratio, rather than individual metabolite concentrations, we can freely vary the ratio without breaking moiety conservation in a GSDA. Furthermore, we showed that by linking the concentrations of the members of two interlinked two-membered moiety-conserved cycles to a “linking metabolite”, we could vary the concentration of this metabolite, within constraints, without breaking moiety conservation. The Python Simulator for Cellular Systems (PySCeS) is a software package developed within our group that provides a variety of tools for the analysis of cellular systems. The RateChar module for PySCeS was previously developed as a tool to perform GSDA on kinetic models of metabolic pathways by automatically generating rate characteristic plots for each variable metabolite in a pathway. The plots generated by RateChar, however, were at times unclear when the models analysed were too complex. Additionally, invalid results where steady-states could not be reached were not filtered out, and therefore appeared together with valid results on the rate characteristic plots generated by RateChar. We therefore set out to improve upon RateChar by building plotting interface that produces clear and error-free rate characteristics. The resulting RCFigure class allows users to interactively change the composition of a rate characteristic plot and it includes automatic error checking. It also provides clearer rate characteristics with e ective use of colour. Using these tools two case studies were undertaken. In the first, GSDA was used to investigate the regulation of aspartate-derived amino acid synthesis in Arabidopsis thaliana. A central result was that the direct interaction of aspartate-semialdehyde (ASA), a metabolite at a branch point in the pathway, with the enzyme that produces it only accounts for 7% of the total response in the flux of supply. Instead, 89% of the observed flux response was due to ASA interacting with of the downstream enzymes for which it is a substrate. This result was unexpected as the ASA producing enzyme had a high elasticity towards ASA. In a second case study moiety-conserved cycles in a model of the pyruvate branches in lactic acid bacteria were linearised using the above mentioned method. This served to illustrate how multiple reaction blocks are connected by these conserved moieties. By performing GSDA on this model, we demonstrated that the interactions of these conserved moieties with the various reaction blocks in the pathway, led to non-monotonic behaviour of the rate characteristics of the supply and demand for the moiety ratios. An example of this is that flux would increase in response to an increase in product for certain ranges. This thesis illustrates the power of GSDA as an entry point in studying metabolic pathways, as it can potentially reveal properties of the regulation and behaviour of metabolic pathways that were not previously known, even if these pathways were subjected to previous analysis and a kinetic model is available. In general it also demonstrates how e ective analysis tools and metabolic models are vital for the study of metabolism.
- ItemAn assessment of the diversity and pathogenicity of Potato leafroll virus in South Africa(Stellenbosch : Stellenbosch University, 2018-03) Roos, Wiets Gideon; Bellstedt, D. U.; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.ENGLISH ABSTRACT: Potato production in South Africa has steadily intensified through improved pivot irrigation. Since potatoes are vegetatively propagated the potato industry is continuously threatened by Potato leafroll virus (PLRV) which is responsible for increasing yield losses in South Africa. Effective management of PLRV is dependent on its accurate detection in seed potato stocks. PLRV is a small spherical plant virus consisting of a protein capsid and an RNA genome of approximately 5900 bases. This virus is phloem restricted and is vectored by, most notably, by the green peach aphid. Plant RNA viruses pose a threat due to their high mutation rate and the ability to adapt rapidly to a changing environment. To effectively manage PLRV infection, detection of virus infection in seed potatoes is paramount. In this study, five field trials were carried out in potato production fields, to compare the commonly used DAS-ELISA with RT-PCR for PLRV detection. From the results obtained it was concluded that DAS-ELISA detection greatly underestimates the number of infected samples when compared to RT-PCR. The hotter climate of the Sandveld region appears to inhibit PLRV accumulation in infected plants and these infections then remain undetected by DAS-ELISA. PLRV isolates were sequenced and phylogenetic and bioinformatic analyses were performed to identify and characterise local variants of PLRV. PLRV isolates found in the Sandveld region were closely related to PLRV isolates from Australia. Some of these isolates had recombined with variants commonly found in Europe, Asia and the Americas as well as with those similar to PLRV isolates from Peru and Germany. Three locally produced cultivars were graft-inoculated with two PLRV isolates that represent the two main variant groups found to assess symptom development and yield reduction. Symptom development in locally produced cultivars was typical for PLRV. A yield loss resulted from this infection with no difference between the Australian type and the European/Australian recombinant type. The proteins produced by the newly sequenced isolates were further analysed in comparison to other isolates found worldwide. The variation in the proteins produced by the newly sequenced isolates was mainly due to recombination between distinct groups of PLRV in the 5’ half of the genome and through mutation in the 3’region of the genome. A differential RT-PCR was designed to distinguish, in a single reaction, between the Australian type and the European/Australian recombinant type of PLRV. This revealed that simultaneous infections with both types occurred commonly, and could explain why recombination has occurred.
