Biosulphidogenic hydrolysis of lignin and lignin model compounds
- Authors: Madikane, Mzekelo
- Date: 2002
- Subjects: Lignin Lignin -- Biodegradation
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3917 , http://hdl.handle.net/10962/d1003976
- Description: Lignin degradation under biosulphidogenic conditions has not been extensively reported in the literature. Although aerobic degradation of lignin is well documented, anaerobic biodegradation has focused mainly on methanogenic systems with biosulphidogenic systems receiving less attention. Sulphate reducing bacteria are known to generate moderately high levels of both sulphide and alkalinity at room temperatures, and these conditions draw some comparison with the Kraft pulping process. In the Kraft pulping process, lignin is degraded chemically at ±170°C under high sulphide and alkaline conditions and may provide a model for understanding biosulphidogenic lignin degrading activity. The aim of this study was to investigate the biosulphidogenic hydrolysis of lignin within the context of the chemical and biological conditions generated by a mixed sulphate reducing bacteria consortia. Bioreactor studies with a mixed sulphate reducing consortia and pine wood powder (both untreated and depectinated) resulted in the generation of comparable levels of sulphide and alkalinity used in the chemical hydrolysis studies. Aromatic compound yields were between 20 to 50% of the chemical hydrolysis studies. This fluctuation may have been due to the utilization of these aromatic compounds as electron donors by the sulphate reducing consortia as evidenced by the high rate of sulphate reduction in both the untreated and depectinated wood bioreactors. Biodegradation of lignin model compounds was investigated in order to elucidate lignin degradation mechanisms. Both mono-aromatic and dimeric lignin model compounds were used as electron donors and carbon sources for the mixed sulphate reducing consortia. Biodegradation and mass spectrometer analysis of mono-aromatic compounds, ferulic acid and ferulic acid ethyl ester resulted in the production of intermediates such as catechol, cyclohexane carboxylic acid and adipic acid. These intermediates were also observed in the degradation of dimeric ferulic acid. Biodegradation of salicin resulted in the production of salicyl alcohol, ortho-cresol and acetate. Biodegradation of benzylic ether resulted in the production of vanillin and acetate as end products. The results of these studies provide evidence for a biosulphidogenic hydrolysis of lignin, and also the utilisation of lignin-derived aromatic compounds as electron donor sources, by a mixed sulphate reducing consortia.
- Full Text:
- Date Issued: 2002
- Authors: Madikane, Mzekelo
- Date: 2002
- Subjects: Lignin Lignin -- Biodegradation
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3917 , http://hdl.handle.net/10962/d1003976
- Description: Lignin degradation under biosulphidogenic conditions has not been extensively reported in the literature. Although aerobic degradation of lignin is well documented, anaerobic biodegradation has focused mainly on methanogenic systems with biosulphidogenic systems receiving less attention. Sulphate reducing bacteria are known to generate moderately high levels of both sulphide and alkalinity at room temperatures, and these conditions draw some comparison with the Kraft pulping process. In the Kraft pulping process, lignin is degraded chemically at ±170°C under high sulphide and alkaline conditions and may provide a model for understanding biosulphidogenic lignin degrading activity. The aim of this study was to investigate the biosulphidogenic hydrolysis of lignin within the context of the chemical and biological conditions generated by a mixed sulphate reducing bacteria consortia. Bioreactor studies with a mixed sulphate reducing consortia and pine wood powder (both untreated and depectinated) resulted in the generation of comparable levels of sulphide and alkalinity used in the chemical hydrolysis studies. Aromatic compound yields were between 20 to 50% of the chemical hydrolysis studies. This fluctuation may have been due to the utilization of these aromatic compounds as electron donors by the sulphate reducing consortia as evidenced by the high rate of sulphate reduction in both the untreated and depectinated wood bioreactors. Biodegradation of lignin model compounds was investigated in order to elucidate lignin degradation mechanisms. Both mono-aromatic and dimeric lignin model compounds were used as electron donors and carbon sources for the mixed sulphate reducing consortia. Biodegradation and mass spectrometer analysis of mono-aromatic compounds, ferulic acid and ferulic acid ethyl ester resulted in the production of intermediates such as catechol, cyclohexane carboxylic acid and adipic acid. These intermediates were also observed in the degradation of dimeric ferulic acid. Biodegradation of salicin resulted in the production of salicyl alcohol, ortho-cresol and acetate. Biodegradation of benzylic ether resulted in the production of vanillin and acetate as end products. The results of these studies provide evidence for a biosulphidogenic hydrolysis of lignin, and also the utilisation of lignin-derived aromatic compounds as electron donor sources, by a mixed sulphate reducing consortia.
