Prediction of interacting motifs within the protein subunits of Picornavirus capsids
- Authors: Ross, Caroline Jane
- Date: 2015
- Subjects: Picornaviruses , Antiviral agents , Poliovirus , Coxsackieviruses , Hepatitis A virus , Foot-and-mouth disease virus , Viral proteins
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4151 , http://hdl.handle.net/10962/d1017912
- Description: The Picornaviridae family contains a number of pathogens which are economically important including Poliovirus, Coxsakievirus, Hepatitis A Virus, and Foot-and-Mouth-Disease-Virus. Recently the emergence of novel picornaviruses associated with gastrointestinal, neurological and respiratory diseases in humans has been reported. Although effective vaccines for viruses such as FMDV, PV and HAV have been developed there are currently no antivirals available for the treatment of picornavirus infections. Picornaviruses proteins are classified as: the structural proteins VP1, VP2, VP3 and VP4 which form the subunits of the viral capsid and the replication proteins which function as proteases, RNA-polymerases, primers and membrane binding proteins. Although the host specificity and viral pathogenicity varies across members of the family, the icosahedral capsid is highly conserved. The capsid consists of 60 protomers, each containing a single copy of VP1, VP2 and VP3. A fourth capsid protein, VP4, resides on the internal side of the capsid. Capsid assembly is integral to life-cycle of picornaviruses; however the process is complex and not fully-understood. The overall aim of the study was to broaden the understanding of the evolution and function of the structural proteins across the Picornaviridae family. Firstly a comprehensive analysis of the phylogenetic relationships amongst the individual structural proteins was performed. The functions of the structural proteins were further investigated by an exhaustive motif analysis. A subsequent structural analysis of highly conserved motifs was performed with respect to representative enteroviruses, Foot-and-Mouth-Disease-Virus and Theiler’s Virus. This was supplemented by the in silico prediction of interacting residues within the crystal structures of these protomers. Findings in this study suggest that the capsid proteins may be evolving independently from the replication proteins through possible inter-typic recombination of functional protein regions. Moreover the study predicts that protomer assembly may be facilitated through a network of multiple subunit-subunit interactions. Multiple conserved motifs and principle residues predicted to facilitate capsid subunit-subunit interactions were identified. It was also concluded that motif conservation may support the theory of inter-typic recombination between closely related virus sub-types. As capsid assembly is critical to the viral life-cycle, the principle interacting motifs may serve as novel drug targets for the antiviral treatment of picornavirus infections. Thus the findings in the study may be fundamental to the development of treatments which are more economically feasible or clinically effective than current vaccinations.
- Full Text:
- Date Issued: 2015
Synthesis of silver nanoparticles and their role against human and Plasmodium falciparum leucine aminopeptidase
- Authors: Mnkandhla, Dumisani
- Date: 2015
- Subjects: Silver , Nanoparticles , Plasmodium falciparum , Leucine aminopeptidase , Antimalarials , Nanotechnology
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4150 , http://hdl.handle.net/10962/d1017911
- Description: Antimalarial drug discovery remains a challenging endeavour as malaria parasites continue to develop resistance to drugs, including those which are currently the last line of defence against the disease. Plasmodium falciparum is the most virulent of the malaria parasites and it delivers its deadliest impact during the erythrocytic stages of the parasite’s life cycle; a stage characterised by elevated catabolism of haemoglobin and anabolism of parasite proteins. The present study investigates the use of nanotechnology in the form of metallic silver nanoparticles (AgNPs) against P. falciparum leucine aminopeptidase (PfLAP), a validated biomedical target involved in haemoglobin metabolism. AgNPs were also tested against the human homolog cytosolic Homo sapiens leucine aminopeptidase (HsLAP) to ascertain their selective abilities. PfLAP and HsLAP were successfully expressed in Escherichia coli BL21(DE3) cells. PfLAP showed optimal thermal stability at 25 °C and optimal pH stability at pH 8.0 with a Km of 42.7 mM towards leucine-p-nitroanilide (LpNA) and a Vmax of 59.9 μmol.ml⁻¹.min⁻¹. HsLAP was optimally stable at 37 °C and at pH 7.0 with a Km of 16.7 mM and a Vmax of 17.2 μmol.ml⁻¹.min⁻¹. Both enzymes exhibited optimal activity in the presence of 2 mM Mn²⁺. On interaction with polyvinylpyrrolidone (PVP) stabilised AgNPs, both enzymes were inhibited to differing extents with PfLAP losing three fold of its catalytic efficiency relative to HsLAP. These results show the ability of AgNPs to selectively inhibit PfLAP whilst having much lesser effects on its human homolog. With the use of available targeting techniques, the present study shows the potential use of nanotechnology based approaches as “silver bullets” that can target PfLAP without adversely affecting the host. However further research needs to be conducted to better understand the mechanisms of AgNP action, drug targeting and the health and safety issues associated with nanotechnology use.
- Full Text:
- Date Issued: 2015
The detection of glyphosate and glyphosate-based herbicides in water, using nanotechnology
- Authors: De Almeida, Louise Kashiyavala Sophia
- Date: 2015
- Subjects: Water -- Glyphosate content , Aquatic herbicides -- South Africa , Aquatic herbicides -- Physiological effect , Nanotechnology , Invasive plants -- South Africa , Genetic toxicology , Thiazoles , Tetrazolium , Immunotoxicology , Colorimetry , Nanofibers
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4163 , http://hdl.handle.net/10962/d1019755
- Description: Glyphosate (N-phosphonomethylglycine) is an organophosphate compound which was developed by the Monsanto Company in 1971 and is the active ingredient found in several herbicide formulations. The use of glyphosate-based herbicides in South Africa for the control of alien invasive plants and weeds is well established, extensive and currently unregulated, which vastly increases the likelihood of glyphosate contamination in environmental water systems. Although the use of glyphosate-based herbicides is required for economic enhancement in industries such as agriculture, the presence of this compound in natural water systems presents a potential risk to human health. Glyphosate and glyphosate formulations were previously considered safe, however their toxicity has become a major focal point of research over recent years. The lack of monitoring protocols for pesticides in South Africa is primarily due to limited financial capacity and the lack of analytical techniques.