- ItemAn assessment of the mutation patterns in South African isolates of Potato leafroll virus and the expression of recombinant viral coat protein genes in Escherichia coli(Stellenbosch : Stellenbosch University, 2007-03) Rothmann, Adri Hilda; Bellstedt, D. U.; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.ENGLISH ABSTRACT: Presently, the observed variation in symptoms of Potato leafroll virus (PLRV) infection in potato cultivars in South Africa cannot be reconciled with PLRV symptoms obtained 10-15 years ago, even if the different interactions between the pathogen and the cultivar are taken into account. In an effort to analyze this variation, mutations in the coat protein (CP) gene of South African isolates of PLRV were assessed. The CP gene of PLRV isolates from different areas within South Africa was amplified by reverse transcription-polymerase chain reaction (RT-PCR), cloned and sequenced. Significant sequence variation in the CP gene was found within the analyzed South African isolates of PLRV. Phylogenetic analysis revealed two major clades with most South African isolates and an Australian and North American isolate grouped together and the remainder grouped with isolates from diverse countries worldwide. The deduced amino acid sequences from representatives of these two clades indicated differences in CP threedimensional structure. In an effort to produce recombinant PLRV CP for the production of antibodies specific for South African isolates of PLRV for use in enzyme-linked immunosorbent assay (ELISA), the CP gene of a South African isolate of PLRV was subcloned into a bacterial expression vector (pET14-b). Expression of full length recombinant PLRV CP was attempted in Escherichia coli strains BL21(DE3)pLysS, Rosetta-gami B(DE3)pLysS and Rosetta-2(DE3)pLysS. As this was not successful, the PLRV CP gene was subcloned in another expression vector (pGEX) for expression as an N-terminal fusion protein with glutathione-S-transferase (GST) in E. coli strains BL21(DE3)pLysS and Rosetta-2(DE3)pLysS. The recombinant GST-PLRV CP fusion protein was purified and used for antibody production in rabbits. Using western blots, the effectiveness of antibodies produced to recombinant GST-PLRV CP fusion protein was assessed for PLRV recognition. It was found that antibodies to the recombinant GST-PLRV CP fusion protein were more effective for the detection of GST than PLRV CP and that production of antibodies to the cleaved PLRV CP product would be necessary if antibodies are required for ELISA applications.
- ItemAtomic force microscopy : a novel tool for the analysis of the mechanism of action of antimicrobial peptides on target membranes(Stellenbosch : Stellenbosch University, 2003-03) Holroyd, Dale; Rautenbach, Marina; Stellenbosch University. Faculty of Science. Dept. of Biochemistry .ENGLISH ABSTRACT: Nanoscale visualisation of live cells and cellular components under physiological conditions has long been a goal in microscopy. The objective of this study was to validate the use of Atomic Force Microscopy (AFM) as a new tool in unravelling the mysteries of antimicrobial peptide mechanism of action. Using the simplest AFM imaging technique, we were able to analyse the influence of haemolytic melittin and anti-bacterial magainin 2 on different target membranes at nanometer resolution, without using fixing agents. First, magainin 2 was synthesised and purified by gel permeation chromatography and high performance liquid chromatography (HPLC). The purity of magainin 2 and melittin, isolated from bee venom (Sigma-Aldrich), was verified with electro spray ionisation mass spectrometry (ESI-MS). Second, dose-response experiments were used to determine the optimum peptide/target cell ratio that would allow interaction with the membrane without causing lysis. Third, peptide/target-cell samples were placed on silica plates and visualised using contact mode AFM. Images obtained of the cells before and after peptide treatment, showed distinct changes in cell membrane surface topology. We observed grooves, lesions, membrane collapse and vesiculation depending on the concentration, type of peptide and target-cell used, allowing us to make conclusions regarding the mechanism of action of melittin and magainin 2. In comparison with model membrane studies, our AFM results show that a peptide can function by more than one mechanism of action depending on the structural composition of the membrane, which appears to have specific segregated lateral organisation. Magainin 2 (non-toxic) selectively targets cell membranes using different mechanisms of action. In this way it can lyse bacterial membranes (anti-bacterial agent) using one mechanism, while using another mechanism to interact with mammalian cells at physiological concentrations, without destroying them. In contrast, melittin (toxic) is non-selective, and uses the same mechanism of interaction with bacterial and mammalian cells. In conclusion, we propose a new holistic model for the mechanism of action of antimicrobial peptides.
