Browsing by Author "Kirby, Bronwyn"
Now showing 1 - 10 of 10
Results Per Page
Sort Options
Item Bioprospecting for bioactive polysaccharides from marine algae endemic to South Africa(University of the Western Cape, 2016) January, Grant Garren; Bauer, Rolene; Kirby, Bronwyn; Naidoo, RenèFucoidan is a marine-derived sulphated polysaccharide with bioactive properties ideal for the food, chemical and pharmaceutical industries. The polysaccharide consists largely of L-fucose, has a highly heterogeneous structure and is of diverse origin. Fucoidan was extracted from Ecklonia maxima, Laminaria pallida and Splachnidium rugosum and the effect of different extraction methods on fucoidan heterogeneity was assessed. Extraction methods employed hot water, hydrochloric acid or calcium chloride salt. Fucoidan yield and purity were determined by various colorimetric assays. Highest fucoidan yield was obtained with the hot water extraction method as seen by highest L-fucose content. Splachnidium rugosum extracts contained ~5 times more L-fucose than Ecklonia maxima and Laminaria pallida extracts. The salt extraction method yielded extracts free of contaminants, however L-fucose content in all extracts was >20 times lower. Acid extraction yielded highest levels of uronic acid contamination and liberated sulphate from the fucoidan polysaccharide. The fucose-to-sulphate ratio for Ecklonia maxima was approximately 1:5, whilst the ratios for Splachnidium rugosum and Laminaria pallida were approximately 1:1 and 1:2, respectively. The acid and salt extraction methods removed all traces of protein contaminants, while the hot water method retained very low levels of protein. The extraction method used to isolate fucoidan was a determining factor in yield and purity. Chemical compositional analyses of hot water extracts were assessed by gas chromatography mass spectroscopy. Splachnidium rugosum and Laminaria pallida extracts consisted largely of L-fucose, while Ecklonia maxima fucoidan was characterized with high glucose abundance. Crude hot water and acid extracts from Splachnidium rugosum tissue were fractionated and purified by (anionic) ion exchange chromatography as bioactivity has been correlated to lower molecular weight forms. In water extracts, ion exchange chromatography resulted in close to 90% decrease in L-fucose, sulphate and uronic acid, while protein content increased by 57%. Similar results were reported for acid extracts; however protein content did not change significantly. These results show that method of extraction may affect the composition of fucoidan post-purification. Hot water extraction is recommended due to higher fucoidan yield, as reflected by L-fucose content, and higher sulphate-to-fucose ratio. High protein content after ion exchange chromatography was however of concern. Since mucilage in Splachnidium rugosum thallus was free of protein, fucoidan was precipitated from mucilage with ethanol. Fucoidan yield of mucilage was >15-fold higher than content in purified hot water extracts with a sulphate-to-fucose ratio of ~1:1. The average molecular weight of native fucoidan in mucilage was estimated at 2367 kDa. The polysaccharide was hydrolysed by gamma-irradiation levels of 10-50 kGy to fractions ranging between 60 and 15.5 kDa. Hot water crude fucoidan extracts from Ecklonia maxima, Laminaria pallida, and Splachnidium rugosum were assessed for anti-oxidant activity by measuring the ability to scavenge free radicals and the capacity to reduce copper ions with 2,2-Diphenyl-1-picrylhydrazyl and Cupric Reducing Anti-oxidant Capacity assays, respectively. Ecklonia maxima crude fucoidan displayed highest anti-oxidant activity and capacity, having the potential to scavenge reactive oxygen species as well as the capacity to reduce copper to less toxic forms in mammalian systems. Splachnidium rugosum showed weakest anti-oxidant activity and lowest reducing capacity. The anti-cancer activity of crude and purified hot water Splachnidium rugosum extracts, as well as non-irradiated (native) and gamma-irradiated fucoidan, and commercially procured fucoidan were assessed for anti-cancer activity against MCF-7 breast cancer cells. Splachnidium rugosum crude and purified fucoidan displayed a half maximal inhibitory concentration of 0.7 mg/mL and 0.029 mg/mL, respectively. Low cytotoxicity of crude and purified Splachnidium rugosum fucoidan against non-cancerous breast epithelial cell line MCF-12A was observed, as seen by half maximal inhibitory concentration values of 2 mg/mL and 0.663 mg/mL, respectively. The cancer specific selectivity of purified Splachnidium rugosum fucoidan was therefore much higher as reflected by 10-fold higher selectivity index than that of crude fucoidan. Native and low molecular weight gamma-irradiated fucoidan also showed bioactive properties including anti-cancer activity as seen by the reduction of cell proliferation in vitro, whereas crude fucoidan showed the ability to scavenge free radicals, and the capacity to reduce copper ions.Item Characterisation of a lignocellulosic degrading bacillus strain isolated from thermophilic compost(University of the Western Cape, 