- Full Text:
- Date Issued: 2002
Elucidation and manipulation of the Hydantoin-Hydrolysing Enzyme System of Agrobacterium tumefaciens RU-OR for the Biocatalytic production of D-amino acids
- Authors: Hartley, Carol Janet
- Date: 2002
- Subjects: Amino acids Agrobacterium tumefaciens
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3916 , http://hdl.handle.net/10962/d1003975
- Description: There is widespread interest in the biocatalytic production of enantiomerically pure D-amino acids for use in the synthesis of antibiotics, insecticides, herbicides, drug carriers and many other pharmaceuticals. Hydantoin-hydrolysing enzyme systems can be successfully utilised to stereoselectively convert racemic hydantoins into enantiomerically pure amino acid products. In fact, the use of microbial D-hydantoinase and D-stereoselective N-carbamoyl amino acid amidohydrolase activity to produce D-p-hydroxyphenylglycine from D,L-5-phydroxyphenylhydantoin has been described as one of the most successful biotechnological applications of enzyme technology developed to date. A need to utilise the novel biodiversity of South African microorganisms for the development of an indigenous process to produce enantiomerically pure amino acids was identified in 1995. Subsequently, the Rhodes Hydantoinase Group was established and several local hydantoin-hydrolysing microorganisms were isolated. The research in this study describes the isolation and selection of Agrobacterium tumefaciens RU-OR, which produced D-stereoselective hydantoinhydrolysing activity. Characterisation of the hydantoin-hydrolysing enzyme system of RU-OR revealed novel biocatalytic properties, and potential for the application of this strain for the biocatalytic production of D-amino acids. A fundamental understanding of the regulation of hydantoin-hydrolysing enzyme activity in A. tumefaciens RU-OR was established, and utilised to produce mutant strains with altered regulation of hydantoin-hydrolysing activity. These strains were used to further elucidate the mechanisms regulating the production of hydantoins-hydrolysing activity in A. tumefaciens RU-OR cells. Overproduction of hydantoinase and N-carbamoyl-D-amino acid amidohydrolase activity in selected mutant strains resulted in efficient conversion of D,L-5-p-hydroxyphenylhydantoin to D-p-hydroxyphenylglycine. Thus the establishment of a primary understanding of the hydantoin-hydrolysing enzyme system in A. tumefaciens RU-OR could be used to manipulate the hydantoin-hydrolysing activity in RU-OR cells to produce an improved biocatalyst. The isolation of A. tumfecaiens RU-OR genes encoding for hydantoin-hydrolysing activity revealed two separate N-carbamoyl-D-amino acid amidohydrolaseencoding genes (ncaR1 and ncaR2) in this bacterium with distinct chromosomal locations, nucleotide coding sequence and predicted primary amino acid sequence. The novel biocatalytic properties of the hydantoin-hydrolysing enzyme system in A. tumefaciens RU-OR and mutant derivatives present fascinating opportunities for further elucidation of the natural function, regulation and biocatalytic potential of hydantoin-hydrolysing enzymes.
- Full Text:
- Date Issued: 2002
- Authors: Hartley, Carol Janet
- Date: 2002
- Subjects: Amino acids Agrobacterium tumefaciens
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3916 , http://hdl.handle.net/10962/d1003975
- Description: There is widespread interest in the biocatalytic production of enantiomerically pure D-amino acids for use in the synthesis of antibiotics, insecticides, herbicides, drug carriers and many other pharmaceuticals. Hydantoin-hydrolysing enzyme systems can be successfully utilised to stereoselectively convert racemic hydantoins into enantiomerically pure amino acid products. In fact, the use of microbial D-hydantoinase and D-stereoselective N-carbamoyl amino acid amidohydrolase activity to produce D-p-hydroxyphenylglycine from D,L-5-phydroxyphenylhydantoin has been described as one of the most successful biotechnological applications of enzyme technology developed to date. A need to utilise the novel biodiversity of South African microorganisms for the development of an indigenous process to produce enantiomerically pure amino acids was identified in 1995. Subsequently, the Rhodes Hydantoinase Group was established and several local hydantoin-hydrolysing microorganisms were isolated. The research in this study describes the isolation and selection of Agrobacterium tumefaciens RU-OR, which produced D-stereoselective hydantoinhydrolysing activity. Characterisation of the hydantoin-hydrolysing enzyme system of RU-OR revealed novel biocatalytic properties, and potential for the application of this strain for the biocatalytic production of D-amino acids. A fundamental understanding of the regulation of hydantoin-hydrolysing enzyme activity in A. tumefaciens RU-OR was established, and utilised to produce mutant strains with altered regulation of hydantoin-hydrolysing activity. These strains were used to further elucidate the mechanisms regulating the production of hydantoins-hydrolysing activity in A. tumefaciens RU-OR cells. Overproduction of hydantoinase and N-carbamoyl-D-amino acid amidohydrolase activity in selected mutant strains resulted in efficient conversion of D,L-5-p-hydroxyphenylhydantoin to D-p-hydroxyphenylglycine. Thus the establishment of a primary understanding of the hydantoin-hydrolysing enzyme system in A. tumefaciens RU-OR could be used to manipulate the hydantoin-hydrolysing activity in RU-OR cells to produce an improved biocatalyst. The isolation of A. tumfecaiens RU-OR genes encoding for hydantoin-hydrolysing activity revealed two separate N-carbamoyl-D-amino acid amidohydrolaseencoding genes (ncaR1 and ncaR2) in this bacterium with distinct chromosomal locations, nucleotide coding sequence and predicted primary amino acid sequence. The novel biocatalytic properties of the hydantoin-hydrolysing enzyme system in A. tumefaciens RU-OR and mutant derivatives present fascinating opportunities for further elucidation of the natural function, regulation and biocatalytic potential of hydantoin-hydrolysing enzymes.
