Formulation of an enzyme cocktail, HoloMix, using cellulolytic and xylanolytic enzyme core-sets for effective degradation of various pre-treated hardwoods
- Authors: Malgas, Samkelo
- Date: 2018
- Subjects: Biomass , Cellulase , Hardwoods , Xylanases
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/62827 , vital:28297 , DOI https://doi.org/10.21504/10962/62827
- Description: Currently, there is a growing interest in utilising hardwoods as feedstocks for bioethanol production due to the vast advantages they have over other feedstocks for fermentable sugar production. In this study, two selected hardwoods, Acacia and Populus spp., were subjected to two pre-treatment processes (Sodium chlorite delignification and Steam explosion) and compared with respect to how these pre-treatments affect their enzymatic saccharification. Hardwoods were selected for this study, because hardwoods are easier to delignify when compared to softwoods, and therefore their polysaccharides are more easily accessible by enzymes for the purpose of producing fermentable sugars. Currently available commercial enzyme mixtures have been developed for optimal hydrolysis of acid-pre-treated corn stover and are therefore not optimal for saccharification of pre-treated hardwoods. In this work, we attempted the empirical design of a hardwood specific enzyme cocktail, HoloMix. Firstly, a cellulolytic core-set, CelMix (in a ratio of Egl 68%: Cel7A 17%: Cel6A 6%: Bgl1 9%), for the optimal release of glucose, and a xylanolytic core-set, XynMix (in a ratio of Xyn2A 60%: XT6 20%: AguA 11%: SXA 9%), for the optimal release of xylose, were formulated using an empirical enzyme ratio approach after biochemically characterising these enzymes. As it is well ̶ known that biomass pre-treatment may result in the generation of compounds that hamper enzymatic hydrolysis and microbial fermentation, the effects of these compounds on CelMix and XynMix were evaluated. Using the optimised CelMix and XynMix cocktails, a HoloMix cocktail was established for optimal reducing sugar, glucose and xylose release from the various pre-treated hardwoods. For delignified biomass, the optimized HoloMix consisted of CelMix to XynMix at 75% to 25% protein loading, while for the untreated and steam exploded biomass the HoloMix consisted of CelMix to XynMix at 93.75% to 6.25% protein loading. Sugar release by the HoloMix at a loading of 27.5 mg protein/g of biomass (or 55 mg protein/g of glucan) after 24 h gave 70-100% sugar yield. Treatment of the hardwoods with a laccase from Agaricus bisporus, especially wood biomass with a higher proportion of lignin, significantly improved saccharification by the formulated HoloMix enzyme cocktails. This study provided insights into the enzymatic hydrolysis of various pre-treated hardwood substrates and assessed whether the same lignocellulolytic cocktail can be used to efficiently hydrolyse different hardwood species. The present study also demonstrated that the hydrolysis efficiency of the optimised HoloMix was comparable to (if not better) than commercial enzyme preparations during hardwood biomass saccharification. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2018
- Full Text:
- Date Issued: 2018
- Authors: Malgas, Samkelo
- Date: 2018
- Subjects: Biomass , Cellulase , Hardwoods , Xylanases
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/62827 , vital:28297 , DOI https://doi.org/10.21504/10962/62827
- Description: Currently, there is a growing interest in utilising hardwoods as feedstocks for bioethanol production due to the vast advantages they have over other feedstocks for fermentable sugar production. In this study, two selected hardwoods, Acacia and Populus spp., were subjected to two pre-treatment processes (Sodium chlorite delignification and Steam explosion) and compared with respect to how these pre-treatments affect their enzymatic saccharification. Hardwoods were selected for this study, because hardwoods are easier to delignify when compared to softwoods, and therefore their polysaccharides are more easily accessible by enzymes for the purpose of producing fermentable sugars. Currently available commercial enzyme mixtures have been developed for optimal hydrolysis of acid-pre-treated corn stover and are therefore not optimal for saccharification of pre-treated hardwoods. In this work, we attempted the empirical design of a hardwood specific enzyme cocktail, HoloMix. Firstly, a cellulolytic core-set, CelMix (in a ratio of Egl 68%: Cel7A 17%: Cel6A 6%: Bgl1 9%), for the optimal release of glucose, and a xylanolytic core-set, XynMix (in a ratio of Xyn2A 60%: XT6 20%: AguA 11%: SXA 9%), for the optimal release of xylose, were formulated using an empirical enzyme ratio approach after biochemically characterising these enzymes. As it is well ̶ known that biomass pre-treatment may result in the generation of compounds that hamper enzymatic hydrolysis and microbial fermentation, the effects of these compounds on CelMix and XynMix were evaluated. Using the optimised CelMix and XynMix cocktails, a HoloMix cocktail was established for optimal reducing sugar, glucose and xylose release from the various pre-treated hardwoods. For delignified biomass, the optimized HoloMix consisted of CelMix to XynMix at 75% to 25% protein loading, while for the untreated and steam exploded biomass the HoloMix consisted of CelMix to XynMix at 93.75% to 6.25% protein loading. Sugar release by the HoloMix at a loading of 27.5 mg protein/g of biomass (or 55 mg protein/g of glucan) after 24 h gave 70-100% sugar yield. Treatment of the hardwoods with a laccase from Agaricus bisporus, especially wood biomass with a higher proportion of lignin, significantly improved saccharification by the formulated HoloMix enzyme cocktails. This study provided insights into the enzymatic hydrolysis of various pre-treated hardwood substrates and assessed whether the same lignocellulolytic cocktail can be used to efficiently hydrolyse different hardwood species. The present study also demonstrated that the hydrolysis efficiency of the optimised HoloMix was comparable to (if not better) than commercial enzyme preparations during hardwood biomass saccharification. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2018
- Full Text:
- Date Issued: 2018
Production, purification and characterization of a multifunctional, thermostable and acido/alkaline stable putative xylanase from the psychrotrophic bacterium, Sphingomonas aerolata
- Authors: Mathibe, Brian Nkanyiso
- Date: 2020
- Subjects: Uncatalogued
- Language: English
- Type: thesis , text , Masters , MSc
- Identifier: http://hdl.handle.net/10962/164478 , vital:41122
- Description: Thesis (MSc)--Rhodes University, Faculty of Science, Biochemistry and Microbiology, 2020
- Full Text:
- Date Issued: 2020
- Authors: Mathibe, Brian Nkanyiso
- Date: 2020
- Subjects: Uncatalogued
- Language: English
- Type: thesis , text , Masters , MSc
- Identifier: http://hdl.handle.net/10962/164478 , vital:41122
- Description: Thesis (MSc)--Rhodes University, Faculty of Science, Biochemistry and Microbiology, 2020
- Full Text:
- Date Issued: 2020
A case-control approach to assess variability in distribution of distance between transcription factor binding site and transcription start site
- Authors: Moos, Abdul Ragmaan
- Date: 2017
- Subjects: Transcription factors , Proteomics , Chromatin , Chromatin immunoprecipitation
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/5315 , vital:20808
- Description: Using the in-silico approach, with ENCODE ChIP-seq data for various transcription factors and different cell types; we systematically compared the distance between the transcription factor binding site (TFBS) and the transcription start (TSS). Our aim was to determine if the same transcription factor binds at a different position relative to the TSS in a normal and an abnormal cell type. We compare distribution of distance of binding sites from the TSS; to make description less verbose we call this “distance” where there is no possibility of confusion. We used a case-control methodology where the distance between the TFBS and the TSS in the normal, non-cancerous or untreated cell type is the control. The distance between the TFBS and the TSS in the cancerous or treated cell type is the case. We use the distance between the TFBS and the TSS in the control as the standard. We compared the distance between the TFBS and the TSS in the case and the control. If the distance between the TFBS and the TSS in the control was greater than the distance between the TFBS and the TSS in the case, we can infer the following. The transcription factor in the case binds closer to the TSS compared to the control. If the distance between the TFBS and the TSS in the control is smaller than the distance between the TFBS and the TSS in the case, we can infer the following. The TF in the case binds further away from the TSS compared to the control. Our method is a screening method whereby we compare ChIP-seq data to determine if there is a difference in the distribution distance between the TFBS and the TSS for normal and abnormal cell types. We used the R package ChIP-Enrich to compare the distribution of distance between ChIP-seq peak and the nearest TSS. ChIP-Enrich produces a histogram with the number of ChIP-seq peaks at a certain distance from the TSS. The results indicate for some transcription factors like GM12878-cMyc and K562-cMyc there is a difference between the distribution of distance between the TFBS and the nearest TSS. cMyc has more binding sites within a distance of 1kb from the TSS in GM12878 when compared to K562. GM12878-CTCF and K562-CTCF have slight differences when comparing their distribution of distance from the TSS. This means CTCF binds almost the same distance from the TSS in both GM12878 and K562. A549-gr treated with dexamethasone is interesting because with increase dose of dexamethasone the distribution of distance from the TSS changes as well.