2011) Munaka, Matshaya; Cowan, D.A; Bauer, R; Tuffin, M; Kirby, BronwynThe negative environmental impact of fossil fuels and growing concerns about petroleum supplies has driven the search for alternative, renewable transportation fuels. An 'ideal' fuel replacement would be a biofuel produced from lignocellulosic biomass. Unfortunately, the presence of lignin in plant cell walls impedes the breakdown of cell wall polysaccharides into simple sugars and the subsequent conversion of these sugars into useable fuels. One of the most common fates of lignin in nature is to be metabolized by lignin peroxidases (LiPs), predominantly of microbial origin. This study aims to isolate and characterise microorganism(s) involved in the degradation of lignocellulose. Thermophilic bacteria were isolated from straw-based compost and screened for lignin peroxidase activity. One isolate, CP11, showed significant lignin peroxidase activity and based on 16S rRNA gene sequence analysis, the isolate was found to be most closely related to Bacillus thermoamylovorans. Morphological, physiological and biochemical characterisation was conducted to determine whether the isolate was a novel species. Morphologically, CP11 was characterised as an endospore-forming, Gram positive rod. In addition, the isolate was found to be a facultative anaerobe, catalase positive and capable of utilising a range of carbon sources including glucose, sucrose and arabinose. Isolate CP11 was moderately thermotolerant and grew between 37°C and 55°C, with an optimum growth temperature of 45°C. Based on its phenotypic characteristics CP11 could be clearly distinguished from its closest phylogenetic neighbours. Preliminary characterisation of the lignin peroxidase was conducted using crude enzyme extract and Azure B dye as the substrate. Activity was detected in the supernatant only and a growth curve was constructed to determine the growth phase of lignin peroxidase production. In order to identify the gene encoding the lignin peroxidase a small insert library was constructed and screened for ligninase activity using Azure B as the substrate.Item The degradation of the endocrine disrupting chemical, bisphenol-A : a comparative study between fungal and bacterial laccases(University of the Western Cape, 2015) Prins, Alaric; Le Roes-Hill, M..; Kirby, BronwynThe degradation of endocrine disrupting chemicals (EDCs) is a topic of high importance and one that research efforts are continually being focused on. These harmful chemicals are known to cause adverse health effects in humans and animals. In particular, bisphenol-A (BPA), a high volume chemical which is mainly used in the manufacturing of polycarbonate plastics and epoxy resins have been shown to be implicated in the development of a variety of health problems. In this study, the ability of two fungal laccases [Trametes versicolor (TvL) and Trametes pubescens (TpL)], and two bacterial laccases [Streptomyces coelicolor (SLAC), and a mutant of SLAC (SLAC- VN)] to degrade or remove BPA from solution was investigated. The commercial preparation of TvL was used for the purposes of this study, while TpL was produced from the native strain. T. pubescens was cultured in shake-flasks, the supernatant harvested and subjected to ammonium sulphate precipitation. SLAC and SLAC-VN were produced from recombinant strains using a standard protocol and the enzymes purified by size-exclusion chromatography. The presence of the laccases were confirmed by the 2,6-dimethoxyphenol assay and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE).The removal or degradation of BPA from solution was determined for the free enzymes, as well as the enzymes in immobilised form. For immobilisation, the enzymes were encapsulated in sodium alginate beads and cross-linked to form cross- linked enzyme aggregates (CLEAs).High levels of BPA removal was exhibited by the fungal laccase, TpL (100% removal)and the bacterial mutant laccase, SLAC-VN (96%) in their free form. When all four laccases were encapsulated in sodium alginate beads, a number of changes to the characteristics of the enzymes were observed. Overall, the level of BPA removal was reduced for all enzymes as when compared to the free laccases, while SLAC-VN removed more BPA than either of the fungal laccases (59% for SLAC-VN versus 57% TvL and 54% for TpL). The encapsulation of the laccases in alginate beads also led to changes in the optimal temperature for BPA removal, with all encapsulated laccase being able to remove BPA optimally at 40°C. The immobilisation of the laccases in CLEA form had the most significant effect on the BPA removal ability of the laccases. The pH range for both fungal laccases was extended beyond the acidic range [for TpL, optimal removal occurred at pH 8.5 compared to pH 4.5 (free) and pH 6.0 (encapsulated)]. Most remarkable, however, was that the formation of CLEAs greatly enhanced the BPA removal ability of SLAC (60% removal compared to 25% when encapsulated).Item Development of genotyping systems for pharmacogenomics profiling(University of the Western Cape, 2016) Eshumani, Fatima A.; Benjeddou, Mongi; Kirby, BronwynGenetic variability in genes