- Full Text:
- Date Issued: 2002
Genetic characterization of conspecific populations of Tilapia Sparrmanii (A.Smith 1840) in the dolomitic sinkholes and springs of the North-West Province (South Africa), and their comparison to Tilapia Guinasana (Trewavas 1936)
- Authors: Nxomani, Clifford David
- Date: 2002
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4079 , http://hdl.handle.net/10962/d1007452
- Description: This study was undertaken to investigate the genetic relationships of allopatric populations of the cichlid fish, Tilapia sparrmanii (A. Smith 1840) inhabiting the sinkholes and springs of the North West Province, South Africa. It also examined the genetic relationships of T sparrmanii to its polychromatic sister species, Tilapia guinasana (Trewavas 1936) which is endemic to the Guinas sinkhole in Namibia. Finally, the study investigated whether there is a genetic basis for T guinasana's colour polymorphism. The research was prompted by the concern of conservation authorities about the possible loss of unique fauna given the high demand for use of the subterranean waters for agricultural, domestic and industrial purposes. Such demands have the potential to drain these habitats. Further concerns related to habitat destruction and the introduction of alien species in the ecosystems inhabited by both fish species. Three approaches were adopted in attempting to answer the above questions. First was the investigation of Sodium dodecylsulphate (SDS)-Polyacrylamide Gel Electrophoresis (PAGE) of total cellular proteins as a fast and relatively inexpensive indicator of genetic relatedness between the fish populations. Secondly, genetic differentiation between the T sparrmanii populations and its relationship to T guinasana were assayed using restriction endonuclease analysis of Polymerase Chain Reaction (PCR)-amplified regions of the cytochrome b gene and the d-Ioop of mitochondrial DNA, coupled with Temperature Gradient Gel Electrophoresis (TGGE) analysis of the same regions. The third approach involved the use of Random Amplified Polymorphic DNA (RAPD) fingerprinting of the populations ofT sparrmanii as an indicator of genetic differentiation between them. RAPD fingerprinting was further used to investigate the genetic relationships between T sparrmanii and T guinasana and to probe the genetic basis of the polychromatism of the latter. SDS-PAGE did not reveal any genetic differentiation between the T sparrmanii populations, nor could the analysis detect variation within them. It however clearly distinguished at a species level between T sparrmanii and T guinasana as well as between these and other fish species, thus indicating its possible utility as an indicator of genetic relatedness at a species level. Mitochondrial studies employing the Restriction Fragment Length Polymorphism (RFLP) of Polymerase Chain Reaction (PCR)-amplified cytochrome b (1.1 kb) and d-Ioop regions (0.9 kb) with six and five restriction enzymes respectively, failed to reveal genetic differences within and between the allopatric populations. TGGE of500 bp of the d-Ioop and 400 bp of the 12sRNA PCR-amplified fragments did not reveal any differences between the populations of T. sparrmanii, nor did the analysis reveal any differences between T. sparrmanii and T. guinasana. The lack of differentiation between the T. sparrmanii populations by these mitochondrial Dna analysis techniques, despite habitat fragmentation, indicated a recent origin of the populations from a common ancestral population. Failure to distinguish between T. sparrmanii and T. guinasana may be related to the sensitivity of the techniques utilized. RAPD fingerprinting analysis indicated that the populations are genetically differentiated from each other. Using a measure of coefficient of variation, the population with the highest variation was the Wondergat population (13.99%), followed by the Klerkskraal popUlation (8.29%), the Malmani and Marico Oog populations (each with 5.88%) and the least variation (4.95 and 4.83%) was with the Amalinda and Molopo Oog populations respectively. This high degree of intra population similarity points to the fact that this differentiation is still confined within the limits of con specificity. The genetic distances between all of the T. sparrmanii populations across all primers ranged from 0.09 to 0.234 and averaged 0.146, a value that falls in the upper end of conspecific population differentiation. Such results indicate populational sub-division below the species level. RAPD fingerprinting therefore proved more sensitive than protein or mitochondrial studies. The differentiation it detected between the populations is a reflection of their adaptation to local conditions of the unique ecosystems they inhabit. A comparison with a subset of primers between T. guinasana and T. sparrmanii confirmed the separate species status of the former from the latter. The mean genetic distance between the T. sparrmanii populations was 0.136, compared to that between T. sparrmanii and T. guinasana which was found to be 0.374. Statistical analysis of the difference between the mean genetic distances indicated significance with 95% confidence. The polychromatism of T guinasana was investigated to determine whether there were significant differences between its five colour morphs. RAPD fingerprinting indicated with 95% confidence that there were significant differences between the colour forms based on the genetic distances computed between them. These genetic differences appeared to correlate with the observed assortative mating between the colour forms of the species. The manifestation of the polychromatism at sexual maturity in T guinasana probably indicates that colouration plays an important role in the breeding process. The genetic uniqueness shown here between the populations of T sparrmanii and the colour forms of T guinasana indicate for protective measures to be put in place if the genetic resources of the isolated fish populations are to be preserved. These must be coupled with a thorough assessment of the temporal and spatial distribution of genetic variability of the populations as a guide to a long-term management strategy for the fish populations and the ecosystems they inhabit. This study therefore has shown that the allopatric populations of T sparrmanii in the sinkholes and springs of the North-West Province are genetically unique, as well as show that the colour forms of T guinasana are genetically distinct.