- Full Text:
- Date Issued: 2017
- Authors: Moos, Abdul Ragmaan
- Date: 2017
- Subjects: Transcription factors , Proteomics , Chromatin , Chromatin immunoprecipitation
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/5315 , vital:20808
- Description: Using the in-silico approach, with ENCODE ChIP-seq data for various transcription factors and different cell types; we systematically compared the distance between the transcription factor binding site (TFBS) and the transcription start (TSS). Our aim was to determine if the same transcription factor binds at a different position relative to the TSS in a normal and an abnormal cell type. We compare distribution of distance of binding sites from the TSS; to make description less verbose we call this “distance” where there is no possibility of confusion. We used a case-control methodology where the distance between the TFBS and the TSS in the normal, non-cancerous or untreated cell type is the control. The distance between the TFBS and the TSS in the cancerous or treated cell type is the case. We use the distance between the TFBS and the TSS in the control as the standard. We compared the distance between the TFBS and the TSS in the case and the control. If the distance between the TFBS and the TSS in the control was greater than the distance between the TFBS and the TSS in the case, we can infer the following. The transcription factor in the case binds closer to the TSS compared to the control. If the distance between the TFBS and the TSS in the control is smaller than the distance between the TFBS and the TSS in the case, we can infer the following. The TF in the case binds further away from the TSS compared to the control. Our method is a screening method whereby we compare ChIP-seq data to determine if there is a difference in the distribution distance between the TFBS and the TSS for normal and abnormal cell types. We used the R package ChIP-Enrich to compare the distribution of distance between ChIP-seq peak and the nearest TSS. ChIP-Enrich produces a histogram with the number of ChIP-seq peaks at a certain distance from the TSS. The results indicate for some transcription factors like GM12878-cMyc and K562-cMyc there is a difference between the distribution of distance between the TFBS and the nearest TSS. cMyc has more binding sites within a distance of 1kb from the TSS in GM12878 when compared to K562. GM12878-CTCF and K562-CTCF have slight differences when comparing their distribution of distance from the TSS. This means CTCF binds almost the same distance from the TSS in both GM12878 and K562. A549-gr treated with dexamethasone is interesting because with increase dose of dexamethasone the distribution of distance from the TSS changes as well.
- Full Text:
- Date Issued: 2017
Bio-prospecting a Soil Metagenomic Library for Carbohydrate Active Esterases
- Authors: Shezi, Ntombifuthi
- Date: 2016
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4172 , http://hdl.handle.net/10962/d1021266
- Description: Lignocellulosic biomass is a promising renewable resource on earth. Plant biomass contains fermentable sugars and other moieties that can be converted to biofuels or other chemicals. Enzymatic hydrolysis of these biopolymers is significant in the liberation of sugars for fermentation into desired products. Owing to its complex structure, synergistic action of enzymes is required for its degradation. Enzymes that are involved in biomass degradation include cellulases, hemicellulases and the accessory enzymes acetyl xylan esterases and ferulic acid esterases. Ferulic acid esterases (FAEs, EC 3.1.1.73), represent a subclass of carboxylester hydrolases (EC 3.1.1.-) that catalyse the release of hydroxycinnamic acids (such as ferulic acid, p-coumaric, ferulic, sinapic and caffeic acid) that are generally found esterified to polysaccharides, such as arabinoxylans. Hydroxycinnamic acids have widespread potential applications due to their antimicrobial, photoprotectant and antioxidant properties, as well as their use as flavour precursors. Therefore, this interesting group of FAEs has a potentially wide variety of applications in agriculture, food and pharmaceutical industries. In the search for novel biocatalysts, metagenomics is considered as an alternative approach to conventional microbe screening, therefore, searching for novel biocatalysts from a soil metagenome that harbours a unique diversity of biocatalyst is significant. The aim of this study was to extract DNA from soil associated with cattle manure and construct a soil metagenomic library using a fosmid based plasmid vector and subsequently functionally screen for ferulic acid esterases using ethyl ferulate as a model substrate. A total of 59 recombinant fosmids conferring ferulic acid esterase phenotypes were identified (Hit rate 1:3122) and the two fosmids that consistently showed high FAE activities were selected for further study. Following nucleotide sequencing and translational analysis, two fae encoding open reading frames (FAE9 and FAE27) of approximately 274 and 322 aa, respectively, were identified. The amino acid sequence of the two ORFs contained a classical conserved esterase/lipase G-x-S-x-G sequence motif. The two genes (fae9 and fae27) were successfully expressed in Escherichia coli BL21 (DE3) and the purified enzymes exhibited respective temperature optima of 50 °C and 40 °C, and respective pH optima of 6.0 and 7.0. Further biochemical characterisation showed that FAE9 and FAE27 have high substrate specificity, following the fact that EFA is the preferred substrate for FAE9 (kcat/Km value of 128 s−1.mM-1) and also the preferred substrate for FAE27 (kcat/Km value of 137 s−1.mM-1). This work proves that soil is a valuable environmental source for novel esterase screening through functional based metagenomic approach. Therefore, this method may be used to screen for other valuable enzymes from environmental sources using inexpensive natural sources to encourage the screening of specific enzymes. Biochemistry of the two isolated enzymes makes these enzymes to be useful in industrial applications due to broad substrate activity that could replace the specialised enzymes to complete plant biomass degradation.
- Full Text:
- Date Issued: 2016
- Authors: Shezi, Ntombifuthi
- Date: 2016
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4172 , http://hdl.handle.net/10962/d1021266
- Description: Lignocellulosic biomass is a promising renewable resource on earth. Plant biomass contains fermentable sugars and other moieties that can be converted to biofuels or other chemicals. Enzymatic hydrolysis of these biopolymers is significant in the liberation of sugars for fermentation into desired products. Owing to its complex structure, synergistic action of enzymes is required for its degradation. Enzymes that are involved in biomass degradation include cellulases, hemicellulases and the accessory enzymes acetyl xylan esterases and ferulic acid esterases. Ferulic acid esterases (FAEs, EC 3.1.1.73), represent a subclass of carboxylester hydrolases (EC 3.1.1.-) that catalyse the release of hydroxycinnamic acids (such as ferulic acid, p-coumaric, ferulic, sinapic and caffeic acid) that are generally found esterified to polysaccharides, such as arabinoxylans. Hydroxycinnamic acids have widespread potential applications due to their antimicrobial, photoprotectant and antioxidant properties, as well as their use as flavour precursors. Therefore, this interesting group of FAEs has a potentially wide variety of applications in agriculture, food and pharmaceutical industries. In the search for novel biocatalysts, metagenomics is considered as an alternative approach to conventional microbe screening, therefore, searching for novel biocatalysts from a soil metagenome that harbours a unique diversity of biocatalyst is significant. The aim of this study was to extract DNA from soil associated with cattle manure and construct a soil metagenomic library using a fosmid based plasmid vector and subsequently functionally screen for ferulic acid esterases using ethyl ferulate as a model substrate. A total of 59 recombinant fosmids conferring ferulic acid esterase phenotypes were identified (Hit rate 1:3122) and the two fosmids that consistently showed high FAE activities were selected for further study. Following nucleotide sequencing and translational analysis, two fae encoding open reading frames (FAE9 and FAE27) of approximately 274 and 322 aa, respectively, were identified. The amino acid sequence of the two ORFs contained a classical conserved esterase/lipase G-x-S-x-G sequence motif. The two genes (fae9 and fae27) were successfully expressed in Escherichia coli BL21 (DE3) and the purified enzymes exhibited respective temperature optima of 50 °C and 40 °C, and respective pH optima of 6.0 and 7.0. Further biochemical characterisation showed that FAE9 and FAE27 have high substrate specificity, following the fact that EFA is the preferred substrate for FAE9 (kcat/Km value of 128 s−1.mM-1) and also the preferred substrate for FAE27 (kcat/Km value of 137 s−1.mM-1). This work proves that soil is a valuable environmental source for novel esterase screening through functional based metagenomic approach. Therefore, this method may be used to screen for other valuable enzymes from environmental sources using inexpensive natural sources to encourage the screening of specific enzymes. Biochemistry of the two isolated enzymes makes these enzymes to be useful in industrial applications due to broad substrate activity that could replace the specialised enzymes to complete plant biomass degradation.