encoding drug metabolizing enzymes, transporters and targets are known to be the main factors of inter-individual differences in therapeutic outcome. Genetic factors are estimated to be responsible for about 15-30% of inter-individual variation in drug disposition and response. Single-nucleotide polymorphisms (SNPs) are the most prevalent class of genetic variation that could explain the variability in drug efficacy and undesired side effects for patients. The aims of this study were to develop and evaluate the performance of robust and high throughput techniques for genotyping ten polymorphisms related to anticancer drugs and ten polymorphisms related to cholesterol lowering drugs. SNaPshot minisequencing and high resolution melt analysis (HRM) genotyping panels were developed, optimized, and their performances were evaluated and compared. SNaPshot minisequencing systems were developed and successfully optimized for the genotyping of ten SNPs associated with anticancer drug therapy, and ten SNPs associated with cholesterol lowering drugs. These systems were used to genotype the selected SNPs in 130 healthy Cape Admixed participants residing in Cape Town, South Africa. Population genetics data obtained for the studied SNPs were analysed using several statistical analysis software tools. Important population genetic parameters were calculated. Among others, allelic and genotypic frequencies were determined and compared with other populations in the world. High resolution melt analysis (HRM) genotyping panels were developed, optimized and their performance were evaluated and compared to the SNaPshot assays. HRM was explored as an alternative inexpensive and rapid methodology to genotype five SNPs related to anticancer therapy and five SNPs related to cholesterol lowering therapy (statins). Unlike the SNaPshot assays, rigorous optimization was required for the detection heterozygous genotypes via HRM. Both assays were validated using direct sequencing and compared to each other. The HRM system is a closed tube, cheap and (theoretically) rapid method for identifying genetic variations. HRM was however found to be more time consuming, needed further optimization, primer redesigning and more evaluation. The developed genotyping systems could be further validated using clinical samples from patients. This could help in optimizing drug therapy for cancer and cholesterol treatment.Item Diversity of dsDNA viruses in a South African hot spring assessed by metagenomics and microscopy(MDPI, 2017) Zablocki, Olivier; van Zyl, Leonardo Joaquim; Kirby, Bronwyn; Trindade, MarlaThe current view of virus diversity in terrestrial hot springs is limited to a few sampling sites. To expand our current understanding of hot spring viral community diversity, this study aimed to investigate the first African hot spring (Brandvlei hot spring; 60 C, pH 5.7) by means of electron microscopy and sequencing of the virus fraction. Microscopy analysis revealed a mixture of regular- and ‘jumbo’-sized tailed morphotypes (Caudovirales), lemon-shaped virions (Fuselloviridae-like; salterprovirus-like) and pleiomorphic virus-like particles. Metavirome analysis corroborated the presence of His1-like viruses and has expanded the current clade of salterproviruses using a polymerase B gene phylogeny. The most represented viral contig was to a cyanophage genome fragment, which may underline basic ecosystem functioning provided by these viruses. Furthermore, a putative Gemmata-related phage was assembled with high coverage, a previously undocumented phage-host association. This study demonstrated that a moderately thermophilic spring environment contained a highly novel pool of viruses and should encourage future characterization of a wider temperature range of hot springs throughout the world.Item Identification and characterization of microorganisms associated with marine macroalgae Splachnidium rugosum(University of the Western Cape, 2014) Albakosh, Mouna Abdalhamed; Kirby, Bronwyn; Bauer, Rolene; Naidoo, ReneMarine macroalgae are known to carry diverse bacterial communities which interact with their hosts in both harmful and beneficial ways. Algae hosts provide the bacteria with a rich source of carbon in the form of carbohydrate polysaccharides such as fucoidan, agar and alginate, which the bacteria enzymatically degrade. Splachnidium rugosum is a brown alga (Phylum: Phaeophyta) that grows exclusively in the Southern Hemisphere along the temperate shores of South Africa, New Zealand and Australia. While several studies have investigated S. rugosum distribution and fucoidan production, the microbiome of S. rugosum remains largely uncharacterized. Thus, the major objective of the present study was to isolate, identify and characterize epiphytic bacterial communities associated with S. rugosum. Algae were sourced from Rooi Els (Western Cape, South Africa) during winter 2012. Culture based methods relied on a range of selective marine media including marine agar, nutrient sea water agar, nutrient agar and thiosulfate-citrate-bile-salts-sucrose agar to determine the composition and uniqueness of bacterial communities associated with