- ItemBioaffinity separation using ligand-modified pluronic and synthetic membranes(Stellenbosch : University of Stellenbosch, 2011-10) Govender, Selvakumaran; Swart, P.; Jacobs, E. P.; Bredenkamp, M. W.; University of Stellenbosch. Faculty of Science. Dept. of Biochemistry.ENGLISH ABSTRACT: A new membrane based affinity separation system that is bio-specific, biocompatible, well characterised and capable of being regenerated or re-used is described. The amphiphilic non-ionic surfactant Pluronic® F108, was covalently derivatised to form two novel bioligands (Pluronic-Biotin and Pluronic-DMDDO) for the bio-specific immobilisation of avidin conjugated proteins and histidine tagged proteins respectively. Pluronic was also used to non-covalently functionalise nonporous membranes for ligand attachment and to simultaneously shield the surfaces from non-specific protein adsorption. Each component of this bioaffinity system (from the membrane matrix to the elution/desorption of the ligate/ligand system) was studied with the aim of producing a well characterised system and key quantitative data for the development of a robust, reliable, re-usable and scalable technology. Specifically, this study describes: 1. The fabrication and partial characterisation of nonporous planar and capillary membranes as model affinity matrices. 2. The development and evaluation of a robust protocol for solvent desorption and accurate colorimetric quantification of Pluronic® F108 and its derivatives. 3. Interfacial analysis of Pluronic adsorption onto nonporous affinity membranes, including the direct solid-state analysis of model, halogenated Pluronic derivatives using nuclear microprobe analysis. 4. Development of a surfactant based protocol for affinity membrane regeneration and re-use. 5. Specific bioaffinity immobilisation of avidin conjugated peroxidase onto biotinylated membranes in the presence of model protein foulants. 6. Cloning and expression of C-terminal hex-histidine tagged human cytochrome b5 into the bacterial expression system E. coli BL-21 DE3. 7. Development and characterisation of an immobilised metal affinity membrane system for metal chelation (Ni2+, Cu2+ and Zn2+) using a new chelator Pluronic- N,N-dicarboxymethyl-3,6-diazaoctanedioate and the bio-specific immobilisation of N-terminal hex-histidine tagged pantothenate kinase.
- ItemBiocatalytic preparation and characterization of alternative substrate of MshB, a mycothiol pathway enzyme(Stellenbosch : Stellenbosch University, 2012-12) Muneri, Ndivhuwo Olga; Strauss, Erick; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.ENGLISH ABSTRACT: Mycobacterium tuberculosis (M. tuberculosis), the causative agent of tuberculosis, utilizes mycothiol (MSH) as the major low molecular weight thiol to protect itself against oxidative stress and thereby to ensure its growth and survival. MSH is a pseudo-disaccharide molecule that contains an α(1→1) glycosidic bond, and is biosynthesised in five enzymatic steps involving the enzymes MshA, MshA2, MshB, MshC and MshD. Owing to the essentiality of MSH to M. tuberculosis, various studies have focused on the MSH biosynthetic and other MSH-dependent enzymes viewed as potential drug targets for the development of antituberculosis agents. In the course of this study two practical challenges affecting the development of inhibitors of one the MSH biosynthesis pathway enzyme, MshB, were addressed. These challenges entail the lack of a high-throughput continuous assay to determine MshB activity, and the poor availability of the natural and alternative MshB substrates. In this study an alternate MshB substrate was characterized and shown to undergo a rearrangement reaction upon deactylation, which allowed the development of a new continuous assay for MshB activity that uses DNTB (Ellman’s reagent). In addition, three new α-thioglycoligases were created from the α-Nacetylglucosaminidase of Clostridium perfringens. These enzymes showed potential as biocatalysts that can be used for the enzymatic synthesis of thioglycoside-based alternative substrates of MshB.