- Full Text:
- Date Issued: 2002
- Authors: Nxomani, Clifford David
- Date: 2002
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4079 , http://hdl.handle.net/10962/d1007452
- Description: This study was undertaken to investigate the genetic relationships of allopatric populations of the cichlid fish, Tilapia sparrmanii (A. Smith 1840) inhabiting the sinkholes and springs of the North West Province, South Africa. It also examined the genetic relationships of T sparrmanii to its polychromatic sister species, Tilapia guinasana (Trewavas 1936) which is endemic to the Guinas sinkhole in Namibia. Finally, the study investigated whether there is a genetic basis for T guinasana's colour polymorphism. The research was prompted by the concern of conservation authorities about the possible loss of unique fauna given the high demand for use of the subterranean waters for agricultural, domestic and industrial purposes. Such demands have the potential to drain these habitats. Further concerns related to habitat destruction and the introduction of alien species in the ecosystems inhabited by both fish species. Three approaches were adopted in attempting to answer the above questions. First was the investigation of Sodium dodecylsulphate (SDS)-Polyacrylamide Gel Electrophoresis (PAGE) of total cellular proteins as a fast and relatively inexpensive indicator of genetic relatedness between the fish populations. Secondly, genetic differentiation between the T sparrmanii populations and its relationship to T guinasana were assayed using restriction endonuclease analysis of Polymerase Chain Reaction (PCR)-amplified regions of the cytochrome b gene and the d-Ioop of mitochondrial DNA, coupled with Temperature Gradient Gel Electrophoresis (TGGE) analysis of the same regions. The third approach involved the use of Random Amplified Polymorphic DNA (RAPD) fingerprinting of the populations ofT sparrmanii as an indicator of genetic differentiation between them. RAPD fingerprinting was further used to investigate the genetic relationships between T sparrmanii and T guinasana and to probe the genetic basis of the polychromatism of the latter. SDS-PAGE did not reveal any genetic differentiation between the T sparrmanii populations, nor could the analysis detect variation within them. It however clearly distinguished at a species level between T sparrmanii and T guinasana as well as between these and other fish species, thus indicating its possible utility as an indicator of genetic relatedness at a species level. Mitochondrial studies employing the Restriction Fragment Length Polymorphism (RFLP) of Polymerase Chain Reaction (PCR)-amplified cytochrome b (1.1 kb) and d-Ioop regions (0.9 kb) with six and five restriction enzymes respectively, failed to reveal genetic differences within and between the allopatric populations. TGGE of500 bp of the d-Ioop and 400 bp of the 12sRNA PCR-amplified fragments did not reveal any differences between the populations of T. sparrmanii, nor did the analysis reveal any differences between T. sparrmanii and T. guinasana. The lack of differentiation between the T. sparrmanii populations by these mitochondrial Dna analysis techniques, despite habitat fragmentation, indicated a recent origin of the populations from a common ancestral population. Failure to distinguish between T. sparrmanii and T. guinasana may be related to the sensitivity of the techniques utilized. RAPD fingerprinting analysis indicated that the populations are genetically differentiated from each other. Using a measure of coefficient of variation, the population with the highest variation was the Wondergat population (13.99%), followed by the Klerkskraal popUlation (8.29%), the Malmani and Marico Oog populations (each with 5.88%) and the least variation (4.95 and 4.83%) was with the Amalinda and Molopo Oog populations respectively. This high degree of intra population similarity points to the fact that this differentiation is still confined within the limits of con specificity. The genetic distances between all of the T. sparrmanii populations across all primers ranged from 0.09 to 0.234 and averaged 0.146, a value that falls in the upper end of conspecific population differentiation. Such results indicate populational sub-division below the species level. RAPD fingerprinting therefore proved more sensitive than protein or mitochondrial studies. The differentiation it detected between the populations is a reflection of their adaptation to local conditions of the unique ecosystems they inhabit. A comparison with a subset of primers between T. guinasana and T. sparrmanii confirmed the separate species status of the former from the latter. The mean genetic distance between the T. sparrmanii populations was 0.136, compared to that between T. sparrmanii and T. guinasana which was found to be 0.374. Statistical analysis of the difference between the mean genetic distances indicated significance with 95% confidence. The polychromatism of T guinasana was investigated to determine whether there were significant differences between its five colour morphs. RAPD fingerprinting indicated with 95% confidence that there were significant differences between the colour forms based on the genetic distances computed between them. These genetic differences appeared to correlate with the observed assortative mating between the colour forms of the species. The manifestation of the polychromatism at sexual maturity in T guinasana probably indicates that colouration plays an important role in the breeding process. The genetic uniqueness shown here between the populations of T sparrmanii and the colour forms of T guinasana indicate for protective measures to be put in place if the genetic resources of the isolated fish populations are to be preserved. These must be coupled with a thorough assessment of the temporal and spatial distribution of genetic variability of the populations as a guide to a long-term management strategy for the fish populations and the ecosystems they inhabit. This study therefore has shown that the allopatric populations of T sparrmanii in the sinkholes and springs of the North-West Province are genetically unique, as well as show that the colour forms of T guinasana are genetically distinct.