- Full Text:
- Date Issued: 2016
An investigation into yeast-baculovirus synergism for the improved control of Thaumatotibia leucotreta, an economically important pest of citrus
- Authors: Van der Merwe, Marcél
- Date: 2021-10-29
- Subjects: Baculoviruses , Cryptophlebia leucotreta , Yeast , Natural pesticides , Citrus Diseases and pests , Biological pest control agents , Pests Integrated control , Thaumatotibia leucotreta
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/191236 , vital:45073
- Description: A mutualistic association between Cydia pomonella and yeasts belonging to the genus Metschnikowia has previously been demonstrated. Larval feeding galleries inoculated with M. andauensis, reduced larval mortality and enhanced larval development. Additionally, adult C. pomonella female oviposition preference was also shown to be influenced by the volatiles produced by M. andauensis. This mutualistic relationship was manipulated for biological control purposes, by combining M. pulcherrima with the baculovirus Cydia pomonella granulovirus. The combination of M. pulcherrima with brown cane sugar and CpGV in laboratory assays and field trials resulted in a significant increase in larval mortality. A similar observation was made when M. pulcherrima was substituted for Saccharomyces cerevisiae. This indicates that yeasts harbour the potential for use in biological control, especially when combined with other well-established biocontrol methods. Thaumatotibia leucotreta is a phytophagous insect endemic to southern Africa. It is highly significant to the South African citrus industry due to its classification as a phytosanitary pest by most international markets. An integrated pest management programme has been implemented to control T. leucotreta. The baculovirus Cryptophlebia leucotreta granulovirus forms one component of this programme and is highly effective. In this study, we proposed to determine which yeast species occur naturally in the gut of T. leucotreta larvae and to examine whether any of the isolated yeast species, when combined with the CrleGV-SA, enhance its effectiveness. Firstly, Navel oranges infested with T. leucotreta larvae were collected from geographically distinct citrus-producing regions across South Africa. This led to the isolation and identification of six yeast species from the gut of T. leucotreta larvae via PCR amplification and sequencing of the internal transcribed spacer region and D1/D2 domain of the large subunit. Six yeast species were identified, viz. Meyerozyma guilliermondii, Hanseniaspora uvarum, Clavispora lusitaniae, Kluyveromyces marxianus, Pichia kudriavzevii and Pichia kluyveri. Additionally, Saccharomyces cerevisiae was included as a control in all trials due to its commercial availability and use in the artificial diet used to rear T. leucotreta. Secondly, larval development and attraction assays were conducted with the isolated yeast species. Thaumatotibia leucotreta larvae that fed on Navel oranges inoculated with M. guilliermondii, P. kluyveri, H. uvarum, and S. cerevisiae had accelerated developmental periods and reduced mortality rates. Additionally, it was demonstrated that T. leucotreta neonates were attracted to YPD broth cultures inoculated with P. kluyveri, H. uvarum, P. kudriavzevii and K. marxianus for feeding. Thirdly, oviposition preference assays were conducted with adult T. leucotreta females to determine whether the isolated yeast species influence their egg-laying in two-choice and multiple-choice tests. Navel oranges were inoculated with a specific yeast isolate, and mated adult females were left to oviposit. Meyerozyma guilliermondii, P. kudriavzevii and H. uvarum were shown to influence adult T. leucotreta female oviposition preference in two-choice tests. However, multiple-choice tests using the aforementioned yeast species did not mimic these results. Lastly, a series of detached fruit bioassays were performed to determine the optimal yeast:virus ratio, test all isolated yeast species in combination with CrleGV-SA and to further enhance yeast/virus formulation through the addition of an adjuvant and surfactant. CrleGV-SA was applied at a lethal concentration that would kill 50 % of T. leucotreta larvae. The optimal yeast concentration to use alongside CrleGV-SA was determined. Pichia kluyveri, P. kudriavzevii, K. marxianus and S. cerevisiae in combination with CrleGV-SA increased larval mortality compared to CrleGV-SA alone. The inclusion of molasses and BREAK-THRU® S 240 to P. kudriavzevii and S. cerevisiae plus CrleGV-SA formulations greatly enhanced their efficacy. Additionally, semi-field trials were initiated using P. kudriavzevii and S. cerevisiae, with promising preliminary results being obtained, although more replicates need to be performed. The experiments performed in this study provide a platform for further research into the application of a yeast/virus combination as a novel control and monitoring option for T. leucotreta in the field. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2021
- Full Text:
- Date Issued: 2021-10-29
- Authors: Van der Merwe, Marcél
- Date: 2021-10-29
- Subjects: Baculoviruses , Cryptophlebia leucotreta , Yeast , Natural pesticides , Citrus Diseases and pests , Biological pest control agents , Pests Integrated control , Thaumatotibia leucotreta
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/191236 , vital:45073
- Description: A mutualistic association between Cydia pomonella and yeasts belonging to the genus Metschnikowia has previously been demonstrated. Larval feeding galleries inoculated with M. andauensis, reduced larval mortality and enhanced larval development. Additionally, adult C. pomonella female oviposition preference was also shown to be influenced by the volatiles produced by M. andauensis. This mutualistic relationship was manipulated for biological control purposes, by combining M. pulcherrima with the baculovirus Cydia pomonella granulovirus. The combination of M. pulcherrima with brown cane sugar and CpGV in laboratory assays and field trials resulted in a significant increase in larval mortality. A similar observation was made when M. pulcherrima was substituted for Saccharomyces cerevisiae. This indicates that yeasts harbour the potential for use in biological control, especially when combined with other well-established biocontrol methods. Thaumatotibia leucotreta is a phytophagous insect endemic to southern Africa. It is highly significant to the South African citrus industry due to its classification as a phytosanitary pest by most international markets. An integrated pest management programme has been implemented to control T. leucotreta. The baculovirus Cryptophlebia leucotreta granulovirus forms one component of this programme and is highly effective. In this study, we proposed to determine which yeast species occur naturally in the gut of T. leucotreta larvae and to examine whether any of the isolated yeast species, when combined with the CrleGV-SA, enhance its effectiveness. Firstly, Navel oranges infested with T. leucotreta larvae were collected from geographically distinct citrus-producing regions across South Africa. This led to the isolation and identification of six yeast species from the gut of T. leucotreta larvae via PCR amplification and sequencing of the internal transcribed spacer region and D1/D2 domain of the large subunit. Six yeast species were identified, viz. Meyerozyma guilliermondii, Hanseniaspora uvarum, Clavispora lusitaniae, Kluyveromyces marxianus, Pichia kudriavzevii and Pichia kluyveri. Additionally, Saccharomyces cerevisiae was included as a control in all trials due to its commercial availability and use in the artificial diet used to rear T. leucotreta. Secondly, larval development and attraction assays were conducted with the isolated yeast species. Thaumatotibia leucotreta larvae that fed on Navel oranges inoculated with M. guilliermondii, P. kluyveri, H. uvarum, and S. cerevisiae had accelerated developmental periods and reduced mortality rates. Additionally, it was demonstrated that T. leucotreta neonates were attracted to YPD broth cultures inoculated with P. kluyveri, H. uvarum, P. kudriavzevii and K. marxianus for feeding. Thirdly, oviposition preference assays were conducted with adult T. leucotreta females to determine whether the isolated yeast species influence their egg-laying in two-choice and multiple-choice tests. Navel oranges were inoculated with a specific yeast isolate, and mated adult females were left to oviposit. Meyerozyma guilliermondii, P. kudriavzevii and H. uvarum were shown to influence adult T. leucotreta female oviposition preference in two-choice tests. However, multiple-choice tests using the aforementioned yeast species did not mimic these results. Lastly, a series of detached fruit bioassays were performed to determine the optimal yeast:virus ratio, test all isolated yeast species in combination with CrleGV-SA and to further enhance yeast/virus formulation through the addition of an adjuvant and surfactant. CrleGV-SA was applied at a lethal concentration that would kill 50 % of T. leucotreta larvae. The optimal yeast concentration to use alongside CrleGV-SA was determined. Pichia kluyveri, P. kudriavzevii, K. marxianus and S. cerevisiae in combination with CrleGV-SA increased larval mortality compared to CrleGV-SA alone. The inclusion of molasses and BREAK-THRU® S 240 to P. kudriavzevii and S. cerevisiae plus CrleGV-SA formulations greatly enhanced their efficacy. Additionally, semi-field trials were initiated using P. kudriavzevii and S. cerevisiae, with promising preliminary results being obtained, although more replicates need to be performed. The experiments performed in this study provide a platform for further research into the application of a yeast/virus combination as a novel control and monitoring option for T. leucotreta in the field. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2021
- Full Text:
- Date Issued: 2021-10-29
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
- 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
A novel Arf GTPase assay for antimalarial drug discovery
- Authors: Swart, Tarryn
- Date: 2021-04
- Subjects: To be added
- Language: English
- Type: thesis , text , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/178558 , vital:42950
- Description: Access restricted until April 2022. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2021
- Full Text:
- Date Issued: 2021-04
- Authors: Swart, Tarryn
- Date: 2021-04
- Subjects: To be added
- Language: English
- Type: thesis , text , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/178558 , vital:42950
- Description: Access restricted until April 2022. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2021
- Full Text:
- Date Issued: 2021-04
Towards a biological profile for South African perinatal remains: osteological and genetic perspectives
- Authors: Thornton, Roxanne
- Date: 2019
- Subjects: Identification , Forensic osteology , Methylation , RNA , Autopsy
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/68102 , vital:29198 , DOI 10.21504/10962/68102