- ItemBiochemical and genetic characterization of bacteria isolated from diseased rainbow trout (Oncorhynchus mykiss) farmed in Lesotho and Mpumalanga province of South Africa(Stellenbosch : Stellenbosch University, 2017-12) Kutu, Vuyokazi; Bellstedt, D. U.; Macey, B. M.; Mouton, A.; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.ENGLISH ABSTRACT: Rainbow trout farms in Mpumalanga Province, South Africa and Lesotho, have periodically suffered significant losses from infections caused by Gram-positive bacteria. Such outbreaks have hampered the development of this industry in both South Africa and Lesotho. A total of 55 bacterial strains had been isolated between 2006-2012 from infected trout farmed in Lesotho and Mpumalanga Province and had been stored for long term by freeze drying. Some isolate identification had been performed and a few were used for vaccine development. Vaccines were however only effective for one or two seasons, highlighting the need to properly characterize these Gram-positive bacteria. The aims of the study were therefore to: (i) investigate the genetic diversity of these bacterial isolates by their phenotype; antimicrobial susceptibility and 16S ribosomal RNA (rRNA) sequencing and phylogenetic analysis, (ii) investigate the different antigenic epitopes that exist within this group of bacterial isolates by development of an enzyme-linked immunosorbent assay (ELISA) utilizing six rabbit produced polyclonal antibodies, produced against six selected bacterial isolates from the 55 isolates investigated in this study. Phenotypic analysis showed that fifty of the isolates were Gram-positive cocci and five were Gram-positive rods. Their growth characterists and antimicrobial susceptibility were extensively characterized. The 16S rRNA analysis indicated the following isolate composition: 49 Lactococcus garvieae, one Lactococcus lactis, three Carnobacterium maltaromaticum and two Weissella species, which is the first report of Weissella from diseased trout from South Africa. Antigenicity analysis showed that there were highly specific epitopes that were limited to very few isolates, but also common epitopes that were shared between isolates of the same genus, but even some epitopes that were shared between different bacterial genera. The patterns of epitope sharing broadly correlated with the 16S rRNA phylogeny, but not entirely which was not unexpected as phylogeny does not indicate the presence or absence of bacterial epitopes. These results address the importance and accuracy of molecular identification of disease causing species and the need to investigate the antigenic differences expressed by these pathogenic bacteria to assist in generating correct information needed for the development of vaccines of high efficacy.
- ItemA biochemical and immunochemical study of ovine adrenal cytochrome P-450 reductase(Stellenbosch : Stellenbosch University, 1992-03) Petersen, Carmen J.; Swart, P.; Bellstedt, D. U.; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.ENGLISH ABSTRACT: This study describes: (a) the isolation · of ovine adrenal cytochrome P-450 reductase, (b) the preparation of , antibodies against this enzyme and (c) comparitive immunochemical studies with ovine' liver and .bovine. adrenal cytochrome P-450 'reductases
- ItemA biochemical and immunological study of horseradish peroxidase(Stellenbosch : University of Stellenbosch, 2009-12) Odendaal, Ruenda; Swart, P.; Lombard, N.; University of Stellenbosch. Faculty of Science. Dept. of Biochemistry.ENGLISH ABSTRACT: This study describes: a) the isolation and purification of horseradish peroxidase isoenzymes from horseradish roots, b) the characterization of various forms and components of the enzyme by cation-exchange and reversed-phase high performance liquid chromatography, c) the preparation of antibodies against horseradish peroxidase isoenzymes, d) immunological studies for the development of an isoenzyme quantification method and e) the formation of an enzyme-melamine conjugate for use in a melamine quantification immunoassay.
- ItemA biochemical investigation into the compounds involved in pigmentation of apple and pear skin and the manipulation thereof(Stellenbosch : Stellenbosch University, 1998) Arends, Arrie Paul; Bellstedt, D. U.; Swart, P.; Stellenbosch University. Faculty of Science. Department of Biochemistry.ENGLISH ABSTRACT: This study describes (a) Identification of the main anthocyanin pigment in the skin of the 'Fuji' apple cultivar and in the pear cultivars 'Bon Rouge' 'Forelle', Red d'Anjou', 'Rosemarie' and 'Flamingo', (b) an investigation of the effect of on-tree bagging on anthocyanin pigment accumulation in the skin of the 'Fuji' apple cultivar by means of high performance liquid chromatography (HPLC) technology. (c) an investigation of the influence of cold storage and ripening on anthocyanin concentration and accumulation in the skin of the pear cultivars, 'Forelle', 'Rosemarie', 'Flamingo', 'Bon Rouge' and Red d'Anjou' pear cultivars by means of HPLC technology and (d) an investigation of the level of induction of dihydroflavonol 4-reductase (DFR) gene in 'Fuji' apple cultivar during bagging trial, through mRNA studies.