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- Date Issued: 2015
The development of biological tools to aid in the genetic investigation of the black (Diceros bicornis) and white (Ceratotherium simum) rhinoceros mitochondrial genomes
- Authors: Parsons, Michelle
- Date: 2015
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/56059 , vital:26769
- Description: The black (Diceros bicornis) and white (Ceratotherium simum) rhinoceros are found in South Africa. A decline in the populations of these species has resulted due to human activities such as habitat fragmentation and poaching. This has contributed to the loss of genetic diversity amongst the black and white rhinoceros. Conservation and anti-poaching efforts are needed to help maintain genetic diversity. These efforts could be improved through the development of non-invasive techniques to examine DNA from threatened animals. The aim of this research was to develop a molecular technique which would allow for the identification of the black and white rhinoceros and to develop a molecular technique which would allow for intraspecies genetic variation to be examined. DNA extractions were performed on matched faecal and tissue samples that were collected from two regions in South Africa. Polymerase chain reaction (PCR) primer sets were designed to investigate several regions of the rhinoceros mitochondrial genome. PCR optimisation was completed for the target regions. Sequencing was conducted on all final PCR products. The cytochrome c oxidase subunit 1 (COIi) gene allowed for the rhinoceros family to be identified. This region was digested with the HindIII restriction enzyme, which allowed for the specific identification of either the black or white rhinoceros. A subsequent region of the cytochrome c oxidase subunit 1 (COIii) as well as the D-loop, hypervariable regions (HV1 and HV2), cytochrome b (cytb) and 16s rRNA regions were investigated. These regions displayed potential for establishing geographic origin for black rhinoceros samples, whereas the D-loop and HV2 show potential for the white rhinoceros. The white rhinoceros displayed sequence variation in the HV2 and cytb region, while variation was observed in the COIi and HV1 for the black rhinoceros. All investigated target regions allowed for the rhinoceros family to be identified. The COI (COIi and COIii), HV2 and cytb regions allowed for the subspecies of rhinoceros to be identified, however the D-loop was not able to identify the white rhinoceros species. The 16s rRNA and HV1 regions allowed for the correct subspecies of rhinoceros to be identified, however as the primers were only compatible for the black rhinoceros therefore a subsequent investigation is required for the white rhinoceros. The establishment of this novel PCR based technique to identify white and black rhinoceros will allow for efficient species identification in wildlife forensic cases. A biological method was established to study intraspecies variation for the white and black rhinoceros; however the investigated target regions did not yield sufficient genetic variation. The core techniques developed in this study will be valuable for future studies that wish to investigate genetic variation in mammal species.
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- Date Issued: 2015
The diversity of root fungi associated with Erica species occurring in the Albany Centre of Endemism
- Authors: Bizabani, Christine
- Date: 2015
- Subjects: Ericaceae , Ericas , Roots (Botany) -- Diseases and pests , Mycorrhizal fungi , Polymerase chain reaction , Fungi -- Classification
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4160 , http://hdl.handle.net/10962/d1018575
- Description: South Africa has the highest species diversity of ericaceous plants belonging to the Erica genus. There are over 850 identified species in the Cape Floral Region. The Albany Centre of Endemism (ACOE) is located within this region and is a hotspot of diversity consisting of various plant genera. The success of Erica plants is ubiquitously attributed to mycorrhizal relationships they engage in with a diverse group of fungi. This symbiosis is known as the ericoid mycorrhizal (ERM) association. The overall aim of this study was to establish the diversity of root fungi associated with Erica plants using morphological, molecular and 454 pyrosequencing techniques. Six Erica species were identified using leaf and flower morphology according to taxonomic keys. The identified plants were Erica cerinthoides, Erica demissa, Erica chamissonis, Erica glumiflora, Erica caffra and Erica nemorosa. Roots from sampled plants were stained and examined microscopically to determine their mycorrhizal status. Ericoid mycorrhizal associations together with dark septate endophyte (DSE) structures and hyphae that did not form any specific structure were observed in all the roots. In addition arbuscular mycorrhizal (AM) structures in the form of vesicles were detected in E. glumiflora and E. cerinthoides. In order to identify the culturable fungi associated with the respective hosts, sterilised roots were placed on various culture media for cultivation. Thereafter isolated fungi were morphologically classified into 67 morphotypes. These were mostly sterile and darkly pigmented. Non-sporulating mycelia of variable colouration such as white, cream-yellowish, beige, green and brown were also observed. Further identification was carried out using molecular techniques. DNA was extracted separately from pure cultures and amplified using ITS1 and ITS4 primers in a polymerase chain reaction (PCR). Thereafter sequencing and Basic Local Alignment Search Tool (BLAST) were used to identify the isolates to generic level. The fungi were taxonomically classified into 54 operational taxonomic units and 94 percent were Ascomycetes and Helotiales was the dominant order. Unclassified Helotiales with affinities to fungi currently identified as Epacrid root fungus was common in all hosts. Other isolates that were identified included Oidiodendron, Meliniomyces, Phialocephala, Cadophora, Lachnum, Leohumicola Cryptosporiopsis, Chaetomium, Acremonium and Epicoccum species. Basidiomycetes were represented by two OTUs belonging to the genus Mycena. Four OTUs comprised fungi that had no significant alignments in the reference databases. Direct root DNA extraction together with 454 pyrosequencing was used to detect the diversity of culturable and unculturable fungi associated with the identified hosts. The ITS2 region was targeted for sequencing. Although Ascomycetes remained the dominant phyla, Basidiomycetes were also detected in all host plants. Glomeromycota was present in E. caffra and E. cerinthoides. Helotiales was dominant in all Erica plants with the exception of E. cerinthoides and E. chamissonis which were dominated by the order Chaetothyriales. The OTUs identified to genus level included Epacris pulchella root fungus, Oidiodendron cf. maius, Acremonium implicatum, Leohumicola, Lachnum, Capronia and Mycena species. Culture-based techniques and pyrosequencing detected similar fungal composition comprising Ascomycetes, while, pyrosequencing was able to detect Glomeromycetes and Basidiomycetes.