- Description: Forensic identification of abandoned and suspected infanticide cases admitted to the South African Forensic Pathology Services is often impossible due to decomposition of the remains. In these cases, investigation of suspected criminal activity is almost never pursued. Ancillary tests in the form of anthropological and molecular analyses can assist with the forensic identification of perinatal remains. To provide fundamental information about bone development of perinatal skeleton, osteological and genetic techniques focusing on the pars basilaris, pars lateralis, sternal rib and left femur were used. Samples were obtained from unidentified and unclaimed remains originating from the Johannesburg Forensic Pathology Service (JFPS). To provide a biological age to individuals in the collection, dental aging was used to categorize remains for comparisons with anthropological and molecular data. A molecular protocol was designed to sex individuals using the X-linked G6PD and Y-linked SRY genes. Bone development was studied using osteometric and morphological data of dry bone remains coupled with bone mineral density analysis (Micro-CT). The methylation levels of CpG rich sites within the promoter region of selected bone-associated genes were incorporated to examine silencing of genes during development. Osteological results support the use of the pars basilaris, pars lateralis and femur for age-at-death estimations as well as provide the foundation for dry bone aging criteria for South African individuals. Data compared with established skeletal aging standards indicated developmental differences between populations. Through the use of animal models and the perinatal sternal rib tissue, insights and precautions into the use of post mortem bone derived RNA for forensic applications is communicated. The methylation status of CpG rich sites within the promoter regions support the hypothesis for interdependent machinery involving selected genes during early bone development. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2019
- Full Text:
- Date Issued: 2019
- Authors: Thornton, Roxanne
- Date: 2019
- Subjects: Identification , Forensic osteology , Methylation , RNA , Autopsy
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/68102 , vital:29198 , DOI 10.21504/10962/68102
- Description: Forensic identification of abandoned and suspected infanticide cases admitted to the South African Forensic Pathology Services is often impossible due to decomposition of the remains. In these cases, investigation of suspected criminal activity is almost never pursued. Ancillary tests in the form of anthropological and molecular analyses can assist with the forensic identification of perinatal remains. To provide fundamental information about bone development of perinatal skeleton, osteological and genetic techniques focusing on the pars basilaris, pars lateralis, sternal rib and left femur were used. Samples were obtained from unidentified and unclaimed remains originating from the Johannesburg Forensic Pathology Service (JFPS). To provide a biological age to individuals in the collection, dental aging was used to categorize remains for comparisons with anthropological and molecular data. A molecular protocol was designed to sex individuals using the X-linked G6PD and Y-linked SRY genes. Bone development was studied using osteometric and morphological data of dry bone remains coupled with bone mineral density analysis (Micro-CT). The methylation levels of CpG rich sites within the promoter region of selected bone-associated genes were incorporated to examine silencing of genes during development. Osteological results support the use of the pars basilaris, pars lateralis and femur for age-at-death estimations as well as provide the foundation for dry bone aging criteria for South African individuals. Data compared with established skeletal aging standards indicated developmental differences between populations. Through the use of animal models and the perinatal sternal rib tissue, insights and precautions into the use of post mortem bone derived RNA for forensic applications is communicated. The methylation status of CpG rich sites within the promoter regions support the hypothesis for interdependent machinery involving selected genes during early bone development. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2019
- Full Text:
- Date Issued: 2019
Evaluation of SNPs of G6PD, with regard to the 3D conformational, structural and stability alterations, in order to investigate the clinical implications and potential applications
- Authors: Sanabria, Natasha Mary-Anne
- Date: 2019
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/76500 , vital:30574
- Description: Expected release date-April 2020
- Full Text: false
- Date Issued: 2019
- Authors: Sanabria, Natasha Mary-Anne
- Date: 2019
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/76500 , vital:30574
- Description: Expected release date-April 2020
- Full Text: false
- Date Issued: 2019
Production, purification, and characterisation of proteases from an ericoid mycorrhizal fungus, Oidiodendron maius
- Authors: Manyumwa, Colleen Varaidzo
- Date: 2018
- Subjects: Ascomycetes , Mycorrhizal fungi , Ericaceae , Proteolytic enzymes , Silver Recycling
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/62833 , vital:28298
- Description: The aim of this study was to produce, purify and characterise proteases from the ericoid mycorrhizal fungus, Oidiodendron maius (CafRU082b/KP119480), as well as to explore their potential application in the recovery of silver from X-ray film. Firstly, the growth of the ericoid mycorrhizal fungus, Oidiodendron maius (CafRU082b), was studied, and its ability to produce proteolytic enzymes was investigated. O. maius proved to grow well in the dark, submerged in Modified Melin Norkran’s liquid medium at a pH of 5 and at 25°C. Pure cultures of the fungus were maintained on Potato Dextrose Agar (PDA). The fungus grew on PDA plates containing different substrates including haemoglobin, casein, gelatin as well as azocasein. Zones of clearance, however, were only observed on plates containing gelatin after treatment with mercuric chloride, HgCl2. Proteases were successfully produced after 14 days when gelatin was incorporated into the growth medium. After production of the proteases, purification and characterisation of the enzymes was performed. Purification of the enzymes was performed by acetone precipitation followed by ultrafiltration with 50 kDa and 30 kDa cut off membrane filters. A final purification fold of approximately 37.6 was achieved. Unusual yields of above 100% were observed after each purification step with the final yield achieved being 196% with a final specific activity of 2707 U/mg. SDS-PAGE revealed a protease band of 35 kDa which was also visible on the zymogram at approximately 36 kDa. The zymogram showed clear hydrolysis bands against a blue background after staining with Coomassie Brilliant Blue. Physico-chemical characterisation of the protease revealed its pH optimum to be pH 3.0 and its temperature optimum 68°C. Another peak was observed on the pH profile at pH 7.0. The protease exhibited high thermostability at temperatures 37°C, 80°C as well as 100°C with the enzyme retaining close to 50% of its initial activity after 4 h of exposure to all three temperatures. All ions tested for their effects on the proteases, except Ca2+, enhanced protease activity. Ca2+ did not exhibit any significant effect on the enzyme’s activity while Zn2+ had the highest effect, enhancing enzyme activity by 305%. The proteases, however, were not significantly inhibited by EDTA, a metal chelating agent and a known metalloprotease inhibitor. The enzyme was classified as an aspartic protease due to complete inhibition by 25 μM of pepstatin A, coupled to its low pH optimum of 3.0. Addition of trans-Epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E-64), a cysteine protease inhibitor, and 2-mercaptoethanol increased protease activity. The proteases exhibited a narrow substrate specificity towards gelatin and no other substrate. Substrate kinetics values were plotted on a Michaelis-Menten Graph and showed that the enzyme had a Vmax of 55.25 U/ml and a Km of 2.7 mg/ml gelatin. A low Km indicated that the protease had a high affinity for gelatin. Silver recovery studies from X-ray film revealed the proteases’ capability to remove silver from X-ray film, leaving the film intact. The recovery of silver was perceived visually, by film observation, as well as by scan electron microscopy (SEM) images, where clearance of the film was observed after incubation with the enzyme. Energy dispersive X-ray spectroscopy (EDS) profiles also confirmed removal of silver from the film, with a Ag peak showing on the profile of the film before treatment with the proteases and no peak after treatment. The crude protease sample was, however, catalytically more efficient compared to the partially purified sample. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2018
- Full Text:
- Date Issued: 2018
- Authors: Manyumwa, Colleen Varaidzo
- Date: 2018
- Subjects: Ascomycetes , Mycorrhizal fungi , Ericaceae , Proteolytic enzymes , Silver Recycling
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/62833 , vital:28298
- Description: The aim of this study was to produce, purify and characterise proteases from the ericoid mycorrhizal fungus, Oidiodendron maius (CafRU082b/KP119480), as well as to explore their potential application in the recovery of silver from X-ray film. Firstly, the growth of the ericoid mycorrhizal fungus, Oidiodendron maius (CafRU082b), was studied, and its ability to produce proteolytic enzymes was investigated. O. maius proved to grow well in the dark, submerged in Modified Melin Norkran’s liquid medium at a pH of 5 and at 25°C. Pure cultures of the fungus were maintained on Potato Dextrose Agar (PDA). The fungus grew on PDA plates containing different substrates including haemoglobin, casein, gelatin as well as azocasein. Zones of clearance, however, were only observed on plates containing gelatin after treatment with mercuric chloride, HgCl2. Proteases were successfully produced after 14 days when gelatin was incorporated into the growth medium. After production of the proteases, purification and characterisation of the enzymes was performed. Purification of the enzymes was performed by acetone precipitation followed by ultrafiltration with 50 kDa and 30 kDa cut off membrane filters. A final purification fold of approximately 37.6 was achieved. Unusual yields of above 100% were observed after each purification step with the final yield achieved being 196% with a final specific activity of 2707 U/mg. SDS-PAGE revealed a protease band of 35 kDa which was also visible on the zymogram at approximately 36 kDa. The zymogram showed clear hydrolysis bands against a blue background after staining with Coomassie Brilliant Blue. Physico-chemical characterisation of the protease revealed its pH optimum to be pH 3.0 and its temperature optimum 68°C. Another peak was observed on the pH profile at pH 7.0. The protease exhibited high thermostability at temperatures 37°C, 80°C as well as 100°C with the enzyme retaining close to 50% of its initial activity after 4 h of exposure to all three temperatures. All ions tested for their effects on the proteases, except Ca2+, enhanced protease activity. Ca2+ did not exhibit any significant effect on the enzyme’s activity while Zn2+ had the highest effect, enhancing enzyme activity by 305%. The proteases, however, were not significantly inhibited by EDTA, a metal chelating agent and a known metalloprotease inhibitor. The enzyme was classified as an aspartic protease due to complete inhibition by 25 μM of pepstatin A, coupled to its low pH optimum of 3.0. Addition of trans-Epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E-64), a cysteine protease inhibitor, and 2-mercaptoethanol increased protease activity. The proteases exhibited a narrow substrate specificity towards gelatin and no other substrate. Substrate kinetics values were plotted on a Michaelis-Menten Graph and showed that the enzyme had a Vmax of 55.25 U/ml and a Km of 2.7 mg/ml gelatin. A low Km indicated that the protease had a high affinity for gelatin. Silver recovery studies from X-ray film revealed the proteases’ capability to remove silver from X-ray film, leaving the film intact. The recovery of silver was perceived visually, by film observation, as well as by scan electron microscopy (SEM) images, where clearance of the film was observed after incubation with the enzyme. Energy dispersive X-ray spectroscopy (EDS) profiles also confirmed removal of silver from the film, with a Ag peak showing on the profile of the film before treatment with the proteases and no peak after treatment. The crude protease sample was, however, catalytically more efficient compared to the partially purified sample. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2018
- Full Text:
- Date Issued: 2018
Investigating the use of Arbuscular Mycorrhizas and Plant Growth Promoting Bacteria to improve the drought tolerance of maize (Zea mays L.)