- ItemA biochemical study of budbreak and plant growth regulators in table grapes(Stellenbosch : Stellenbosch University, 2002-03) Lombard, Petrus Johannes; Bellstedt, D. U.; Cook, N. C.; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.ENGLISH ABSTRACT: The cultivation of table grapes in the warmer areas of South Africa, indeed worldwide, is complicated by rest breaking problems in spring due to delayed budbreak. In order to overcome these problems rest breaking agents, mainly hydrogen cyanamide, are applied. However, instead of alleviating the problem, additional problems such as uneven budbreak and reduced production are often induced. This study was initiated to further understand the physiological processes occurring during budbreak and how the application of hydrogen cyanamide influences these processes. The following aspects were investigated in this study: a. The effect of hydrogen cyanamide on tissue cytokinin (specifically zeatin riboside) levels of Sultanina table grape vines after application at different times before natural budbreak was studied over two seasons. In 1997, hydrogen cyanamide was applied at three weeks before induced budbreak and in 1998 at six weeks before induced budbreak. One year-old canes were sampled weekly after hydrogen cyanamide application, divided into distal and proximal sections, then further divided into buds, bark and wood tissues and the zeatin riboside (ZR) levels determined. A relatively high amount of chilling coupled to late hydrogen cyanamide application in 1997 led to a large effect on ZR release, but did not lead to significant shifting of the budbreak pattern. Zeatin riboside peaks were observed in buds, internode wood and bark of treated vines compared to control vines. The peaks were higher in distal portions compared to proximal portions in all tissues. The relatively lower chilling and earlier application of hydrogen cyanamide in 1998 had a larger effect on the budbreak pattern while the bud ZR peak was shifted earlier. The distal portion bud ZR . peak was again higher than the proximal portion bud ZR peak. In 1997, as sampling was not initiated early enough, bud ZR peaks were only observed after budbreak, while in 1998 bud ZR peaks were observed before and after budbreak. The effect of these ZR increases on the development of inflorescence primordia, subsequent bunch development and ultimately production, are discussed. b. Free xylem sap was sampled at cane and spur pruned lengths from unpruned canes of Sultanina from budswell until after budbreak in 1999 and from three table grape cultivars, i.e Sultanina, Alphonse Lavalleé and Sunred Seedless, in 2001 and ZR levels determined. The ZR levels in the buds of these three table grape cultivars, pruned to different cane lengths were also determined. One year old canes of these cultivars, were each pruned to long canes (14 buds) and short spurs (2 buds). The ZR content in buds of these canes at distal and proximal positions were determined weekly from budswell until after budbreak in 1999. Xylary ZR peaks occurred before 50% budbreak. Spur xylary ZR levels of all three cultivars followed a similar pattern, although at lower ZR levels than that of the canes. This is similar to previous studies on xylary ZR levels of apple shoots. The high levels of free ZR found in xylem sap at the distal portions of canes support the hypothesis of a cumulative ZR build-up effect as cane length increases. Spur pruning resulted in earlier budbreak and a higher final budbreak than cane pruning. The proximal portions of shoots, whether spur pruned or the proximal portions of canes, showed elevated ZR levels in all cultivars. This difference in ZR levels in bud tissue of different portions of the cane would suggest a difference in ZR consumption or turnover. The results of this study have important management implications for the cultivation of vines in warmer areas in which hydrogen cyanamide is used to alleviate budbreak problems.
- ItemA biochemical study of the effect of ultraviolet treatment on bovine milk and Cheddar cheese(Stellenbosch : Stellenbosch University, 2015-12) Cilliers, Frans Pieter; Swart, Pieter; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.ENGLISH ABSTRACT: This study describes: 1. The evaluation of a novel, patented thin-film, turbulent-flow Ultravioletdisinfection system as an alternative processing method to thermal pasteurisationfor the disinfection of bovine milk. 2. The microbial, biochemical and sensory characterization of bovine milk treated by heat and Ultraviolet light and then used for the commercial production of Cheddarcheese. 3. The microbial, biochemical and sensory characterization of commercial Cheddarcheese produced from bovine milk treated by heat and Ultraviolet light.