- Full Text:
- Date Issued: 2002
Heterologous expression of the helicoverpa armigera stunt virus in Saccharomyces cerevisiae
- Authors: Venter, Philip Arno
- Date: 2002
- Subjects: Helicoverpa armigera Saccharomyces cerevisiae
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3895 , http://hdl.handle.net/10962/d1003811
- Description: Lepidopteran insects like Helicoverpa armigera, more commonly known as the cotton bollworm, are economically important pests of a wide variety of crops throughout the world. The Helicoverpa armigera stunt virus (HaSV), a tetravirus with a bipartite single-stranded positive-sense RNA genome, has great potential as a biological pesticide against H. armigera. The larger genomic strand of this virus (RNA1) encodes the viral replicase, while the other (RNA2) encodes the 71 kDa capsid protein precursor (p71). 240 copies of p71 assemble into a procapsid with the concomitant encapsidation of the viral RNA. This is followed by a complex maturation event that is characterized by the autoproteolytic cleavage of p71 into the 64 kDa capsid protein (P64) and a 7 kDa peptide (p7). The rearrangements that occur during maturation results in the formation of mature HaSV capsids that can thereupon deliver RNA to other susceptible host cells. The principal objective of the research described in this study was to demonstrate that this virus could be assembled in Saccharomyces cerevisiae. S. cerevisiae expression vectors were constructed for the production of p71. This protein was detected in cell lysates from two different strains of S. cerevisiae, both containing either chromosomal or episomal copies of an expression cassette for P71. A number of factors relating to the expression of P71 (e.g. strains used, expression loci and expression rate) and the preparation of protein extracts from S. cerevisiae (e.g. the presence of various protease inhibitors and salt concentrations) were examined to attain optimal levels of soluble p71. A small fraction of the optimized soluble p71 was shown to be in the form of virus-like particles (VLPs), with a yield of ≤10⁷ VLPs from a 1.5l culture of P71⁺ cells. These particles were exclusively in the procapsid form, had a similar buoyant density to that of wild-type HaSV and could undergo maturation when the pH was reduced to 5. S. cerevisiae vectors were constructed for the episomal expression of the HaSV genomic RNAs. These vectors directed the transcription of RNA1 and RNA2 transcripts, which had similar sizes to those of the HaSV genomic RNAs. Mature HaSV particles were purified from cells, transgenic for P71, RNA1 and RNA2, by way of two different virus purification protocols that were developed during this study. RT-PCR analyses on RNA-extracts from these particles demonstrated that RNA transcripts, which were produced in trans with p71, could be encapsidated by HaSV capsids in S. cerevisiae. A droplet-feed bioassay on H. armigera larvae demonstrated that the S. cerevisiae-derived HaSV particles caused impaired larval development. This response was correlated with the detection of HaSV RNA2 in RNA extractions from larvae that were used in this bioassay. The results that were generated through the course of this study, provided proof for the concept of the non-host production of infectious HaSV particles from S. cerevisiae. This work could serve as a foundation for future research on the development of an expression system for the large-scale production of this virus as a biopesticide.
- Full Text:
- Date Issued: 2002
- Authors: Venter, Philip Arno
- Date: 2002
- Subjects: Helicoverpa armigera Saccharomyces cerevisiae
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3895 , http://hdl.handle.net/10962/d1003811
- Description: Lepidopteran insects like Helicoverpa armigera, more commonly known as the cotton bollworm, are economically important pests of a wide variety of crops throughout the world. The Helicoverpa armigera stunt virus (HaSV), a tetravirus with a bipartite single-stranded positive-sense RNA genome, has great potential as a biological pesticide against H. armigera. The larger genomic strand of this virus (RNA1) encodes the viral replicase, while the other (RNA2) encodes the 71 kDa capsid protein precursor (p71). 240 copies of p71 assemble into a procapsid with the concomitant encapsidation of the viral RNA. This is followed by a complex maturation event that is characterized by the autoproteolytic cleavage of p71 into the 64 kDa capsid protein (P64) and a 7 kDa peptide (p7). The rearrangements that occur during maturation results in the formation of mature HaSV capsids that can thereupon deliver RNA to other susceptible host cells. The principal objective of the research described in this study was to demonstrate that this virus could be assembled in Saccharomyces cerevisiae. S. cerevisiae expression vectors were constructed for the production of p71. This protein was detected in cell lysates from two different strains of S. cerevisiae, both containing either chromosomal or episomal copies of an expression cassette for P71. A number of factors relating to the expression of P71 (e.g. strains used, expression loci and expression rate) and the preparation of protein extracts from S. cerevisiae (e.g. the presence of various protease inhibitors and salt concentrations) were examined to attain optimal levels of soluble p71. A small fraction of the optimized soluble p71 was shown to be in the form of virus-like particles (VLPs), with a yield of ≤10⁷ VLPs from a 1.5l culture of P71⁺ cells. These particles were exclusively in the procapsid form, had a similar buoyant density to that of wild-type HaSV and could undergo maturation when the pH was reduced to 5. S. cerevisiae vectors were constructed for the episomal expression of the HaSV genomic RNAs. These vectors directed the transcription of RNA1 and RNA2 transcripts, which had similar sizes to those of the HaSV genomic RNAs. Mature HaSV particles were purified from cells, transgenic for P71, RNA1 and RNA2, by way of two different virus purification protocols that were developed during this study. RT-PCR analyses on RNA-extracts from these particles demonstrated that RNA transcripts, which were produced in trans with p71, could be encapsidated by HaSV capsids in S. cerevisiae. A droplet-feed bioassay on H. armigera larvae demonstrated that the S. cerevisiae-derived HaSV particles caused impaired larval development. This response was correlated with the detection of HaSV RNA2 in RNA extractions from larvae that were used in this bioassay. The results that were generated through the course of this study, provided proof for the concept of the non-host production of infectious HaSV particles from S. cerevisiae. This work could serve as a foundation for future research on the development of an expression system for the large-scale production of this virus as a biopesticide.