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- Date Issued: 2015
The effect of extracellular Hsp90β and TGF-β1 on colon cancer biology
- Authors: Perks, Tamarin
- Date: 2015
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/55896 , vital:26753
- Description: The TGF-β signaling pathway is known to be one of the most commonly mutated pathways in human cancers, while Hsp90 is a bone fide drug target that is involved in regulating the conformation and activity of many oncoproteins. The role of intracellular Hsp90 in cancer has thus far been established and there is a growing link between extracellular Hsp90 and cancer metastasis, as well as the role of TGF-β in metastasis. This study aimed to analyse the interaction between Hsp90 (both intracellular and extracellular) and the TGF-β machinery in cancer cells, as well as to determine the effect of these proteins on cellular responses on the biology of cancer cells. This was achieved by studying the expression of Hsp90; TGF-βRII and TGF-β1 in cancer cell lines of various origins using flow cytometry, ELISA, and western blot analysis. The genetically paired SW480 and SW620 colon cancer cell lines, derived from a primary tumour and lymph node metastasis, respectively, were selected for further study due to differences in expression levels and activation of the TGF-β1 pathway. SW480 cells expressed double the level of TGF-βRII compared to SW620 cells, while SW620 expressed two times more extracellular TGF-β1 than SW480 cells. A direct interaction between TGF-β1 and Hsp90β was determined in vitro, and confirmed in vivo in SW620 cells. Growth, adhesion and migration were analysed in SW480 and SW620 cells. SW480 cells adhered significantly faster than SW620 cells, while SW620 cells had a greater rate of migration. Inhibiting the TGF-β pathway, specifically TGF-βRI, using SB 431542, as well as inhibiting Hsp90 with novobiocin, caused an increase in migration in SW480 cells. Only the addition of TGF-β1 in combination with Hsp90 as well as SB 431542 caused an increase in migration in SW620 cells. The canonical TGF-β1/TGF-βRI/TGF-βRII pathway may be constitutively active in SW620 cells and the inhibition of TGF-βRI may suggest an alternate pathway or receptor in both SW480 and SW620 cells.
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- Date Issued: 2015
The effect of GH family affiliations of mannanolytic enzymes on their synergistic associations during the hydrolysis of mannan-containing substrates
- Authors: Malgas, Samkelo
- Date: 2015
- Subjects: Lignocellulose , Biomass energy , Ethanol as fuel , Polysaccharides , Sugar -- Inversion , Glycosidases , Galactoglucomannans , Oligosaccharides
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4148 , http://hdl.handle.net/10962/d1017909
- Full Text:
- Date Issued: 2015
The isolation, genetic characterisation and biological activity of a South African Phthorimaea operculella granulovirus (PhopGV-SA) for the control of the Potato Tuber Moth, Phthorimaea operculella (Zeller)
- Authors: Jukes, Michael David
- Date: 2015
- Subjects: Potato tuberworm , Potatoes -- Diseases and pests -- South Africa , Baculoviruses , Natural pesticides , Biological pest control agents , Potato tuberworm -- Biological control , Restriction enzymes, DNA
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4147 , http://hdl.handle.net/10962/d1017908
- Description: The potato tuber moth, Phthorimaea operculella (Zeller), is a major pest of potato crops worldwide causing significant damage to both field and stored tubers. The current control method in South Africa involves chemical insecticides, however, there is growing concern on the health and environmental risks of their use. The development of novel biopesticide based control methods may offer a potential solution for the future of insecticides. In this study a baculovirus was successfully isolated from a laboratory population of P. operculella. Transmission electron micrographs revealed granulovirus-like particles. DNA was extracted from recovered occlusion bodies and used for the PCR amplification of the lef-8, lef-9, granulin and egt genes. Sequence data was obtained and submitted to BLAST identifying the virus as a South African isolate of Phthorimaea operculella granulovirus (PhopGV-SA). Phylogenetic analysis of the lef-8, lef-9 and granulin amino acid sequences grouped the South African isolate with PhopGV-1346. Comparison of egt sequence data identified PhopGV-SA as a type II egt gene. A phylogenetic analysis of egt amino acid sequences grouped all type II genes, including PhopGV-SA, into a separate clade from types I, III, IV and V. These findings suggest that type II may represent the prototype structure for this gene with the evolution of types I, III and IV a result of large internal deletion events and subsequent divergence. PhopGV-SA was also shown to be genetically more similar to South American isolates (i.e. PhopGV-CHI or PhopGV-INDO) than it is to other African isolates, suggesting that the South African isolate originated from South America. Restriction endonuclease profiles of PhopGV-SA were similar to those of PhopGV-1346 and PhopGV-JLZ9f for the enzymes BamHI, HindIII, NruI and NdeI. A preliminary full genome sequence for PhopGV-SA was determined and compared to PhopGV-136 with some gene variation observed (i.e. odv-e66 and vp91/p95). The biological activity of PhopGV-SA against P. operculella neonate larvae was evaluated with an estimated LC₅₀ of 1.87×10⁸ OBs.ml⁻¹ being determined. This study therefore reports the characterisation of a novel South African PhopGV isolate which could potentially be developed into a biopesticide for the control of P. operculella.