- ItemThe biochemical study of the R- and S-enantiomers of 2-(4-acetoxyphenyl)-2-chloro-Nmethylethylammonium chloride (Compound A).(Stellenbosch : Stellenbosch University, 2017-03) Swart, Liezel Maria; Swart, Pieter; Swart, Amanda C.; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.ENGLISH ABSTRACT: The study describes: . The synthesis of a racemic mixture of Compound A (2-(4-acetoxyphenyl)-2-chloro-N-methylethylammonium chloride), a synthetic analogue of the active compound isolated from the African shrub, Salsola tuberculataformis Botschantzev. . The development of a strategy to separate the R- and S-enantiomers of Compound A. . The isolation and purification of a highly purified substrate-free cytochrome P450 11β-hydroxylase (CYP11B1) from ovine adrenals. . The isolation of a partially purified mixture of the mitochondrial electron transport chain, adrenodoxin reductase and adrenodoxin from ovine adrenals. . The investigation into the mechanism of action of the R- and S-enantiomers of Compound A on the spectroscopic properties of substrate-free CYP11B1. . The investigation into the influence of the R- and S-enantiomers of Compound A on the mitochondrial electron transport chain by using cytochrome c as an electron acceptor.
- ItemA biochemical study of tissue type plasminogen activator in bovine milk(Stellenbosch : Stellenbosch University, 2007-03) Cilliers, Frans Pieter; Swart, Pieter; Hofmeyr, Jannie; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.ENGLISH ABSTRACT: This study describes: 1. The isolation and the purification of tissue type plasminogen activator and urokinase plasminogen activator in bovine milk. 2. The biochemical characterisation of tissue type plasminogen activator in bovine milk. 3. An investigation of the influence of the addition of purified tissue type plasminogen activator to ultra high temperature milk, Gouda cheese and yoghurt.
- ItemBiofilms as multifunctional surface coatings and adaptive systems: a biomimetic approach(Stellenbosch : Stellenbosch University, 2016-12) Loots, Ruenda; Cloete, Thomas Eugene; Swart, Pieter; Wolfaardt, Gideon M.; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.ENGLISH ABSTRACT: Biomimicry is an emerging scientific discipline that promotes nature-inspired innovation for sustainable solutions. Several patterns and survival strategies are repeated in Nature and these have been extrapolated into a hierarchical set of biomimetic principles that can be used to investigate the complexity of natural systems. A biomimetic approach was used to review biofilm literature and create a novel framework based on these principles to describe microbial biofilms on a molecular, structural and systems level. By reinterpreting current biofilm knowledge within a biomimetic framework, this study demonstrates that microorganisms use life-friendly chemistry to integrate biofilm development with growth, giving rise to resource-efficient systems. Furthermore, these structured microbial communities are responsive to their local environment, adapt to changes and, ultimately, evolve to survive. Subsequently, the application of biomimetic principles to biofilms was investigated using various analytical techniques. Two gfp-labelled Pseudomonas strains and an environmental multi-species community were selected for this study. Microscopic and spectroscopic techniques were used for biochemical investigations of single-species biofilm composition and structure. The distribution of biomolecules in Pseudomonas biofilms was investigated using protein- and glycoconjugate-specific fluorescent stains and confocal laser scanning microscopy (CLSM). CLSM was also used to investigate structural adaptations of Pseudomonas biofilms to changes in nutrient availability and hydrodynamic conditions. Attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy was used to explore biochemical adaptations of single- and multi-species biofilms cultivated in different nutrient media. ATR-FTIR spectra, visual observations and the quantification of biofilm parameters by digital image analysis of CLSM images support the hypothesis that biofilms are resource-efficient, self-organised systems that are built from the bottom up using life-friendly chemical principles. Both Pseudomonas strains adapted to environmental conditions by changing the three-dimensional structure of their biofilms, specifically in terms of biomass, substratum area coverage, average thickness and the surface area of biofilms exposed to the bulk liquid. In order to study biofilms as a system and investigate the responsiveness of a biofilm community as a whole, a relatively new approach was used to monitor biofilm responses in real time by measuring CO2 production as an indication of whole-biofilm metabolism. A CO2 evolution measurement system (CEMS) was combined with metabolic assays and direct plate count methods to monitor biofilm metabolism and biofilm-derived planktonic