S. rugosum. Epiphytic isolates were identified to species level by 16S rRNA gene sequence analysis and encompassed 39 Gram-negative and 2 Grampositive bacterial taxa. Isolates were classified into four phylogenetic groups, Gamma - Proteobacteria, Alpha-Proteobacteria, Firmicutes and Bacteriodetes. Bacteria belonging to the phylum Gamma-Proteobacteria were the most abundant, with Vibrio and Pseudoalteromonas being the dominant genera. Three isolates with low sequence identity (˂97%) to their closest relatives could possibly represent novel species. These isolates were grouped into the genera Shewanella, Sphingomonas and Sulfitobacter. All bacterial isolates (41) were screened for antimicrobial activity against the following test strains: Escherichia coli, Bacillus cereus, Staphylococcus epidermidis, Mycobacterium smegmatis Micrococcus luteus and Pseudomonas putida. Fifteen isolates (36%) displayed antimicrobial activity against one or more of the test strains, while one isolate (Pseudomonas species) showed broad spectrum antimicrobial activity against all the test strains except for E. coli. This study provides the first account of the diversity and composition of bacterial populations on the surface of S. rugosum, and demonstrates the ability of these bacteria to produce antimicrobial compounds. Despite recent advances in metagenomics, this study highlights the fact that traditional culturing technologies remain a valuable tool for the discovery of novel bioactive compounds of bacterial origin.Item Isolation, identification and characterisation of novel actinobacteria from Zambian hot-springs(2011) Mavengere, Natasha Robertha; Cowan, D. A.; Kirby, Bronwyn; Tuffin, I.M.Actinomycetes are ubiquitous in many environments such as soil, activated sludge and water.Besides the genus Streptomyces, which has been extensively exploited, members of other genera including Micromonospora have been shown to be a promising source of novel secondary metabolites and enzymes.The biocatalytic conversion of 5-monosubstituted hydantoin derivatives to optically pure amino acids involves two reaction steps. The first step, catalysed by a hydantoinase, yields an N-carbamylamino acid intermediate, which is subsequently broken down by an Ncarbamoylase to the amino acid. This process has been successfully applied in industry for the production of optically pure amino acids which are used in the synthesis of pharmaceuticals,insecticides, hormones, and food additives. The need for novel hydantoinases to hydrolyse a wider variety of substrates is increasing. This thesis describes the search for a novel hydantoinase from environmental isolates obtained from two Zambian hot-springs. The aim of this study was to isolate, characterise and screen novel actinobacteria for industrially relevant enzymes including hydantoinases. Fifty one actinobacteria were isolated. Isolates were characterized by a polyphasic approach using standard methods, combining phylogenetic analysis of the 16S rRNA gene, chemotaxonomic and phenotypic characterization. Results revealed that these sites were dominated by actinobacteria belonging to the family Micromonosporaceae, and a potentially novel Verrucosispora species was identified. Screening the isolate identified a Streptomyces species which has hydantoinase, carbamoylase, amidase and nitrilase activities.The Streptomyces sp. hydantionase was cloned and functionally expressed in E.coli. The recombinant enzyme showed 49 % similarity to a crystallised hydantoinase from a Bacillus species. Homology modelling revealed that the enzyme had the TIM barrel topology which is characteristic of hydantoinases. Amino acid residues predicted to be involved in the catalytic activity as well as substrate orientation were identified. The partially purified hydantoinase was characterised and showed optimally activity at 45 °C and pH 8. This study revealed that hot springs may represent a previously unexplored source of novel actinobacterial diversity. However, it also revealed that novel secondary metabolites are not only limited to novel organisms but that some of the answers for the challenges we face today maybe found in organisms we have already encountered and characterised.Item Microbial diversity of the Namib Desert salt pans(University of the Western Cape, 2015) Cloete, Melissa; Trindade, I. M.; Kirby, BronwynSalt pans are a characteristic feature of many dry deserts. The microbial communities inhabiting salt pans are thought to be particularly complex and are generally dominated by halophilic microorganisms. Although saline pools are frequently found within the hyper-arid Namib Desert, the microbial communities of these saline sites have been scarcely investigated. The aim of the present study was to characterise the archaeal, bacterial and cyanobacterial diversity inhabiting these extreme saline pools using three culture independent molecular techniques (DGGE, T-RFLP and 16S rRNA clone libraries). The physiochemical results, mainly the conductivity readings recorded from the sampling sites, indicated that the Gobabeb (103.0mS/cm) region was less saline than the two Swakopmund [(Sps01) (150.0mS/cm) and Sps02 (180.0mS/cm)] sites. Results obtained from DGGE and T-RFLP data were in agreement for both bacterial and cyanobacterial analysis indicating that the Gobabeb site was more diverse than the two Swakopmund sites (Sps01 and Sps02). In comparison, the archaeal community profiles for DGGE and T-RFLP analysis were in agreement illustrating that the archaeal community were more abundant in the two extreme Swakopmund saline sites. Phylogenetic data obtained from 16S rRNA gene clone libraries identified halophilic phylotypes (Rhodothermaceae, Idiomarinaceae Puniceicoccaceae and Cyanobacteria/Chloroplast, Family VII) normally associated with salt rich sites. In addition, a large number of unclassified taxa were identified. To conclude, the study highlighted the presence of a rich microbial diversity present within the salt pans of the Namib Desert and establishes a platform for future investigations.Item Response of microbial communities to synthetic winery wastewater in biological sand filters(University of the Western Cape, 2017) Palmer, Zaida; Kirby, BronwynThere has been a significant increase in the use of constructed wetlands globally for the bioremediation of wastewater (Welz et al., 2011). In South Africa, the wine industry generates more than one billion litres of wastewater annually and this is disposed of by irrigation (Burton et al., 2007). A more cost effective and simple system need to be employed for the treatment of winery effluent and a possible solution would be the use of biological sand filters (BSFs). The microbial communities present in these BSFs play an important role in the biodegradation of the organic wastewater pollutants. Physicochemical and microbiological studies have been used to determine the performance of BSFs for the remediation of winery effluent since 2009. In this study, changes in the bacterial and fungal communities in different spatial niches was analysed through the use of molecular fingerprinting techniques [terminal restriction fragment length polymorphism (T-RFLP)] of the 16S rRNA gene and fungal ITS gene as a response to the exposure to synthetic winery wastewater and to perform functional studies using q-PCR on selected genes. Changes in the bacterial and fungal community profiles were observed at the different niches after amendment with synthetic winery wastewater. This result was confirmed by performing the phylogenetic analysis on the bacterial population present within the BSF systems. Sand organisms including Clostridium, Sarcina, Streptomyces, Actinobacteria were among the expected species present in the sand samples at the deep inlet of the BSFs. Through the study by Burton et al, (2007), one could hypothesise which organisms mentioned above were able to metabolise the components of the synthetic winery wastewater to secondary metabolites. Amplification of the functional gene through the use of qPCR of catechol 2,3-dioxygenase was successful. Increase in the amount of copy numbers between the samples showed that the increase in expression of the catechol 2,3-dioxygenase meant that there was an increase in the amount of organisms degrading the catehol build-up in the BSF systems.Item Virome assembly and annotation: A surprise in the Namib Desert(Frontiers Research Foundation, 2017) Hesse, Uljana; Van Heusden, Peter; Kirby, Bronwyn; Olonade, Israel; van Zyl, Leonardo Joaquim; Trindade, MarlaSequencing, assembly, and annotation of environmental virome samples is challenging. Methodological biases and differences in species abundance result in fragmentary read coverage; sequence reconstruction is further complicated by the mosaic nature of viral genomes. In this paper, we focus on biocomputational aspects of virome analysis, emphasizing latent pitfalls in sequence annotation. Using simulated viromes that mimic environmental data challenges we assessed the performance of five assemblers (CLC-Workbench, IDBA-UD, SPAdes, RayMeta, ABySS). Individual analyses of relevant scaffold length fractions revealed shortcomings of some programs in reconstruction of viral genomes with excessive read coverage (IDBA-UD, RayMeta), and in accurate assembly of scaffolds ?50 kb (SPAdes, RayMeta, ABySS). The CLC-Workbench assembler performed best in terms of genome recovery (including highly covered genomes) and correct reconstruction of large scaffolds; and was used to assemble a virome from a copper rich site in the Namib Desert. We found that scaffold network analysis and cluster-specific read reassembly improved reconstruction of sequences with excessive read coverage, and that strict data filtering for non-viral sequences prior to downstream analyses was essential. In this study we describe novel viral genomes identified in the Namib Desert copper site virome. Taxonomic affiliations of diverse proteins in the dataset and phylogenetic analyses of circovirus-like proteins indicated links to the marine habitat. Considering additional evidence from this dataset we hypothesize that viruses may have been carried from the Atlantic Ocean into the Namib Desert by fog and wind, highlighting the impact of the extended environment on an investigated niche in metagenome studies.