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- Date Issued: 2015
The role of Stress Inducible Protein 1 (STI1) in the regulation of actin dynamics
- Authors: Beckley, Samantha Joy
- Date: 2015
- Subjects: Heat shock proteins , Molecular chaperones , Actin , Microfilament proteins , Cell migration , Adenosine triphosphatase , Metastasis
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/193941 , vital:45409
- Description: Stress-inducible protein 1 (STI1) otherwise known as Hop (Hsp70/Hsp90 organising protein) is a highly conserved abundant co-chaperone of the Hsp70 and Hsp90 chaperones. STI1 acts as an adapter protein, where it regulates the transfer of protein substrates from Hsp70 to Hsp90 during the assembly of a number of chaperone-client protein complexes. The role of STI1 associating independently with non-chaperone proteins has become increasingly prominent. Recent data from colocalisation and co-sedimentation analyses in our laboratory suggested a direct interaction between STI1 and the cytoskeletal protein, actin. However, there was a lack of information on the motifs which mediated this interaction, as well as the exact role of STI1 in the regulation of cytoskeletal dynamics. Two putative actin binding motifs, DAYKKK (within the TPR2A domain) and a polyproline region (after the DP1 domain), were identified in mammalian STI1. Our data from in vitro interaction studies including surface plasmon resonance and high speed co-sedimentation assays suggested that both TPR1 and TPR2AB were required for the STI1-actin interaction, and peptides corresponding to either the DAYKKK or the polyproline motif, alone or in combination, could not block the STI1-actin interaction. Full length mSTI1 was shown to have ATPase activity and when combined with actin an increase in ATPase activity was seen. Ex vivo studies using STI1 knockdown shRNA HEK293T cells and non-targeting control shRNA HEK293T cells showed a change of F-actin morphology as well as reduction in levels of actin-binding proteins profilin, cofilin and tubulin in the STI1 knockdown cells. These data extend our understanding of the role of STI1 in regulating actin dynamics and may have implications for cell migration. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2015
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- Date Issued: 2015
Towards a Mobile Bioethanol Unit for point of source conversion of sugar sources to bioethanol: design and feasibility study for South Africa
- Authors: Cech, Alexandra Louise
- Date: 2015
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/59141 , vital:27439
- Description: Restricted access-thesis embargoed for 5 years
- Full Text:
- Date Issued: 2015
The characterization of DNAJC3: elucidating the function of the TPR domains
- Authors: Mutsvunguma, Lorraine Zvichapera
- Date: 2014
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/55874 , vital:26751
- Description: DNAJC3 is a novel member of the DNAJ family with two domains linked to co-chaperone functions, namely the tetratricopeptide repeat (TPR) and J domain. Out of the two domains, the TPR domains are the least characterized. Therefore, the aim of this study was to characterize and elucidate additional functions of DNAJC3 TPR domains through in silico, in vitro and ex vivo approaches. Through multiple sequence and structural alignment as well as electrostatic potential analysis, DNAJC3 TPR domain were found to be most similar to TPR-containing proteins with Hsp90 or Hsp70 independent functions. In vitro pull down assays illustrated that DNAJC3 TPR domains did not interact with either cytosolic Hsp90 and Hsp70 or Grp78 and Grp94 directly, however a potential indirect interaction with Grp94 and Hsp90 was observed in mammalian lysates, via pull down assays; suggesting the formation of a complex between the proteins mediated by a specific substrate. DNAJC3 TPR domains were found to bind indiscriminately to both native and heat denatured substrates in a dose dependent manner. DNAJC3 TPR domains bound to β-galactosidase with greater affinity than malate dehydrogenase (MDH), suggesting that DNAJC3 TPR domains might exhibit substrate specificity that has not been reported before. Preliminary ex vivo analysis of DNAJC3 in mammalian cells showed that induced stress conditions did not alter the cytosolic or endoplasmic reticulum (ER) localization, or levels of DNAJC3 protein, suggesting that the protein is not stress inducible. However, protein levels of DNAJC3 were dramatically reduced by Hsp90 inhibitor novobiocin at 500 μM. Transient knockdown DNAJC3 did not change the protein levels of either Grp78 or Grp94, but decreased the protein levels of Hsp70/Hsp90 organizing protein HOP. On the other hand, protein levels of DNAJC3 were increased in HOP depleted cells. In conclusion, this study was the first to experimentally demonstrate that DNAJC3 TPR domains do not interact directly with Hsp90, Hsp70, Grp78 or Grp94, and therefore DNAJC3 is unlikely to participate in traditional co-chaperone interactions with those proteins via its TPR domain. However, the J domain is known to interact with Grp78. The discovery that DNAJC3 TPR domains resemble that of TPR-containing proteins with functions independent of Hsp90 or Hsp70 suggests that DNAJC3 might link the Hsp70/Grp78 chaperone machinery to non co-chaperone related functions, which requires further analysis.
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- Date Issued: 2014
Isolation, expression and purification of the hydantoin hydrolysing enzymes of agrobacterium tumefaciens
- Authors: Clark, Sally-Ann
- Date: 2003
- Subjects: Agrobacterium tumefaciens , Amino acids Hydantoin Enzymes
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4140 , http://hdl.handle.net/10962/d1016233
- Description: The production of enantiomerically pure amino acids is of industrial importance as they are used in the synthesis of a number of pharmaceuticals, insecticides and herbicides and biologically active peptides and hormones. A number of microorganisms have been identified which possess hydantoin hydrolysing enzymes that stereoselectively convert racemic hydantoins into anantiomerically pure amino acids. Consequently these microorganisms and their enzymes are sought after as biocatalysts for the production of amino acids. The isolation of novel hydantoin hydrolising enzymes with unique or improved biocatalytic characteristics is of importance for the development of potential biocatalysts to be used in the production of enantiomerically pure amino acids. The genes encoding an N-carbamoyl-amino acid amidohydrolase, an enzyme involved in the hydrolysis of hydantoin, was isolated by screening a genomic DNA library of Agrobacterium tumefacience RU-AE01. Nucleotide sequence analysis of the region upstream of this gene revealed a fragment of a gene encoding the hydantoinase enzyme. I this study, a DNA probe consisting of the gene encoding the N-carbamoyl amino acid amidohydrolase, on a large enough fragment of the genomic DNA library which would allow for the simultaneous isolation the hydantoinase gene located upstream. Recombinant expression of the genes encoding hydantoin hydrolysing enzymes has been used to facilitate the production and purification of these enzymes for their use as biocatalysts. Two genes (ncaR1 and ncaR2) encoding different N-carbamoyl-amino acid amidohydrolases with distinct nucleotide and deduced amino acid sequences were isolated from the genome of A, tumefaciens RU-OR. In this study, the heterologous expression of ncaR1 and ncaR2 was explored. Investigation into the optimisation of the heterologous expression of ncaR1 showed that reducing the growth temperature of the recombinant E. coli producing NcaR1 resulted in a two-fold increase in N-carbamoyl-amino acid amidohydrolase activity and solubility. Furthermore, NcaR1 was produced with a C-terminal 6xHis tag, but NcaR1-6xHis did not possess N-carbamoyl amino acid amidohydrolase activity. Furthermore, purification of NcaR-6xHis under native conditions using affinity chromatography performed, and used for the production of antibodies.