cell yield in response to environmental changes, i.e. changes in nutrient source and concentration or exposure to antimicrobial compounds (either streptomycin or a solution containing isothiazolone). The metabolic responses of biofilms, measured as CO2 production rates, showed that both single- and multi-species biofilms are able to respond rapidly to changes in nutrient availability or exposure to biocides and antibiotics. Multi-species biofilms generally recover faster after environmental changes or antimicrobial exposures, indicating that diversity adds to biofilm resilience and adaptability. Regardless of the conditions, single- and multi-species biofilms are able to maintain some level of metabolic activity, as well as release high numbers of planktonic cells into the effluent. The maintenance of biofilm-derived planktonic cell yield supports the hypothesis that biofilms are active proliferation sites in order to ensure survival – a feature of biofilms that is often overlooked in biofilm research. This study contributes to the growing field of biomimicry by applying biomimetic principles in biofilm research for the first time. A biomimetic approach can inform novel anti-biofilm strategies, promote biofilm-inspired innovation and explain complex microbial ecological phenomena. Within a biomimetic framework, the increasing degrees of complexity in biofilms are organised in a new way, demonstrating that the biochemical, structural and functional complexity of microbial communities are interconnected and need to be considered together in biofilm studies. To this end, the usefulness of CEMS as a non-destructive technique to study real-time biofilm responses is demonstrated.
- ItemBioinformatic characterisation of genes associated with coenzyme A biosynthesis in mycoplasmas and expression and isolation of dephospho-coenzyme A kinase from Mycoplasma sp. Ms02(Stellenbosch : Stellenbosch University, 2018-03) Ras, Tertius Alwyn; Botes, Annelise; Strauss, Erick; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.ENGLISH ABSTRACT: The South African ostrich industry is internationally the leading provider of ostrich products. The increasing popularity of ostrich products has resulted in the adjustment of production strategies, which includes intensifying rearing conditions by using feedlot systems. However, this intensive rearing strategy creates an ideal environment for pathogens, such as mycoplasmas, to spread. There are three Mycoplasma species that infect ostriches, which are associated with respiratory diseases. These mycoplasma infections can result in production losses, which not only have an economic impact on the ostrich industry but also significant socio-economic implications. Hence, there is a need for specific and cost-effective treatment against these ostrich-infecting mycoplasmas. The enzymes involved in the biosynthesis pathway of coenzyme A have long been regarded as potential targets for drug development. These enzymes could, therefore, offer a solution to the control of mycoplasma infections in ostriches. Since the coenzyme A biosynthetic pathway is relatively unexplored in mycoplasmas, the first aim of this study was to determine the presence or absence of enzyme-encoding genes involved in this pathway in Mycoplasma species. This was done using a bioinformatics approach. Of the 62 Mycoplasma species investigated, there were eight species (13%) found to have none of the enzyme-encoding genes, while the remaining species had at least one. Additionally, twelve enzyme-encoding gene homologues were identified and their predicted identities confirmed by evaluating the conserved and functional motifs and domains. The enzyme-encoding gene found to be most common amongst the investigated species was that of dephosphocoenzyme A kinase (DPCK), the final enzyme in the biosynthesis pathway. Furthermore, there was no correlation between the number of identified coenzyme A biosynthetic pathway enzyme-encoding genes in a species and the phylogeny of the respective proteins. There was also no correlation with the 16S rRNA phylogenetic groupings. Given the common presence of the DPCK-encoding gene, the second aim of this study was to recombinantly express the DPCK of the ostrich-infecting Mycoplasma sp. Ms02 (Ms02) and isolate the protein using a His-tag. The Ms02 DPCK-encoding gene was successfully amplified, cloned and mutated by site-directed mutagenesis to allow for expression in a nonmycoplasma host. However, the soluble expression and isolation of the Ms02 DPCK protein proved to be challenging. Using a variation of methods, the protein was eventually solubilised using a sarkosyl treatment method. A pure isolate of the Ms02 DPCK protein could, however, not be attained when using immobilised metal affinity chromatography (IMAC) purification. Subsequent activity testing of the isolated DPCK enzyme, using an HPLC-based method, also showed no activity.