- Full Text:
- Date Issued: 2002
Identification of Cowdria ruminantium proteins that induce specific cellular immune responses
- Authors: Van Kleef, Mirinda
- Date: 2002
- Subjects: Ruminants--Pathogens Heartwater
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4061 , http://hdl.handle.net/10962/d1004373
- Description: Cowdria ruminantium (Cowdria) is an obligate intracellular pathogen that causes heartwater in ruminants. Cellular immunity and the type I cytokine IFN-γ have been implicated in protective immunity to heartwater. The aim of this thesis was to identify proteins of the Welgevonden isolate of Cowdria that induce lymphocyte proliferation and IFN-γ production. Differential centrifugation was found to be the simplest and most efficient method of Cowdria purification. Cowdria organisms were fractionated into their constituent proteins of between 11 and 168 kDa by continuous flow electrophoresis. The resulting fractions were tested for their ability to stimulate lymphocyte proliferation in vitro. In an attempt to simulate the natural infective process, peripheral blood mononuclear cells (PBMC) were obtained from two cattle rendered immune by infection and treatment and assayed in proliferation assays with the proteins fractions. In a parallel study, four cattle were immunised with inactivated Cowdria to determine whether their lymphocytes responded similarly. Cowdria-specific proliferation was detected for only a brief period after immunisation by infection with live organisms. This response was only detected again two to three years later. In contrast, PBMC from animals immunised with inactivated organisms were continuously responsive for at least three years. Only Cowdria proteins with molecular masses of 11, 12, 14 to 17 and 19 to 23 kDa induced proliferative responses in PBMC obtained from all six animals. Cell surface phenotypic analysis of Cowdria specific T-cell lines indicated that CD4⁺ lymphocytes were enriched over time with a concomitant increase in antigen-specific proliferation and IFN-γ production. Proteins of molecular masses 13 to 18 kDa induced CD4⁺ lymphocyte proliferation and IFN-γ production by T-cell lines from all the animals tested. Antibodies raised in a chicken and in rabbits to these low molecular weight proteins had low titres and specificity. Two-dimensional electrophoresis indicated that proteins within a single molecular weight range comprised several components with different pIs, revealing the complexity of the Cowdria proteome. This complicates the search for potentially protective antigens. Nevertheless, since they cause proliferation and IFN-γ production by lymphocytes from immunised cattle, these low molecular weight proteins merit further investigation as potential vaccine antigens. , Author: Mirinda van Kleef neé Rossouw
- Full Text:
- Date Issued: 2002
- Authors: Van Kleef, Mirinda
- Date: 2002
- Subjects: Ruminants--Pathogens Heartwater
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4061 , http://hdl.handle.net/10962/d1004373
- Description: Cowdria ruminantium (Cowdria) is an obligate intracellular pathogen that causes heartwater in ruminants. Cellular immunity and the type I cytokine IFN-γ have been implicated in protective immunity to heartwater. The aim of this thesis was to identify proteins of the Welgevonden isolate of Cowdria that induce lymphocyte proliferation and IFN-γ production. Differential centrifugation was found to be the simplest and most efficient method of Cowdria purification. Cowdria organisms were fractionated into their constituent proteins of between 11 and 168 kDa by continuous flow electrophoresis. The resulting fractions were tested for their ability to stimulate lymphocyte proliferation in vitro. In an attempt to simulate the natural infective process, peripheral blood mononuclear cells (PBMC) were obtained from two cattle rendered immune by infection and treatment and assayed in proliferation assays with the proteins fractions. In a parallel study, four cattle were immunised with inactivated Cowdria to determine whether their lymphocytes responded similarly. Cowdria-specific proliferation was detected for only a brief period after immunisation by infection with live organisms. This response was only detected again two to three years later. In contrast, PBMC from animals immunised with inactivated organisms were continuously responsive for at least three years. Only Cowdria proteins with molecular masses of 11, 12, 14 to 17 and 19 to 23 kDa induced proliferative responses in PBMC obtained from all six animals. Cell surface phenotypic analysis of Cowdria specific T-cell lines indicated that CD4⁺ lymphocytes were enriched over time with a concomitant increase in antigen-specific proliferation and IFN-γ production. Proteins of molecular masses 13 to 18 kDa induced CD4⁺ lymphocyte proliferation and IFN-γ production by T-cell lines from all the animals tested. Antibodies raised in a chicken and in rabbits to these low molecular weight proteins had low titres and specificity. Two-dimensional electrophoresis indicated that proteins within a single molecular weight range comprised several components with different pIs, revealing the complexity of the Cowdria proteome. This complicates the search for potentially protective antigens. Nevertheless, since they cause proliferation and IFN-γ production by lymphocytes from immunised cattle, these low molecular weight proteins merit further investigation as potential vaccine antigens. , Author: Mirinda van Kleef neé Rossouw