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- Date Issued: 2003
Biological generation of reactive alkaline species and their application in a sustainable bioprocess for the remediation of acid and metal contaminated wastewaters
- Authors: Van Hille, Robert Paul
- Date: 2002
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:21049 , http://hdl.handle.net/10962/6129
- Description: This project focused on the development of an integrated biological system for the treatment of acidic and metal-laden effluents, based on the sustainable biological generation of reactive alkaline species. Initial studies concentrated on the binding and accumulation of heavy metals by biomass of the cyanobacteria, Spirulina sp. Metal binding was rapid, with saturation reached in 30 minutes, and followed an affinity series of Pb > Cu > Zn >>Fe. The binding capacity of the Spirulina for each of the metals was relatively low when compared to a range of other biosorbents. The toxicity thresholds of the algae was determined for copper and zinc. These were low (10umoles/g) and as such, the algae were not suitable for application in a treatment system in which they came into direct contact with the toxic metals. The algae were able to increase the pH of the surrounding medium. This occurred as a result of the accumulation of inorganic carbon, from bicarbonate, as a response to low concentrations of carbon dioxide in the medium. The resulting release of a hydroxide ion into solution led to the increase in pH. The increase in pH was shown to be due to a reduction in acidity, rather than an increase in alkalinity. The enzyme carbonic anhydrase was shown to be pivotal in this system. Attempts to determine the enzyme activity directly were unsuccessful, due to the inherent inaccuracy of the assay system. An indirect method of determining enzyme activity, by measuring changes in the carbonate species equilibrium, was developed. Under optimal conditions Spirulina was able to reduce the acidity by an amount equivalent to the addition of 3670umoles NaOH g·' h·'. Predictive modelling showed that this enhanced the potential of the medium to effect metal precipitation. For the algal system to be sustainable, a readily available source of bicarbonate was needed. This was achieved by the oxidation of organic carbon, under sulphidogenic conditions, by a bacterial consortium isolated from the anaerobic component of a facultative pond. The consortium was shown to consist of sulphate reducing (most likely Desulvovibrio and Desulfotomaculum)and acetogenic bacteria. Sulphate removal rates of 500mg 1·' day·' and 135mg 1·' day·' were achieved in a 21 agitated and 281 upflow reactor respectively. The bicarbonate generation rate in the 281 reactor was calculated as 4033umoles 1·' day·', which proved sufficient to act as a feed for the algal system. Sparging the anaerobic digester overflow with air and nitrogen resulted in a reduction in the aqueous sulphide concentration. Using nitrogen, a 70% recovery of sulphide, as H2S gas, was achieved in 60 minutes, while with air, this dropped to 40%, due to the oxidation of the aqueous sulphide. The stripping ofH2S resulted in an increase in pH. The H2S gas was used for the selective precipitation of copper and lead in the integrated system. The dynamics of metal precipitation was investigated. For simple reactions, between individual IV metal and base species, it was possible to generate an accurate predictive model and confirm the precipitating species using wavelength dispersive X-ray spectroscopy (WDS). In more complex systems, where precipitation of the artificial acid mine drainage was examined, the predictive modelling and WDS could not accurately describe the system. The addition of aqueous sulphide to copper and iron resulted in the formation of metastable, amorphous precipitates, which remained in suspension. Ageing of the copper precipitate resulted in the evolution of a stable crystalline structure (covellite) and the aggregation and settling of the precipitate. In the case of iron, the amorphous precipitate underwent oxidation before a stable iron sulphide could evolve and the settled precipitate was an iron oxide or oxyhydroxide. The artificial acid mine drainage was treated with sulphide, hydroxide, anaerobic digester overflow and algal overflow. The best metal removal was achieved with the sulphide and hydroxide, while the algal overflow outperformed the anaerobic digester overflow. The precipitate generated by the addition of sulphide was the most compact, followed by the algal overflow, the anaerobic digester overflow and the hydroxide. Efficient precipitation of all the heavy metals, except manganese, was achieved using the algal overflow at an acidity to alkalinity ratio of 1 :2. This ratio was selected for use in the pilot system. The Spirulina based pilot system was effectively used to treat an effluent from the Black Mountain base metal mine. The necessity to maintain the algae in suspension and avoid biomass washout were practical considerations which counted against this system. The replacement of the Spirulina by Oscillatoria, which adhered to a solid support, overcame these problems. The integrated biological system was able to effectively treat an artificial acid mine drainage for 90 days, reducing the concentration of all metals, except manganese, to below the acceptable environmental risk levels. The treatment of the final effluent in a second anaerobic digester reduced the manganese concentration to 4.5uM and proved that the sulphate reducing bacteria could be cultivated on enriched, partially treated acid mine drainage. The integrated biological treatment system performed well, effectively treating an effluent modelled closely on the quality of the water being discharged from the East Rand Basin. The cost of such a system would be considerably less than a "high tech" physico-chemical system. This, coupled with the potential long term sustainability of a biological system, would make it a potentially attractive option for the treatment of future acid mine drainage discharges.
