Masters Degrees (Biochemistry)

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    Antifungal 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.
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    Characterisation & purification of class IIa bacteriocins from lactiplantibacillus plantarum
    (Stellenbosch : Stellenbosch University, 2024-03) Beukes, Cheyenne Tamika; Beukes, Mervyn; Rautenbach, Marina; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
    ENGLISH ABSTRACT: The rapid emergence of antibiotic resistance, coupled with the scarcity in the development of new drugs to combat resistance are threatening the food, medical and agricultural industries. This demands the development of novel alternative treatments, such as antimicrobial substances to target specific pathogenic micro-organisms. Lactic acid bacteria (LAB) have attracted attention in this research field, as LAB secrete antimicrobial peptides bacteriocins. Bacteriocins are ribosomally synthesized antimicrobial peptides that display promising potential as natural food preservatives and source of microbial inhibitors. The producer organism chosen for further research was Lactiplantibacillus plantarum (L. plantarum), a member of the LAB known to commonly produce multiple bacteriocins. The goal of this study was to perform the in-silico gene mining in conjunction with the in vitro molecular screening with the aim to isolate, and characterise bacteriocins from the producer organism, L. plantarum. The results obtained from the various assays and characterisation of bacteriocins will contribute to expanding the knowledge in the field. With potential application of such bacteriocins, specifically in the food industry as food preservative or health care sector, as further encouragement in this field. A combinatorial approach was adopted for the in silico gene mining of organisms of interest, alongside the in vitro studies. In silico screening methods included the use of bacteriocin genome mining tool version 4 (BAGEL4), National Centre for Biotechnology Information (NCBI) and Basic Local Alignment Search Tool (BLAST) for rapid identification of putative operons within the genomes of selected producer organisms. Initial in vitro screening of class IIa LAB from our local culture collection was conducted through bioassays. This included the colony-spot, spot-on-lawn, and microtiter plate growth inhibition assays, to monitor inhibitory activity. A producer organism was selected for subsequent isolation and purification studies. Inhibition zones surrounding colony spots were compared for a broad range of indicator organisms, including Listeria monocytogenes (L. monocytogenes), L. plantarum Ta10c, Micrococcus luteus (M. luteus), Staphylococcus aureus (S. aureus), Streptococcus milleri (S. milleri), and Enterococcus faecalis (E. faecalis). The highest sensitivity was observed towards L. monocytogenes. Further, another bioassay was used to confirm initial colony-spot and spot-on-lawn assays results. This included the microtiter plate growth inhibition assay providing inhibition results from continuous monitoring, instead of an end-point result. Production of bacteriocins was optimized with regards to type of media, pH and temperature. The optimal conditions were found to be in Man, Rogosa and Sharpe (MRS) medium (Neogen, USA), pH of 7, and at a temperature of 37°C. Genomic DNA (gDNA) of the producer organism was extracted, amplified through PCR with universal 16S rDNA primers, and the amplicons sequenced. A BLAST search was conducted for homology comparison to the NCBI DNA database, to confirm the producer organism as L. plantarum. Purification of bacteriocins was initiated with the use of Amberlite XAD-16N, for separation based on hydrophobic interactions; followed by cation exchange chromatography, for separation based on ionic charge; and, reversed-phase high-performance liquid chromatography (RP-HPLC), for separation based on hydrophobicity. Mass Spectrometry analysis at each purification step revealed three prominent peaks at 789, 1124.6282 and 2185 Daltons (Da). These display potential masses of bacteriocins as they fall within the range of molecular masses linked to inhibitory activity observed.
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    Heterologous expression and partial characterisation of enzymes predicted in silico by deep feed-forward neural networks
    (Stellenbosch : Stellenbosch University, 2024-03) Foster, Bryce John; Patterton, Hugh-George; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
    ENGLISH ABSTRACT: Inverse protein folding (IPF) involves the prediction of amino acid sequences that will fold into a specified three-dimensional (3D) structure. The implementation of advanced algorithms has expedited progress in structural bioinformatics, with increasing application of machine learning (ML) approaches. This has directly contributed to the unparalleled success of modern protein structure prediction methods that are capable of predicting the 3D atomic coordinates of complex structures from only their protein sequences with near-experimental accuracy. Comparable progress has been seen for reverse folding predictors, which are now largely reliant on ever-evolving neural network architectures. IPF tools that are guided by the physical principles of protein folding offer insight and potential for rational protein design and enzyme engineering that have long been unattainable. However, the outputs of many of these tools have only been assessed in silico by means of ML folding algorithms—consequently, a gap exists between their potential and realised application. SeqPredNN is an in-house feed-forward IPF neural network trained using features extracted from the subset of Protein Data Bank entries that have less than 90% identity. Similarly, ProteinMPNN is a deep learning-based protein sequence design method. Unlike ProteinMPNN, SeqPredNN is yet to be applied in vitro, and therefore lacks experimental validation. This study used both SeqPredNN and ProteinMPNN to predict novel protein sequences for Bacillus subtilis lipase A (LipA) and Streptomyces griseus trypsin (SGT). The SeqPredNN sequence recovery rates were <40%, while the ProteinMPNN predictions were >60% identical to the native sequences. Following the re-introduction of all residues deemed necessary for catalysis, the curated sequences were folded using AlphaFold2. The resulting conformations for both IPF tools were remarkably similar to the native X-ray crystal structures. Further molecular dynamics simulations and ligand docking showed that, despite vastly different amino acid sequences, the IPF enzymes were expected to possess physicochemical properties largely comparable to those of the native counterparts. To validate this experimentally, the novel proteins were produced in recombinant Escherichia coli and Pichia pastoris strains. Noticeably lower levels of heterologous protein expression were observed for the IPF variants, particularly for SeqPredNN LipA, compared to the native proteins. Furthermore, catalytic activity was significantly reduced or completely lost in the predicted enzymes. This is likely due to modifications of the electrostatic surface potential and active site topology that no longer facilitate correct substrate interactions, which are deemed to be repercussions of the highly unique protein sequences. Preliminary characterisation via circular dichroism yielded empirical secondary structure compositions that differed from the expected values, adding to the lack of congruence between the computational and experimental results. Importantly, this study provides the first experimental implementation of SeqPredNN, and emphasises critical considerations for difficult protein design targets. However, the ultimate utility of a reverse folding tool lies in its ability to predict protein sequences that will achieve structures capable of performing intended functions. Accordingly, while these tools offer an unprecedented starting point for rational protein modification, a more holistic approach with continued experimental validation may be required for greater success.