- Authors: Moore, Nicolle Maureen
- Date: 2016
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/54587 , vital:26591
- Description: Maize (Zea mays L.) is a direct staple food crop in Africa and remains an essential component of global food security, with maize crops accounting for over 60% of the total harvested area of annual food crops. Stress caused by drought and high soil salinity limits crop growth and productivity more than any other single environmental factor, with grain yield reductions up to 76% depending on the severity of the drought and the plant growth stage. Arbuscular mycorrhizal (AM) fungi and Plant Growth Promotion Rhizobacteria (PGPR) have previously been shown to improve tolerance of plants to drought stress through a number of chemical and physiological processes. The aim of this investigation was to determine whether mycorrhizal fungi and rhizobacteria adapted to drought and saline conditions and possessing plant growth promoting (PGP) traits were able to stimulate plant growth responses when applied to Zea mays seeds growing under greenhouse conditions Bacterial isolates selected were tolerant to concentrations of NaCl up to 600 mM and maintained 50% growth at low water potentials (-1.44 MPa). They were positive for Indole Acetic Acid (IAA) production, phosphate solubilisation and secretion of siderophores. Bacterial isolates showing plant growth promoting potential were identified using 16S rDNA gene sequencing as Achromobacter xylosoxidans strains A8 and C54 and Klebsiella oxytoca strain M1. Mixed inoculum was prepared from indigenous communities of mycorrhizas in soils sampled from the Cerebos Salt Pan and the Kalahari Desert. Mycorrhizal diversity was investigated using 454-Pyrosequencing which revealed that the community composition was dominated by species in the Ambispora, Glomus and Paraglomus genera with a rare component represented by species in the Redeckera, Archaeospora and Geosiphon genera. Microscopic examination of plant roots at the end of the trial revealed the presence of diagnostic mycorrhizal structures within the root cells, confirming that colonization was successful. Plant growth response to microbial inoculation was assessed by monitoring changes in plant photosynthetic capacity over the duration of a 7 week pot trial. A significant difference in photosynthetic and biomass data was observed between drought and well-watered groups but no mycorrhizal or bacterial treatment effect was evident within the groups, despite the high levels of colonization by mycorrhizas. These results suggest that the beneficial effects of mycorrhizal colonization may be primarily attributed to improved nutrient and mineral uptake in conditions where nutrients are limiting, resulting in improved growth. The improved growth may then have secondary effects on the plant‟s ability to withstand drought. Having controlled for nutrient deficiency, it was not evident in this study that mycorrhizal fungi were able to stimulate a change in plant physiology and confer drought tolerance under the conditions imposed.
- Full Text:
- Date Issued: 2016
- Authors: Moore, Nicolle Maureen
- Date: 2016
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/54587 , vital:26591
- Description: Maize (Zea mays L.) is a direct staple food crop in Africa and remains an essential component of global food security, with maize crops accounting for over 60% of the total harvested area of annual food crops. Stress caused by drought and high soil salinity limits crop growth and productivity more than any other single environmental factor, with grain yield reductions up to 76% depending on the severity of the drought and the plant growth stage. Arbuscular mycorrhizal (AM) fungi and Plant Growth Promotion Rhizobacteria (PGPR) have previously been shown to improve tolerance of plants to drought stress through a number of chemical and physiological processes. The aim of this investigation was to determine whether mycorrhizal fungi and rhizobacteria adapted to drought and saline conditions and possessing plant growth promoting (PGP) traits were able to stimulate plant growth responses when applied to Zea mays seeds growing under greenhouse conditions Bacterial isolates selected were tolerant to concentrations of NaCl up to 600 mM and maintained 50% growth at low water potentials (-1.44 MPa). They were positive for Indole Acetic Acid (IAA) production, phosphate solubilisation and secretion of siderophores. Bacterial isolates showing plant growth promoting potential were identified using 16S rDNA gene sequencing as Achromobacter xylosoxidans strains A8 and C54 and Klebsiella oxytoca strain M1. Mixed inoculum was prepared from indigenous communities of mycorrhizas in soils sampled from the Cerebos Salt Pan and the Kalahari Desert. Mycorrhizal diversity was investigated using 454-Pyrosequencing which revealed that the community composition was dominated by species in the Ambispora, Glomus and Paraglomus genera with a rare component represented by species in the Redeckera, Archaeospora and Geosiphon genera. Microscopic examination of plant roots at the end of the trial revealed the presence of diagnostic mycorrhizal structures within the root cells, confirming that colonization was successful. Plant growth response to microbial inoculation was assessed by monitoring changes in plant photosynthetic capacity over the duration of a 7 week pot trial. A significant difference in photosynthetic and biomass data was observed between drought and well-watered groups but no mycorrhizal or bacterial treatment effect was evident within the groups, despite the high levels of colonization by mycorrhizas. These results suggest that the beneficial effects of mycorrhizal colonization may be primarily attributed to improved nutrient and mineral uptake in conditions where nutrients are limiting, resulting in improved growth. The improved growth may then have secondary effects on the plant‟s ability to withstand drought. Having controlled for nutrient deficiency, it was not evident in this study that mycorrhizal fungi were able to stimulate a change in plant physiology and confer drought tolerance under the conditions imposed.
- Full Text:
- Date Issued: 2016
Exploring the structural integrity of a picornavirus capsid
- Authors: Upfold, Nicole Sarah
- Date: 2020
- Subjects: Picornaviruses , Immunoglobulins , Capsids (Virology) , Viruses Morphology , RNA viruses
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/131837 , vital:36758 , DOI https://doi.org/10.21504/10962/131837
- Description: Picornaviruses are a diverse family of small RNA viruses that cause a broad range of human and veterinary diseases. Despite decades of research into the molecular biology of these pathogens, no antivirals and few vaccines are commercially available for the treatment and prevention of picornavirus infections. The capsids of these non-enveloped viruses are involved in many important aspects of the picornavirus lifecycle, such as cell attachment and entry, uncoating, and protection of the viral RNA. Although the structures of many picornavirus capsids have been solved, a broader understanding of the molecular determinants that are required for structural integrity and stability is imperative for an improved understanding of the basic biology of these viruses, and for designing effective control strategies. Collectively, this thesis aims to elucidate the molecular determinants of structural stability and integrity in the Theiler’s murine encephalomyelitis virus capsid (TMEV). To study the TMEV GDVII capsid using biochemical techniques, neutralising polyclonal antibodies were generated against GDVII particles. The antibodies recognised linear epitopes in the C-terminus of the VP1 protein, but not those present in VP2 or VP3. The VP1 C-terminal residues were mapped to a loop above the putative receptor binding pit on the capsid surface, which prompted an investigation into the potential binding site of the TMEV co-receptor, heparan sulphate. Molecular docking revealed that heparan interacts with residues of the receptor binding pocket, as well as residues of the adjoining VP1 C-terminal loop. These findings suggest that the antibodies neutralise virus infection by preventing attachment of the virus to the co-receptor and possibly the unknown primary receptor. Few studies have identified the specific residues and interactions at subunit interfaces that significantly contribute to picornavirus capsid stability, assembly, and function. A novel in-silico screen was developed for the prediction of hotspot residues at protein-protein interfaces of a virus capsid. This screen can be applied to elucidate the residues that contribute significantly to the intraprotomer, interprotomer and interpentamer interfaces of any picornavirus capsid, on condition that the structure of the virus is available. The screen was applied to TMEV GDVII resulting in the identification of hotspots, several of which correspond to residues that are known to be important for aspects of the virus lifecycle, such as those that contribute to pH stability or form part of receptor binding sites. This observation suggests that residues involved in specific capsid functions may also play a role in capsid stability. Many of the residues identified as hotspots in TMEV corresponded to those required for assembly, uncoating, and virus growth in representative picornaviruses from various genera, suggesting that the residues that regulate capsid stability may be somewhat conserved across the family. Hotspots identified at the interpentamer interfaces of TMEV were individually substituted to alanine to further explore their importance to the TMEV lifecycle. All the amino acid substitutions prevented completion of the virus lifecycle as no CPE was observed following transfection of susceptible cells. Immunofluorescence experiments demonstrated that virus protein synthesis and RNA replication were not inhibited by substitution of the hotspot residues, but that infectivity was severely impeded. This confirmed that the residues were required for some aspect of the virus lifecycle, such as capsid assembly, or were critical for maintaining the conformational stability of the TMEV particles. Virus capsids become unstable and are prone to dissociation under certain conditions such as extreme pH and non-physiological temperatures. The thermostability of TMEV was explored by selecting GDVII virions with improved thermal tolerance through serial passage and heat exposure. Thermostable virions that could tolerate temperatures above 57 °C had reduced infective titres compared to the wild type TMEV suggesting that the virus adapted to thermal stress at the expense of viral fitness. Sequencing the capsid encoding regions of the mutant virions revealed a pair of amino acid substitutions that were present in all mutants. Additional substitutions that were unique to viruses selected at different temperatures were also identified. Most of the substitutions were located within the intraprotomer interfaces of the virus, unlike previous studies on enteroviruses where mutations were mostly localised to the receptor binding pocket. This thesis provides the first analysis of the structural determinants of TMEV capsid stability. The generation of tools to further explore the capsid structures of TMEV and other picornaviruses provides an opportunity for future studies which may contribute to the development of novel control strategies against this important family of viruses. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2020
- Full Text: false
- Date Issued: 2020
- Authors: Upfold, Nicole Sarah
- Date: 2020
- Subjects: Picornaviruses , Immunoglobulins , Capsids (Virology) , Viruses Morphology , RNA viruses
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/131837 , vital:36758 , DOI https://doi.org/10.21504/10962/131837