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- Date Issued: 2002
The independent high rate algal pond as a unit operation in tertiary wastewater treatment
- Authors: Clark, Stewart James
- Date: 2002
- Subjects: Algae -- Biotechnology , Sewage -- Purification -- Biological treatment
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4092 , http://hdl.handle.net/10962/d1007805
- Description: The development of the High Rate Algal Pond (HRAP) as an independent tertiary treatment unit operation for phosphate and nitrate removal is reported. A novel Integrated Algal Ponding System (lAPS) design is proposed for nutrient removal from the effluents of both a conventional domestic sewage treatment plant and from an Advanced Integrated Wastewater Ponding System (AIWPS). The viability of an independently operated HRAP has been identified and termed the Independent High Rate Algal Pond (l-HRAP). A 500 m² pilot 1- HRAP was operated in such a way as to facilitate the precipitation of calcium phosphate, known to be controlled by pH (greater than 9.4) and resulting in final phosphate levels of less than 1 mg.L⁻¹ as P0₄-P. The incorporation of the I-HRAP into a denitrification process was also investigated. Continuously fed column reactors, utilising algal biomass as a carbon source, showed that the heterotrophic bacterial community dominant in the anaerobic algal sludge were denitrifying the nitrate in the feed. It was demonstrated that as the cultures were stressed (using increased nitrate concentrations, anaerobiosis and light starvation) total polysaccharide (TPS) concentrations increased, with a notable increase 111 the exopolysaccharide (EPS) fraction. These experiments corroborated the hypothesis that harvested microalgal biomass can be manipulated to produce, and release, exopolymeric substances under stress conditions, and which may serve as carbon source for denitrification. In both batch flask studies and in laboratory-scale reactor systems, harvested microalgal biomass from an HRAP was shown to produce exopolymeric substances under stress conditions. Initial high loading-rates of greater than 20 mg.L⁻¹ NO₃-N resulted in double the amount of exopolysaccharide production than in flasks with initial low loading-rates (less than 5 mg.L⁻¹ NO₃-N). Making use of an upflow anaerobic sludge blanket-type degrading-bed reactor, and an anaerobic, flooded trickle filter (ANTRIC) receiving HRAP effluent, the relationship between denitrification and the changes in polysaccharide content was investigated. This phenomenon has considerable beneficial implications in biological wastewater treatment systems where high nitrate concentration in the final effluent is a potential mitigating factor. Identification of the heterotrophic bacteria active in the denitrification process was attempted. This study presents a first report on the development and operation of the I-HRAP and has been followed by a technical-scale pilot plant evaluation of the process in the tertiary treatment of domestic wastewaters.
- Full Text:
- Date Issued: 2002
Thermophilic lignin degrading enzymes from actinomycetes for biotechnological applications
- Authors: Mhlanga, Chido Yvonne Lois
- Date: 2002 , 2013-05-16
- Subjects: Actinomycetales -- Biotechnology , Lignin
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4085 , http://hdl.handle.net/10962/d1007628 , Actinomycetales -- Biotechnology , Lignin
- Description: Phenolic residues which accumulate in the environment as a result of agro-industrial practices has resulted in the need to find and use Eco-Friendly techniques, rather than the traditional methods of burning or burying this kind of waste. Bioremediation and bioconversion are attractive alternatives using whole cell or enzyme-based systems. The aims of this project were to isolate and uses thermophilic Actinomycetes, which produce thermo-tolerant oxidoreductase enzymes, which can be used to bioconvert a model industrial phenolic waste commonly genersated in the wine-making industry of South Africa. Current research in bioconversion and bioremediation focuses on mesophilic microbes in that their enzymes can catalyse reactions at higher temperatures without affecting its activity and lower contamination levels. Three novel Actinomycete isolates were isolated (RU-A0l , RU-A03 and RU-A06) from a compost site and characterized using a combination of conventional identification techniques and 16S rDNA methodology to identity the three isolates. All three isolates belong to the Streptomyces clade. In addition, five known Actinomycetes were selected from an internation culture collection and also screened for oxidoreductase activity in comparision to the three novel isolates. Although the five isolates were selected based on their ability to produce oxidoreductase enzymes, unexpectedly, no activity was detected. Screening assays for peroxidase, polyphenol oxidase and laccase on RU-AO 1, RU-A03 and RU-A06, showed that all three isolated produced peroxidases and peroxidases but no laccase. Substrate specificity studies revealed that the most suitable substrates to determine peroxidase and polyphenol oxidase activity on these isolates were catechol for polyphenol oxidase, 2,4-dichlorophenol for peroxidases and veratryl alcohol for lignin peroxidases. Previous studies have indicated that peroxidases and polyphenol oxidases are produced in Actinomycetes during the primary stage of growth. This was the case with RU-AOI , RU-A03 and RU-A06. Growth rates were higher that other Actinomycetes, with maxImum biomass being reached at 36 hours for the isolates RU-AOI and RU-A06 and 48 hours for isolate RUA03. pH studies showed that the three isolates were adaptable and could grow over a broad pH range. Catabolism studies of phenolic model compounds showed that the three isolates were capable of catabolizing the model phenolic compounds within a period of 24 hours. Further studies were carried out to determine the effect of these microbes and their enzymes in whole cell and enzyme-based systems on a model phenolic waste, graoe waste consisting of compressed grape skins, pips and stalks. Whole cell studies showed that the isolates were capable of bioconverting the waste at a maximum concentration of 30% grape waste (vol:vol). Peroxidase and polyphenol oxidase activity increased indicating induction of these enzymes in the presence of phenolic compounds, with a maximum increase of up to 15.9 fold increase in extracellular lignin peroxidase activity in RU-AO1. HPLC and phenolic determination assays indicated that bioconversion of the phenolic grape waste had occurred in the presence of the three isolates. Attempts were made to isolate and identify a peroxidase or phenol oxidase gene from one the isolates. As bacteria, Actinomycetes are amendable to gene manipulation making them suitable candidates for methods such as site directed evolution in comparison to fungi. Two clones were selected for sequencing based on positive activity results when assayed for peroxidase activity. However the resultant sequences did not identify a functional gene sequence. Southern Blotting was then carried out to determine the nature of the peroxidase gene. Previous studies have been focused on the catalase-peroxidase gene (CalC gene) found Actinomycetes and other bacteria. A probe was developed from the CalC gene. No hybridization occurred with any of the enzyme restricted DNA from the three isolates. The implications of these results are that the peroxidase genets in the three isolates are in fact lignin peroxidase in nature. This project has the potential in the bioconversion of phenolic wastes and is the first description of the use of thermophilic Actinomycetes in the bioconversion of an industrial phenolic waste.