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    Investigation into the assimilation of free amino nitrogen from bread waste during yeast proliferation
    (Stellenbosch : Stellenbosch University, 2023-12) Botha, Dewald Du Plessis; Stefan, Hayward; Strauss, Eric; Tait, Timo; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
    ENGLISH ABSTRACT: Bread is a staple food in many economies. It is nutrient rich, cheap, and modern bakeries provide it in excess to be able to meet supply within stringent quality control parameters. Overproduction and consumer underutilisation, however, often result in accumulated bread going to waste if suitable valorisation methods are not available. Yet, unused bread is often returned to production facilities unaltered and may easily be processed to a dried commodity with a stable shelf life. Dried bread crumb is especially applicable to the agricultural industry as a feed additive, but it is also attractive in the biotechnology sector to sustain microbial growth for industrial and pharmaceutical applications. Ethanol is an attractive end-product due to its applicability in various industries, whether related to food and beverages, chemicals, or pharmaceutical sectors. This study therefore aims to establish a process to liberate sugars and free amino compounds from bread waste, relevant to alcoholic fermentation in yeast. Dried bread waste was processed enzymatically to hydrolyse polysaccharides to fermentable sugars and, following fermentation and distillation, alcohol yields of 10% alcohol by volume (ABV) was achieved. Moreover, high performance liquid chromatography coupled to UV/Vis detection was used to establish an analytical method for the quantification amino acids. Analysis of these nitrogen containing compounds revealed that a substantial amount of protein and essential amino acids are left following distillation to support a secondary fermentation. After proteolytic digestion of the dried wash, a nutrient rich mixture was obtained that promoted and supported the growth of yeast in the presence of a suitable carbohydrate source such as sucrose. This nutrient source was shown to be comparable to conventional commercial alternatives. Although the crude yeast nutrient resulted in slower exponential phase it ultimately proved to be superior in sustaining greater yeast biomass. This thesis describes the successful generation of a yeast nutrient from a bread waste product which was obtained after valorisation of bread waste in a bioethanol production process, thus creating a solution to bread waste that may with further development be waste free.
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    Resisting the molecular evolution of fitness: an investigation into the role of ploidy in the perturbed adaptability observed in an HTZ1 knockout
    (Stellenbosch : Stellenbosch University, 2023-12) Reid, Jessica Laura; Patterton, Hugh; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
    ENGLISH ABSTRACT: Shifts in ploidy or gaining and or loss of a set of chromosomes, have occurred in the evolution of many fungi, plant, and animal species and is thought to contribute to speciation. Despite the important role of ploidy in evolution, little is known about how it contributes to adaptation and speciation. Previously it was shown that deletion of the histone variant Htz1p in S. cerevisiae resulted in perturbed evolvability under oxidative stress. Considering the histone variant’s documented roles in mechanisms such as genome integrity and chromosome segregation, this study sought to uncover if the perturbed evolvability of the strain might be caused by increased ploidy. To address this, we employ flow cytometry and ~300 generations of experimental evolution to analyse the DNA ploidy levels in several S. cerevisiae strains. Initial interest was given to two HTZ1 null mutants, one commercially available (Y11703) and another newly synthesized (PB1012). It was expected that both strains would exhibit similar, near-diploid phenotypes. However, it was found that only Y11703 exhibited the expected diploid phenotype. In parallel, we tested whether the reintroduction of the HTZ1 gene into a null mutant background would revert the cell to its parental ploidy state. To investigate this, two HTZ1 recovery strains were synthesised to observe the effects of introducing the gene in Y11703 under its native promoter and the inducible GAL1 promoter. These two strains presented little deviation from the flow cytometry behaviour exhibited by Y11703, presenting no indication of any reduction in its DNA ploidy level, but, unexpectedly, a slight increase in some cases. Although unexpected, this study adds to the intriguing relationship between Htz1p and ploidy alterations in S. cerevisiae. However, more research is needed to elucidate the mechanisms underlying these observations more completely. Future investigations may explore the molecular pathways connecting Htz1p to ploidy maintenance and its impact on evolvability. Understanding these processes may provide valuable insights into the broader context of eukaryote adaptation and speciation.