- Description: Picornaviruses are a diverse family of small RNA viruses that cause a broad range of human and veterinary diseases. Despite decades of research into the molecular biology of these pathogens, no antivirals and few vaccines are commercially available for the treatment and prevention of picornavirus infections. The capsids of these non-enveloped viruses are involved in many important aspects of the picornavirus lifecycle, such as cell attachment and entry, uncoating, and protection of the viral RNA. Although the structures of many picornavirus capsids have been solved, a broader understanding of the molecular determinants that are required for structural integrity and stability is imperative for an improved understanding of the basic biology of these viruses, and for designing effective control strategies. Collectively, this thesis aims to elucidate the molecular determinants of structural stability and integrity in the Theiler’s murine encephalomyelitis virus capsid (TMEV). To study the TMEV GDVII capsid using biochemical techniques, neutralising polyclonal antibodies were generated against GDVII particles. The antibodies recognised linear epitopes in the C-terminus of the VP1 protein, but not those present in VP2 or VP3. The VP1 C-terminal residues were mapped to a loop above the putative receptor binding pit on the capsid surface, which prompted an investigation into the potential binding site of the TMEV co-receptor, heparan sulphate. Molecular docking revealed that heparan interacts with residues of the receptor binding pocket, as well as residues of the adjoining VP1 C-terminal loop. These findings suggest that the antibodies neutralise virus infection by preventing attachment of the virus to the co-receptor and possibly the unknown primary receptor. Few studies have identified the specific residues and interactions at subunit interfaces that significantly contribute to picornavirus capsid stability, assembly, and function. A novel in-silico screen was developed for the prediction of hotspot residues at protein-protein interfaces of a virus capsid. This screen can be applied to elucidate the residues that contribute significantly to the intraprotomer, interprotomer and interpentamer interfaces of any picornavirus capsid, on condition that the structure of the virus is available. The screen was applied to TMEV GDVII resulting in the identification of hotspots, several of which correspond to residues that are known to be important for aspects of the virus lifecycle, such as those that contribute to pH stability or form part of receptor binding sites. This observation suggests that residues involved in specific capsid functions may also play a role in capsid stability. Many of the residues identified as hotspots in TMEV corresponded to those required for assembly, uncoating, and virus growth in representative picornaviruses from various genera, suggesting that the residues that regulate capsid stability may be somewhat conserved across the family. Hotspots identified at the interpentamer interfaces of TMEV were individually substituted to alanine to further explore their importance to the TMEV lifecycle. All the amino acid substitutions prevented completion of the virus lifecycle as no CPE was observed following transfection of susceptible cells. Immunofluorescence experiments demonstrated that virus protein synthesis and RNA replication were not inhibited by substitution of the hotspot residues, but that infectivity was severely impeded. This confirmed that the residues were required for some aspect of the virus lifecycle, such as capsid assembly, or were critical for maintaining the conformational stability of the TMEV particles. Virus capsids become unstable and are prone to dissociation under certain conditions such as extreme pH and non-physiological temperatures. The thermostability of TMEV was explored by selecting GDVII virions with improved thermal tolerance through serial passage and heat exposure. Thermostable virions that could tolerate temperatures above 57 °C had reduced infective titres compared to the wild type TMEV suggesting that the virus adapted to thermal stress at the expense of viral fitness. Sequencing the capsid encoding regions of the mutant virions revealed a pair of amino acid substitutions that were present in all mutants. Additional substitutions that were unique to viruses selected at different temperatures were also identified. Most of the substitutions were located within the intraprotomer interfaces of the virus, unlike previous studies on enteroviruses where mutations were mostly localised to the receptor binding pocket. This thesis provides the first analysis of the structural determinants of TMEV capsid stability. The generation of tools to further explore the capsid structures of TMEV and other picornaviruses provides an opportunity for future studies which may contribute to the development of novel control strategies against this important family of viruses. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2020
- Full Text: false
- Date Issued: 2020
Molecular cloning and expression of equine CYP1A2 in Escherichia coli
- Authors: Mkabayi, Lithalethu
- Date: 2017
- Subjects: Escherichia coli , Escherichia coli infections in animals , Cytochrome P-450 , Cytochromes , Horses -- Effect of drugs on
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/4830 , vital:20734
- Description: Information regarding drug metabolism in veterinary species, especially horses, remains fragmented and incomplete. This information is essential for detection of metabolites of potential performance-enhancing substances in horseracing and for veterinary drug development. Equine liver microsomes have been used to study metabolism of a limited number of drugs, but these provide little information about individual drug metabolizing enzymes. Recombinant CYP enzyme systems are commonly used to determine contribution of individual CYP to metabolism of specific drugs. A limited number of recombinant equine CYPs have been expressed in insect cells and mammalian cell lines. However, there are no reports of recombinant equine CYP1A2 enzyme. In this study, equine CYP1A2 was identified, codon-optimized, cloned and expressed in E. coli BL21 cells. Multiple sequence alignments of equine CYP1A2 revealed an amino acid sequence identity of 83.69% to its human homolog which has previously been expressed in E. coli. The enzyme was expressed using both auto-induction and IPTG induction. Expressed equine CYP1A2 had a size of about 55 kDa, and was insoluble after cell lysis. Sarkosyl- solubilized CYP1A2 was purified using nickel affinity chromatography and gel filtration. For activity reconstitution, yeast NADPH-cytochrome P450 reductase was first expressed in E. coli BL21 cells and exhibited activity of 0.13 U/ml. Activity assay with Glo-P450 CYP1A2 assay kit indicated that CYP1A2 was inactive. Despite numerous attempts to obtain the activity, the CYP1A2 remained inactive. Although expression of equine CYP1A2 in E. coli produced non- catalytically active enzyme, this study could be used as the first step in an effort to fully develop a recombinant equine CYP1A2 system.
- Full Text:
- Date Issued: 2017
- Authors: Mkabayi, Lithalethu
- Date: 2017
- Subjects: Escherichia coli , Escherichia coli infections in animals , Cytochrome P-450 , Cytochromes , Horses -- Effect of drugs on
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/4830 , vital:20734
- Description: Information regarding drug metabolism in veterinary species, especially horses, remains fragmented and incomplete. This information is essential for detection of metabolites of potential performance-enhancing substances in horseracing and for veterinary drug development. Equine liver microsomes have been used to study metabolism of a limited number of drugs, but these provide little information about individual drug metabolizing enzymes. Recombinant CYP enzyme systems are commonly used to determine contribution of individual CYP to metabolism of specific drugs. A limited number of recombinant equine CYPs have been expressed in insect cells and mammalian cell lines. However, there are no reports of recombinant equine CYP1A2 enzyme. In this study, equine CYP1A2 was identified, codon-optimized, cloned and expressed in E. coli BL21 cells. Multiple sequence alignments of equine CYP1A2 revealed an amino acid sequence identity of 83.69% to its human homolog which has previously been expressed in E. coli. The enzyme was expressed using both auto-induction and IPTG induction. Expressed equine CYP1A2 had a size of about 55 kDa, and was insoluble after cell lysis. Sarkosyl- solubilized CYP1A2 was purified using nickel affinity chromatography and gel filtration. For activity reconstitution, yeast NADPH-cytochrome P450 reductase was first expressed in E. coli BL21 cells and exhibited activity of 0.13 U/ml. Activity assay with Glo-P450 CYP1A2 assay kit indicated that CYP1A2 was inactive. Despite numerous attempts to obtain the activity, the CYP1A2 remained inactive. Although expression of equine CYP1A2 in E. coli produced non- catalytically active enzyme, this study could be used as the first step in an effort to fully develop a recombinant equine CYP1A2 system.
- Full Text:
- Date Issued: 2017
An investigation into the bacterial communities associated with pyrroloiminoquinone-producing South African latrunculid sponges
- Authors: Hilliar, Storm Hannah
- Date: 2018
- Subjects: Sponges South Africa Algoa Bay , Betaproteobacteria , Spirochaeta , Symbiosis , Bacterial communities
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/62112 , vital:28128
- Description: Marine sponges belonging to the family Latrunculiidae are known for their production of cytotoxic pyrroloiminoquinone alkaloids and the South African coast provides a unique environment for the exploitation of these potent bioactive compounds. The isolation of structurally similar pyrroloiminoquinone compounds from unrelated, non poriferan sources has led to the suggestion that South African latrunculid pyrroloiminoquinones may be secondary metabolites produced by sponge associated microbial symbionts. Previous studies investigating the bacterial communities of South African latrunculid sponges have shown the conservation of distinct microbial populations with unusual bacterial taxa dominated by a novel betaproteobacterial and spirochete species. This study describes the further investigation into these associated bacterial communities, their conservation and sponge microbiome comparisons across spatial, temporal and environmental scales. The bacterial communities associated with seven latrunculid species representing three genera (Tsitsikamma, Cyclacanthia and Latrunculia) were characterized as well as a Mycale and Tethya rubra species. Latrunculid sponge microbiomes were significantly different from those associated with sympatric outlier sponge species and the surrounding environment. The bacterial communities associated with latrunculid sponges appear host specific with the conservation of two dominant bacterial symbionts which mirror the phylogeny of their host species. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2018
- Full Text:
- Date Issued: 2018
- Authors: Hilliar, Storm Hannah
- Date: 2018
- Subjects: Sponges South Africa Algoa Bay , Betaproteobacteria , Spirochaeta , Symbiosis , Bacterial communities
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/62112 , vital:28128