- Full Text:
- Date Issued: 2002
Removal and recovery of gold and platinum from aqueous solutions utilising the non-viable biomass Asolla filiculoides
- Authors: Antunes, Ana Paula Martins
- Date: 2002
- Subjects: Azolla filiculoides Metal wastes -- Recycling Gold -- Recycling Platinum -- Recycling
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3894 , http://hdl.handle.net/10962/d1003726
- Description: Waste water from the mining industry is generally extremely complex and contains numerous species which influence the adsorption of the metals to any biomass. A variety of factors need to be addressed before treatment is considered viable. It is also beneficial to establish the binding characteristics of the metal of interest to maximise its interaction with the biomass to be utilised. Azalia filiculaides was investigated in the adsorption of gold(III), lead(II), iron(ID), copper(II) and platinum (IV). In batch studies, the optimum biomass and initial gold(III) concentrations were found to be 5 gIL and 8 mgIL respectively. The adsorption of gold(ID) is principally pH-dependent with optimal removal at pH 2. Lead(II), iron(III) and copper(II) did not compete with gold(III) adsorption under equimolar and simulated effluent conditions. Halides, with increasing affinity for gold (chloride < bromide < iodide), can affect gold uptake with the soft base, iodide, exhibiting the most inhibition (25%) and the hard base, chloride, O%. Mercaptoethanol (soft base) showed no interference in gold(III) adsorption while the presence of sulphate (hard base) and sulphite (borderline base) showed that concentrations in excess of 1 0 mM may adversely affect gold(ill) uptake, most likely due to competition for cationic sites on the biomass. Column studies, better suited to high volume treatment, indicated that a flow-rate of 5 mL/min and an initial gold(ill) concentration of 5 mgIL was optimal. Competitive effects between lead, iron, copper and gold again showed little or no interference. The halides, chloride, bromide and iodide, affect gold(ill) uptake similarly to the batch studies, while the bases mercaptoethanol and sulphate minimally affect gold(III) binding with sulphite severely hampering adsorption (70% inhibition). To optimise gold desorption, preliminary batch studies indicated that a ratio of 1:1 of adsorbentdesorbent was optimal, whilst gas purging of thiourea with oxygen, air and nitrogen decreased gold elution in proportion to decreased amounts of oxygen. A series of desorbents were utilised, in column studies, to optimise and determine the speciation of bound gold. The presence of an oxidant with thiourea enhanced desorption greater than 3 fold when compared with thiourea alone. Thiourea desorption studies, aided by the oxidant, suggest that gold is present in the + I and 0 oxidation states. Ultimately thiourea, perchloric acid and hydrochloric acid was found to be the most optimal elutant for gold (J 00% recovery). For selective metal recovery oflead and copper, pre-washing the plant material with water, utilising an acid (0.3 M nitric acid), pumping in an up-flow mode, and recycling the desorbent six times was found to be optimal elutant for gold (J 00% recovery). Cost analysis of utilising elutant versus incinerating the biomass for gold recovery indicated the latter as the most economical. Over a 5 cycle adsorption and desorption series, acid desorption before each adsorption cycle was found to result in greater than 92% desorption for lead and 96% for copper. Gold recovery was 97% with incineration. A preliminary study with gold effluent (Mine C) indicated that nickel and sulphate was removed in batch and column studies. Gold removal was found to be 100% and 4% in batch and column studies respectively. Adsorption of gold in the effluent study was accompanied by the release ofHt. Modifying the plant material with various reagents failed to identify the primary binding sites and the role of polysaccharides, proteins and lipids in gold(ill) uptake. The mode of gold binding is suggested as being initially ionic, this is very rapid, with the interaction of the anionic complex, [AuCI₄]". with the cationic biomass (PH 2). This eventually leads to the displacement of the chloride ligand(s) initiating covalent binding. Spectral studies of the chemical interaction between gold and the representative tannins indicated the protonated hydroxy groups to be responsible. All evidence suggests that the binding mechanisms of gold are not simple. Preliminary adsorption studies of platinum by Azalia filiculaides were conducted. Batch studies indicated that J gIL biomass concentration, initial platinum concentration of 20 mgIL and pH 2 are optimal, while the column studies indicated a flow-rate of! 0 rnL/min and initial platinum concentration of 20 mgIL as optimal. In the platinum effluent study, platinum showed a removal of 23 % and 2 J % for the batch and column studies respectively. Again adsorption was accompanied by //' release. Azalia filiculaides demonstrated its feasibility in the removal of gold and platinum from simulated as well as waste water solutions. Its potential viability as a biosorbent was demonstrated by the high recovery from synthetic solutions of greater than 99% for gold (2-10 mgIL), and greater than 89% for platinum (20 mgIL).