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- Date Issued: 2002
The Rhodes BioSure process in the treatment of acid mine drainage wastewaters
- Authors: Corbett, Christopher John
- Date: 2001 , 2013-05-03
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4077 , http://hdl.handle.net/10962/d1007405
- Description: While sulphate-enriched wastewaters are generated in a number of industrial processes, such as tanning, paper manufacture and metals processing, the principal contributors to large-scale pollution from this source in South Africa are the gold and coal mining industries. Both biological and physico-chemical processes, set in train by mining operations, give rise to the oxidation of sulphur species, and the resultant generation of AMD. The Vaal River system is most affected and receives large tonnages of mining related salinity as both direct discharges, and in diffuse runoff flows. The long-term burden of this problem, and sustaining ongoing treatment over the time-frames involved will almost certainly resort to the community inhabiting the area, notwithstanding progressive mine closure legislation and comprehensive regulation governing the polluterpays principle. The volume and time-frame of the AMD problem, and the need for a long-term and sustainable response has focused interest in biological treatment approaches. These have concentrated on active and passive treatment systems, both of which rely on microbial activity related to the biological sulphur cycle. Notwithstanding the reactor type, and the particular treatment approach used, widespread application of active AMD treatment has not yet been seen on any large scale. Singular factors constraining process development are bioreactor design, cost of bioreactor construction, and the cost of the carbon source and electron donor for the biological sulphate reduction process. The SRB are able to utilise only a limited range of small organic molecules. The studies reported here were motivated by the need to evaluate low-cost options and the treatment of high volume AMD flows. This has focussed research activity on bioprocess developments using complex organic compounds derived from waste streams as electron donor sources, and the integration of AMD treatment with other waste treatment objectives. The co-disposal of organic wastes with AMD treatment would enable the development of an 'integrated resource management' approach to the problem, including sustainability of treatment operations over the long time-frames involved. Apart from the cost advantages accrued to waste treatment, the recovery of the treated water as a resource to the wider community provides a potentially important value-added function to the combined operation. , KMBT_363 , Adobe Acrobat 9.53 Paper Capture Plug-in
- Full Text:
- Date Issued: 2001
Capillary membrane-immobilised polyphenol oxidase and the bioremediation of industrial phenolic effluent
- Authors: Edwards, Wade
- Date: 1999
- Subjects: Membranes (Technology) , Effluent quality , Pollutants , Phenols , Water -- Purification
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4095 , http://hdl.handle.net/10962/d1008458
- Description: Waste-generating industrialisation is intrinsically associated with population and economic proliferation. This places considerable emphasis on South Africa's water shortage due to the integral relationship between population growth rate and infrastructure development. Of the various types of industry-generated effluents, those containing organic pollutants such as phenols are generally difficult to remediate. Much work has been reported in the literature on the use of enzymes for the removal of phenols from these waste-streams but little application of this bioremediation approach has reached practical fruition. This study focuses on integrating and synergistically combining the advantages of enzyme-mediated dephenolisation of synthetic and industrial effluent with that of membrane teclmology. The ability of the enzyme polyphenol oxidase to convert phenol and a number of its derivatives to chemically reactive o-quinones has been reported extensively in the literature. These o-quinones can then physically be removed from solution using various precipitation or adsorption techniques. The enzyme is, however, plagued by a product-induced phenomenon known as suicide inactivation, which renders it inactive and thus limits its application as a bioremediation tool. Integrating membrane technology with the enzyme's catalytic ability by immobilising polyphenol oxidase onto polysulphone and poly(ether sulphone) capillary membranes enabled the physical removal of these inhibitory products from the micro-environment of the immobilised enzyme which therefore increased the phenol conversion capability of the immobilised biocatalyst. Under non-immobilised conditions it was found that when exposed to a mixture of various phenols the substrate preference of the enzyme is a function of the R-group. Under immobilised conditions, however, the substrate preference of the enzyme becomes a function of certain transport constraints imposed by the capillary membrane itself. Furthermore, by integrating a quinone-removal process in the enzyme-immobilised bioreactor configuration, a 21-fold increase in the amount of substrate converted per Unit enzyme was observed when compared to the conversion capacity of the inunobilised enzyme without the product removal step. Comparisons were also made using different membrane bioreactor configurations (orientating the capillaries transverse as opposed to parallel to the module axis) and different immobilisation matrices (poly(ether sulphone) and polysulphone capillary membranes). Conversion efficiencies as high as 77% were maintained for several hours using the combination of transverse-flow modules and novel polysulphone capillary membranes. It was therefore concluded that immobilisation of polyphenol oxidase on capillary membranes does indeed show considerable potential for future development.