- Description: Marine sponges belonging to the family Latrunculiidae are known for their production of cytotoxic pyrroloiminoquinone alkaloids and the South African coast provides a unique environment for the exploitation of these potent bioactive compounds. The isolation of structurally similar pyrroloiminoquinone compounds from unrelated, non poriferan sources has led to the suggestion that South African latrunculid pyrroloiminoquinones may be secondary metabolites produced by sponge associated microbial symbionts. Previous studies investigating the bacterial communities of South African latrunculid sponges have shown the conservation of distinct microbial populations with unusual bacterial taxa dominated by a novel betaproteobacterial and spirochete species. This study describes the further investigation into these associated bacterial communities, their conservation and sponge microbiome comparisons across spatial, temporal and environmental scales. The bacterial communities associated with seven latrunculid species representing three genera (Tsitsikamma, Cyclacanthia and Latrunculia) were characterized as well as a Mycale and Tethya rubra species. Latrunculid sponge microbiomes were significantly different from those associated with sympatric outlier sponge species and the surrounding environment. The bacterial communities associated with latrunculid sponges appear host specific with the conservation of two dominant bacterial symbionts which mirror the phylogeny of their host species. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2018
- Full Text:
- Date Issued: 2018
Chitin hydrolysis with chitinolytic enzymes for the production of chitooligomers with antimicrobial properties
- Authors: Oree, Glynis
- Date: 2019
- Subjects: Chitin -- Biotechnology , Enzymes -- Biotechnology , Hydrolysis , Chitooligomers -- Biotechnology
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/67887 , vital:29165
- Description: There are many diseases and illnesses in the world that require new drug treatments and chitin has been shown to produce chitooligomeric derivatives which exhibit promising antimicrobial and immune-enhancing properties. However, the rate-limiting step is associated with the high recalcitrance of chitinous substrates, and low hydrolytic activities of chitinolytic enzymes, resulting in low product release. To improve and create a more sustainable and economical process, enhancing chitin hydrolysis through various treatment procedures is essential for obtaining high enzyme hydrolysis rates, resulting in a higher yield of chitooligomers (CHOS). In literature, pre-treatment of insoluble biomass is generally associated with an increase in accessibility of the carbohydrate to hydrolytic enzymes, thus generating more products. The first part of this study investigated the effect of alkali- (NaOH) and acid pre-treatments (HCl and phosphoric acid) on chitin biomass, and chemical and morphological modifications were assessed by the employment of scanning electron microscopy (SEM), Fourier Transform Infrared Spectroscopy (FTIR), Energy-Dispersive X-ray spectrometery (EDX) and x-ray diffraction (XRD). Data obtained confirmed that pre-treated substrates were more chemically and morphologically modified. These results confirmed the fact that pre-treatment of chitin disrupts the structure of the biomass, rendering the polymer more accessible for enzymatic hydrolysis. The commercial chitinases from Bacillus cereus and Streptomyces griseus (CHB and CHS) are costly. Bio-prospecting for other chitin-degrading enzymes from alternate sources such as Oidiodendron maius, or the recombinant expression of CHOS, was a more economically feasible avenue. The chit1 gene from Thermomyces lanuginosus, expressed in Pichia pastoris, produced a large range CHOS with a degree of polymerisation (DP) ranging from 1 to above 6. TLC analysis showed that O. maius exhibited chitin-degrading properties by producing CHOS with a DP length of 1 to 3. These two sources were therefore successful in producing chitin-degrading enzymes. The physico-chemical properties of commercial (CHB and CHS) and expressed (Chit1) chitinolytic enzymes were investigated, to determine under which biochemical conditions and on which type of biomass they can function on optimally, for the production of value-added products such as CHOS. Substrate affinity assays were conducted on the un-treated and pre-treated biomass. TLC revealed that chitosan hydrolysis by the commercial chitinases produced the largest range of CHOS with a DP length ranging from 1 to 6. A range of temperatures (35-90oC) were investigated and CHB, CHS and Chit1 displayed optimum activities at 50, 40 and 45 oC, respectively. Thermostability studies that were conducted at 37 and 50oC revealed that CHB and CHS were most stable at 37oC. Chit1 showed great thermostablity at both temperatures, rendering this enzyme suitable for industrial processes at high temperatures. pH optima studies demonstrated that the pH optima for CHB, CHS and Chit1 was at a pH of 5.0, with specific activities of 33.459, 46.2 and 5.776 μmol/h/mg, respectively. The chain cleaving patterns of the commercial enzymes were determined and exo-chitinase activity was exhibited, due to the production of CHOS that were predominantly of a DP length of 2. Enzyme binary synergy studies were conducted with commercial chitinases (CHB and CHS) on colloidal chitin. Studies illustrated that the simultaneous combination of CHB 75%: CHS 25% produced the highest specific activity (3.526 μmol/h/mg), with no synergy. TLC analysis of this enzyme combination over time revealed that predominantly chitobiose was produced. This suggested that the substrate crystallinity and morphology played an important role in the way the enzymes cleaved the carbohydrate. Since CHOS have shown great promise for their antimicrobial properties, the CHOS generated from the chitinous substrates were tested for antimicrobial properties on Bacillus subtilis, Escherichia coli, Klebsiella and Staphlococcus aureus. This study revealed that certain CHOS produced have inhibitory effects on certain bacteria and could potentially be used in the pharamceutical or medical industries. In conclusion, this study revealed that chitinases can be produced and found in alternate sources and be used for the hydrolysis of chitinous biomass in a more sustainabe and economically viable manner. The chitinases investigated (CHB, CHS and Chit1) exhibited different cleaving patterns of the chitinous substrates due to the chemical and morphological properties of the biomass. CHOS produced from chitinous biomass exhibited some inhibitory effects on bacterial growth and show potential for use in the medical industry.
- Full Text:
- Date Issued: 2019
- Authors: Oree, Glynis
- Date: 2019
- Subjects: Chitin -- Biotechnology , Enzymes -- Biotechnology , Hydrolysis , Chitooligomers -- Biotechnology
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/67887 , vital:29165
- Description: There are many diseases and illnesses in the world that require new drug treatments and chitin has been shown to produce chitooligomeric derivatives which exhibit promising antimicrobial and immune-enhancing properties. However, the rate-limiting step is associated with the high recalcitrance of chitinous substrates, and low hydrolytic activities of chitinolytic enzymes, resulting in low product release. To improve and create a more sustainable and economical process, enhancing chitin hydrolysis through various treatment procedures is essential for obtaining high enzyme hydrolysis rates, resulting in a higher yield of chitooligomers (CHOS). In literature, pre-treatment of insoluble biomass is generally associated with an increase in accessibility of the carbohydrate to hydrolytic enzymes, thus generating more products. The first part of this study investigated the effect of alkali- (NaOH) and acid pre-treatments (HCl and phosphoric acid) on chitin biomass, and chemical and morphological modifications were assessed by the employment of scanning electron microscopy (SEM), Fourier Transform Infrared Spectroscopy (FTIR), Energy-Dispersive X-ray spectrometery (EDX) and x-ray diffraction (XRD). Data obtained confirmed that pre-treated substrates were more chemically and morphologically modified. These results confirmed the fact that pre-treatment of chitin disrupts the structure of the biomass, rendering the polymer more accessible for enzymatic hydrolysis. The commercial chitinases from Bacillus cereus and Streptomyces griseus (CHB and CHS) are costly. Bio-prospecting for other chitin-degrading enzymes from alternate sources such as Oidiodendron maius, or the recombinant expression of CHOS, was a more economically feasible avenue. The chit1 gene from Thermomyces lanuginosus, expressed in Pichia pastoris, produced a large range CHOS with a degree of polymerisation (DP) ranging from 1 to above 6. TLC analysis showed that O. maius exhibited chitin-degrading properties by producing CHOS with a DP length of 1 to 3. These two sources were therefore successful in producing chitin-degrading enzymes. The physico-chemical properties of commercial (CHB and CHS) and expressed (Chit1) chitinolytic enzymes were investigated, to determine under which biochemical conditions and on which type of biomass they can function on optimally, for the production of value-added products such as CHOS. Substrate affinity assays were conducted on the un-treated and pre-treated biomass. TLC revealed that chitosan hydrolysis by the commercial chitinases produced the largest range of CHOS with a DP length ranging from 1 to 6. A range of temperatures (35-90oC) were investigated and CHB, CHS and Chit1 displayed optimum activities at 50, 40 and 45 oC, respectively. Thermostability studies that were conducted at 37 and 50oC revealed that CHB and CHS were most stable at 37oC. Chit1 showed great thermostablity at both temperatures, rendering this enzyme suitable for industrial processes at high temperatures. pH optima studies demonstrated that the pH optima for CHB, CHS and Chit1 was at a pH of 5.0, with specific activities of 33.459, 46.2 and 5.776 μmol/h/mg, respectively. The chain cleaving patterns of the commercial enzymes were determined and exo-chitinase activity was exhibited, due to the production of CHOS that were predominantly of a DP length of 2. Enzyme binary synergy studies were conducted with commercial chitinases (CHB and CHS) on colloidal chitin. Studies illustrated that the simultaneous combination of CHB 75%: CHS 25% produced the highest specific activity (3.526 μmol/h/mg), with no synergy. TLC analysis of this enzyme combination over time revealed that predominantly chitobiose was produced. This suggested that the substrate crystallinity and morphology played an important role in the way the enzymes cleaved the carbohydrate. Since CHOS have shown great promise for their antimicrobial properties, the CHOS generated from the chitinous substrates were tested for antimicrobial properties on Bacillus subtilis, Escherichia coli, Klebsiella and Staphlococcus aureus. This study revealed that certain CHOS produced have inhibitory effects on certain bacteria and could potentially be used in the pharamceutical or medical industries. In conclusion, this study revealed that chitinases can be produced and found in alternate sources and be used for the hydrolysis of chitinous biomass in a more sustainabe and economically viable manner. The chitinases investigated (CHB, CHS and Chit1) exhibited different cleaving patterns of the chitinous substrates due to the chemical and morphological properties of the biomass. CHOS produced from chitinous biomass exhibited some inhibitory effects on bacterial growth and show potential for use in the medical industry.
- Full Text:
- Date Issued: 2019
Elucidation of a novel role for HSP70/HSP90 organising protein (Hop) in mRNA processing
- Dingle, Laura Margaret Kirkpatrick
- Authors: Dingle, Laura Margaret Kirkpatrick
- Date: 2020
- Language: English
- Type: thesis , text , Doctoral , Ph.D
- Identifier: http://hdl.handle.net/10962/59173 , vital:27449 , doi:10.21504/10962/59173
- Description: Thesis (PhD.)--Rhodes University, Faculty of Science, Biochemistry and Microbiology, 2020.
- Full Text:
- Date Issued: 2020
- Authors: Dingle, Laura Margaret Kirkpatrick
- Date: 2020
- Language: English
- Type: thesis , text , Doctoral , Ph.D
- Identifier: http://hdl.handle.net/10962/59173 , vital:27449 , doi:10.21504/10962/59173
- Description: Thesis (PhD.)--Rhodes University, Faculty of Science, Biochemistry and Microbiology, 2020.