- Full Text:
- Date Issued: 2002
- Authors: Antunes, Ana Paula Martins
- Date: 2002
- Subjects: Azolla filiculoides Metal wastes -- Recycling Gold -- Recycling Platinum -- Recycling
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3894 , http://hdl.handle.net/10962/d1003726
- Description: Waste water from the mining industry is generally extremely complex and contains numerous species which influence the adsorption of the metals to any biomass. A variety of factors need to be addressed before treatment is considered viable. It is also beneficial to establish the binding characteristics of the metal of interest to maximise its interaction with the biomass to be utilised. Azalia filiculaides was investigated in the adsorption of gold(III), lead(II), iron(ID), copper(II) and platinum (IV). In batch studies, the optimum biomass and initial gold(III) concentrations were found to be 5 gIL and 8 mgIL respectively. The adsorption of gold(ID) is principally pH-dependent with optimal removal at pH 2. Lead(II), iron(III) and copper(II) did not compete with gold(III) adsorption under equimolar and simulated effluent conditions. Halides, with increasing affinity for gold (chloride < bromide < iodide), can affect gold uptake with the soft base, iodide, exhibiting the most inhibition (25%) and the hard base, chloride, O%. Mercaptoethanol (soft base) showed no interference in gold(III) adsorption while the presence of sulphate (hard base) and sulphite (borderline base) showed that concentrations in excess of 1 0 mM may adversely affect gold(ill) uptake, most likely due to competition for cationic sites on the biomass. Column studies, better suited to high volume treatment, indicated that a flow-rate of 5 mL/min and an initial gold(ill) concentration of 5 mgIL was optimal. Competitive effects between lead, iron, copper and gold again showed little or no interference. The halides, chloride, bromide and iodide, affect gold(ill) uptake similarly to the batch studies, while the bases mercaptoethanol and sulphate minimally affect gold(III) binding with sulphite severely hampering adsorption (70% inhibition). To optimise gold desorption, preliminary batch studies indicated that a ratio of 1:1 of adsorbentdesorbent was optimal, whilst gas purging of thiourea with oxygen, air and nitrogen decreased gold elution in proportion to decreased amounts of oxygen. A series of desorbents were utilised, in column studies, to optimise and determine the speciation of bound gold. The presence of an oxidant with thiourea enhanced desorption greater than 3 fold when compared with thiourea alone. Thiourea desorption studies, aided by the oxidant, suggest that gold is present in the + I and 0 oxidation states. Ultimately thiourea, perchloric acid and hydrochloric acid was found to be the most optimal elutant for gold (J 00% recovery). For selective metal recovery oflead and copper, pre-washing the plant material with water, utilising an acid (0.3 M nitric acid), pumping in an up-flow mode, and recycling the desorbent six times was found to be optimal elutant for gold (J 00% recovery). Cost analysis of utilising elutant versus incinerating the biomass for gold recovery indicated the latter as the most economical. Over a 5 cycle adsorption and desorption series, acid desorption before each adsorption cycle was found to result in greater than 92% desorption for lead and 96% for copper. Gold recovery was 97% with incineration. A preliminary study with gold effluent (Mine C) indicated that nickel and sulphate was removed in batch and column studies. Gold removal was found to be 100% and 4% in batch and column studies respectively. Adsorption of gold in the effluent study was accompanied by the release ofHt. Modifying the plant material with various reagents failed to identify the primary binding sites and the role of polysaccharides, proteins and lipids in gold(ill) uptake. The mode of gold binding is suggested as being initially ionic, this is very rapid, with the interaction of the anionic complex, [AuCI₄]". with the cationic biomass (PH 2). This eventually leads to the displacement of the chloride ligand(s) initiating covalent binding. Spectral studies of the chemical interaction between gold and the representative tannins indicated the protonated hydroxy groups to be responsible. All evidence suggests that the binding mechanisms of gold are not simple. Preliminary adsorption studies of platinum by Azalia filiculaides were conducted. Batch studies indicated that J gIL biomass concentration, initial platinum concentration of 20 mgIL and pH 2 are optimal, while the column studies indicated a flow-rate of! 0 rnL/min and initial platinum concentration of 20 mgIL as optimal. In the platinum effluent study, platinum showed a removal of 23 % and 2 J % for the batch and column studies respectively. Again adsorption was accompanied by //' release. Azalia filiculaides demonstrated its feasibility in the removal of gold and platinum from simulated as well as waste water solutions. Its potential viability as a biosorbent was demonstrated by the high recovery from synthetic solutions of greater than 99% for gold (2-10 mgIL), and greater than 89% for platinum (20 mgIL).
- Full Text:
- Date Issued: 2002
The biotransformation of phenolic pollutants using polyphenol oxidase
- Authors: Boshoff, Aileen
- Date: 2002
- Subjects: Polyphenol oxidase Sewage -- Purification
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3976 , http://hdl.handle.net/10962/d1004035
- Description: The potential of using mushroom polyphenol oxidase (EC 1.14.18.1) as a biocatalyst for the biotransformation of phenols to produce catechols in an aqueous medium was investigated. Polyphenol oxidase is characterised by two distinct reactions i.e., the ortho-hydroxylation of phenols to catechols (cresolase activity) and the subsequent oxidation of catechols to orthoquinones (catecholase activity). In order to facilitate the development of a process to produce catechols, the accumulation of catechol as a true intermediate product released in the reaction system needed to be investigated, as its release had been disputed due to the oxidation of catechols to o-quinones. Using LC-MS, catechol products were successfully identified as true intermediate products formed during biocatalytic reactions in water.
- Full Text:
- Date Issued: 2002
- Authors: Boshoff, Aileen
- Date: 2002
- Subjects: Polyphenol oxidase Sewage -- Purification
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3976 , http://hdl.handle.net/10962/d1004035
- Description: The potential of using mushroom polyphenol oxidase (EC 1.14.18.1) as a biocatalyst for the biotransformation of phenols to produce catechols in an aqueous medium was investigated. Polyphenol oxidase is characterised by two distinct reactions i.e., the ortho-hydroxylation of phenols to catechols (cresolase activity) and the subsequent oxidation of catechols to orthoquinones (catecholase activity). In order to facilitate the development of a process to produce catechols, the accumulation of catechol as a true intermediate product released in the reaction system needed to be investigated, as its release had been disputed due to the oxidation of catechols to o-quinones. Using LC-MS, catechol products were successfully identified as true intermediate products formed during biocatalytic reactions in water.
- Full Text:
- Date Issued: 2002
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