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- Date Issued: 1999
The development of an immobilised-enzyme bioprobe for the detection of phenolic pollutants in water
- Authors: Russell, Ingrid Margaret
- Date: 1999
- Subjects: Pollutants -- Biodegradation , Pollutants , Chemical reactors , Membrane reactors , Fungi -- Biotechnology
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4069 , http://hdl.handle.net/10962/d1006211 , Pollutants -- Biodegradation , Pollutants , Chemical reactors , Membrane reactors , Fungi -- Biotechnology
- Description: The possibility of developing an immobilised-enzyme bioprobe, based on mushroom polyphenol oxidase, for the purely biological detection and quantification of phenolic pollutants in water was investigated. Polyphenol oxidase catalyses the bioconversion of many phenolic compounds into quinone-related coloured products. Thus, in an immobilised form, the enzyme serves as a visible indicator of the presence and concentration of phenolic pollutants in water. The objective of this research was to develop a portable, disposable bioprobe incorporating polyphenol oxidase for this purpose. The intensity of the colour changes produced by the enzyme on reaction with p-cresol, p-chlorophenol and phenol was found to increase proportionally with increasing concentrations of these substrates in solution. Immobilisation of the enzyme on various supports did not appear to significantly affect the catalytic activity of the enzyme. The enzyme was immobilised by adsorption and cross-linking on polyethersulphone, nitrocellulose and nylon membranes with the production of various colour ranges on reaction with the phenolic substrates. The most successful immobilisation of the enzyme, in terms of quantity and distribution of enzyme immobilised and colour production, was obtained with the enzyme immobilised by adsorption on nylon membranes in the presence of 3-methyl-2-benzothiazolinone hydrazone (MBTH). The enzyme, immobilised using this method, produced ranges of maroon colours in phenolic solutions and orange colours in cresylic solutions. The colour intensities produced were found to increase proportionally with increasing substrate concentration after 5 minutes exposure to the substrates. The bioprobe had a broad substrate specificity and was sensitive to substrate concentrations down to 0.05 mg/L. The enzyme activity of the bioprobe was not significantly affected in a pH range from 4 to 10 and in a temperature range from 5-25⁰C. The bioprobe activity was not affected by various concentrations of salt and metal ions and the bioprobe was able to detect and semi-quantify phenolic substrates in industrial effluent samples. These features of the bioprobe indicate that the commercialisation of such a bioprobe is feasible and this technology has been patented (Patent No. SA 97/0227). , KMBT_363 , Adobe Acrobat 9.54 Paper Capture Plug-in
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- Date Issued: 1999
Preliminary investigation of the molecular pathogenicity determinants of Xanthomonas campestris pv. zeae
- Authors: Downing, T G
- Date: 1998
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4066 , http://hdl.handle.net/10962/d1004922
- Description: Xanthomonas campestris pv. zeae was shown to posses a vast range of potential plant cell wall degrading enzymes including at least one protease, a carboxymethylcellulase, pectin lyase, polygalacturonase and a B-endoglucanase. Replicon stability and transfer system efficiencies were determined for a range of oriC, and oriT -tra combinations, and suitable vectors were constructed and identified for insertional inactivation by homologous recombination. Ideal suicide replicons were found to be pACYC184 and p15A, while P, Wand Q group replicons were supported by X campestris pv. zeae. Group P and group N transfer systems were shown to be highly efficient in intra-genus matings between Escherichia coli and X campestris pv. zeae, with the exception of P-tra systems in trans for the delivery of Tn5. Various cloning vectors were tested for stability and mobility. Tn5 was shown to transpose at high frequencies into the genome of the bacterial plant pathogen, and insertion was relatively random. Suitable screening assays were established to allow rapid isolation of mutants with potential virulence or pathogenic deviations, after mutagenesis. Two non-pathogenic mutants were identified, one of which was a putative hrp·, while the other was a leaky virulence. A single mutant showing 40% reduced protease activity was also shown to exhibit reduced virulence indicating a minor role for the protease in pathogenicity. The majority of virulence mutants showed altered growth in different levels of nutritional availability and complexity. Nutritional viability (the ability to acquire and use nutrients at a sufficient rate to grow fast enough to overcome host defences) was shown to be essential for virulence and possibly pathogenicity. Wild-type in-planta behaviour was analysed and growth and spread patterns typical for pathogenic response identified. Chief amongst these was the requirement for a threshold level of cells per leaf area or length, before symptoms could develop. Occlusion of vascular bundles was shown not to be the primary factor in the pathogenicity of X campestris pv. zeae. Threshold levels for lesion development indicate the absence of a diffusable lesion forming element, and possibly the requirement of cell density for induction of certain functions. , KMBT_363
- Full Text:
- Date Issued: 1998
An investigation into the potential immunogenicity of various extracts of the South African bont tick Amblyomma hebraeum
- Authors: Adamson, Deborah Jane
- Date: 1993
- Subjects: Amblyomma -- South Africa , Ticks -- South Africa , Ticks -- Control -- South Africa
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4127 , http://hdl.handle.net/10962/d1015640
- Description: Rabbits and goats were inoculated with crude, membrane-associated and soluble components extracted from unengorged adult females and nymphs of the bont tick Amblyomma hebraeum. Inoculation provided some protection against nymphal infestation, however it had little effect on adult feeding. Histological examination of adults fed on inoculated hosts showed evidence of gut damage. Skin provocation testing with tick extracts elicited a Type I immediate hypersensitivity which was influenced by antihistamine. A delayed skin reaction was also evident. Whether this was attributable to Type III Arthus reaction or Type IV cell-mediated hypersensitivity was not determined. A comparative histological study of sites of tick extract injection, on inoculated and naive hosts, demonstrated the role of eosinophils in the hosts response to tick feeding. Serological examination revealed elevated anti-A hebraeum lgG titres following inoculation. These titres were found to decrease in the ten weeks after inoculation, despite the hosts being repeatedly infested with A hebraeum. Although the IgG titres of naive control hosts increased after each tick infestation, they failed to reach the titres achieved through inoculation. Western blot analysis of serum from inoculated hosts recognized most of the A. hebraeum proteins against which it was screened.
- Full Text:
- Date Issued: 1993