- Full Text:
- Date Issued: 2020
In silico analysis of human Hsp90 for the identification of novel anti-cancer drug target sites and natural compound inhibitors
- Authors: Penkler, David Lawrence
- Date: 2015
- Subjects: Heat shock proteins , Cancer -- Treatment , Molecular chaperones , Homeostasis , Carcinogenesis , Chemotherapy , Ligand binding (Biochemistry) , Protein-protein interactions
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4162 , http://hdl.handle.net/10962/d1018938
- Description: The 90-KDa heat shock protein (Hsp90) is part of the molecular chaperone family, and as such it is involved in the regulation of protein homeostasis within cells. Specifically, Hsp90 aids in the folding of nascent proteins and re-folding of denatured proteins. It also plays an important role in the prevention of protein aggregation. Hsp90’s functionality is attributed to its several staged, multi-conformational ATPase cycle, in which associated client proteins are bound and released. Hsp90 is known to be associated with a wide array of client proteins, some of which are thought to be involved in multiple oncogenic processes. Indeed Hsp90 is known to be directly involved in perpetuating the stability and function of multiple mutated, chimeric and over-expressed signalling proteins that are known to promote the growth and survival of cancer cells. Hsp90 inhibitors are thus thought to be promising therapeutic agents for cancer treatment. A lack of a 3D structure of human Hsp90 however has restricted Hsp90 inhibitor development in large to in vivo investigations. This study, aims to investigate and calculate hypothetical homology models of the full human Hsp90 protein, and to probe these structural models for novel drug target sites using several in silico techniques. A multi-template homology modelling methodology was developed and in conjunction with protein-protein docking techniques, two functionally important human Hsp90 structural models were calculated; the nucleotide free “v-like” open and nucleotide bound closed conformations. Based on the conservation of ligand binding, virtual screening experiments conducted on both models using 316 natural compounds indigenous to South Africa, revealed three novel putative target sites. Two binding pockets in close association with important Hsp90-Hop interaction residues and a single binding pocket on the dimerization interface in the C-terminal domain. Targeted molecular docking experiments at these sites revealed two compounds (721395-11-5 and 264624-39-7) as putative inhibitors, both showing strong binding affinities for at least one of the three investigated target sites. Furthermore both compounds were found to only violate one Lipinski’s rules, suggesting their potential as candidates for further drug development. The combined work described here provides a putative platform for the development of next generation inhibitors of human Hsp90.
- Full Text:
- Date Issued: 2015
- Authors: Penkler, David Lawrence
- Date: 2015
- Subjects: Heat shock proteins , Cancer -- Treatment , Molecular chaperones , Homeostasis , Carcinogenesis , Chemotherapy , Ligand binding (Biochemistry) , Protein-protein interactions
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4162 , http://hdl.handle.net/10962/d1018938
- Description: The 90-KDa heat shock protein (Hsp90) is part of the molecular chaperone family, and as such it is involved in the regulation of protein homeostasis within cells. Specifically, Hsp90 aids in the folding of nascent proteins and re-folding of denatured proteins. It also plays an important role in the prevention of protein aggregation. Hsp90’s functionality is attributed to its several staged, multi-conformational ATPase cycle, in which associated client proteins are bound and released. Hsp90 is known to be associated with a wide array of client proteins, some of which are thought to be involved in multiple oncogenic processes. Indeed Hsp90 is known to be directly involved in perpetuating the stability and function of multiple mutated, chimeric and over-expressed signalling proteins that are known to promote the growth and survival of cancer cells. Hsp90 inhibitors are thus thought to be promising therapeutic agents for cancer treatment. A lack of a 3D structure of human Hsp90 however has restricted Hsp90 inhibitor development in large to in vivo investigations. This study, aims to investigate and calculate hypothetical homology models of the full human Hsp90 protein, and to probe these structural models for novel drug target sites using several in silico techniques. A multi-template homology modelling methodology was developed and in conjunction with protein-protein docking techniques, two functionally important human Hsp90 structural models were calculated; the nucleotide free “v-like” open and nucleotide bound closed conformations. Based on the conservation of ligand binding, virtual screening experiments conducted on both models using 316 natural compounds indigenous to South Africa, revealed three novel putative target sites. Two binding pockets in close association with important Hsp90-Hop interaction residues and a single binding pocket on the dimerization interface in the C-terminal domain. Targeted molecular docking experiments at these sites revealed two compounds (721395-11-5 and 264624-39-7) as putative inhibitors, both showing strong binding affinities for at least one of the three investigated target sites. Furthermore both compounds were found to only violate one Lipinski’s rules, suggesting their potential as candidates for further drug development. The combined work described here provides a putative platform for the development of next generation inhibitors of human Hsp90.
- Full Text:
- Date Issued: 2015
Characterisation of the HSP70-HSP90 organising protein gene and its link to cancer
- Authors: Weeks, Stacey
- Date: 2015
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/56006 , vital:26764
- Description: HOP (Heat shock protein 70/ Heat shock protein 90 organising protein) is a co-chaperone essential for client protein transfer from HSP70 to HSP90 within the HSP90 chaperone machine and has been found to be up-regulated in various cancers. However, minimal in vitro information can be found on the regulation of HOP expression. The aim of this study was to analyse the HOP gene structure across known orthologues, identify and characterise the HOP promoter, and identify the regulatory mechanisms influencing the expression of HOP in cancer. We hypothesized that the expression of HOP in cancer cells is likely regulated by oncogenic signalling pathways linked to cis-elements within the HOP promoter. An initial study of the evolution of the HOP gene speciation was performed across identified orthologues using Mega5.2. The evolutionary pathway of the HOP gene was traced from the unicellular organisms to fish, to amphibian and then to land mammal. The synteny across the orthologues was identified and the co-expression profile of HOP analysed. We identified the putative promoter region for HOP in silico and in vitro. Luciferase reporter assays were utilized to demonstrate promoter activity of the upstream region in vitro. Bioinformatic analysis of the active promoter region identified a large CpG island and a range of putative cis-elements. Many of the cis-elements interact with transcription factors which are activated by oncogenic pathways. We therefore tested the regulation of HOP levels by rat sarcoma viral oncogene homologue (RAS). Cancer cell lines were transfected with mutated RAS to observe the effect of constitutively active RAS expression on the production of HOP using qRT-PCR and Western Blot analyses. Additionally, inhibitors of the RAS signalling pathway were utilised to confirm the regulatory effect of mutated RAS on HOP expression. In cancer cell lines containing mutated RAS (Hs578T), HOP was up-regulated via a mechanism involving the MAPK signalling pathway and the ETS-1 and C/EBPβ cis-elements within the HOP promoter. These findings suggest for the first time that Hop expression in cancer may be regulated by RAS activation of the HOP promoter. Additionally, this study allowed us to determine the murine system to be the most suited genetic model organism with which to study the function of human HOP.
- Full Text:
- Date Issued: 2015
- Authors: Weeks, Stacey
- Date: 2015
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/56006 , vital:26764
- Description: HOP (Heat shock protein 70/ Heat shock protein 90 organising protein) is a co-chaperone essential for client protein transfer from HSP70 to HSP90 within the HSP90 chaperone machine and has been found to be up-regulated in various cancers. However, minimal in vitro information can be found on the regulation of HOP expression. The aim of this study was to analyse the HOP gene structure across known orthologues, identify and characterise the HOP promoter, and identify the regulatory mechanisms influencing the expression of HOP in cancer. We hypothesized that the expression of HOP in cancer cells is likely regulated by oncogenic signalling pathways linked to cis-elements within the HOP promoter. An initial study of the evolution of the HOP gene speciation was performed across identified orthologues using Mega5.2. The evolutionary pathway of the HOP gene was traced from the unicellular organisms to fish, to amphibian and then to land mammal. The synteny across the orthologues was identified and the co-expression profile of HOP analysed. We identified the putative promoter region for HOP in silico and in vitro. Luciferase reporter assays were utilized to demonstrate promoter activity of the upstream region in vitro. Bioinformatic analysis of the active promoter region identified a large CpG island and a range of putative cis-elements. Many of the cis-elements interact with transcription factors which are activated by oncogenic pathways. We therefore tested the regulation of HOP levels by rat sarcoma viral oncogene homologue (RAS). Cancer cell lines were transfected with mutated RAS to observe the effect of constitutively active RAS expression on the production of HOP using qRT-PCR and Western Blot analyses. Additionally, inhibitors of the RAS signalling pathway were utilised to confirm the regulatory effect of mutated RAS on HOP expression. In cancer cell lines containing mutated RAS (Hs578T), HOP was up-regulated via a mechanism involving the MAPK signalling pathway and the ETS-1 and C/EBPβ cis-elements within the HOP promoter. These findings suggest for the first time that Hop expression in cancer may be regulated by RAS activation of the HOP promoter. Additionally, this study allowed us to determine the murine system to be the most suited genetic model organism with which to study the function of human HOP.
- Full Text:
- Date Issued: 2015
The Role of HOP in Emerin-Mediated Nuclear Structure
- Authors: Kituyi, Sarah Naulikha
- Date: 2017
- Subjects: Heat shock proteins , Nuclear structure , Nuclear membranes , Cancer Treatment , Molecular chaperones , Cytoskeleton , Cytoplasm
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/59230 , vital:27485 , DOI 10.21504/10962/59230
- Description: A vital component of the integral nuclear membrane is emerin, a Lamin Emerin and Man1 (LEM) domain protein whose concentration determines the levels of partner proteins that together constitute the structure of the nuclear envelope. Deficiencies in any of these proteins causes the failure of the structure and assembly and disassembly of the nuclear envelope, which disrupts chromosome segregation and nuclear compartmentalization that are both associated with disease. Emerin also localizes in the cytoplasm where it is implicated in the structure of the cytoskeleton via interaction with tubulin and actin and thus its deficiency may equally contribute to the collapse of the cytoskeleton. The Hsp70-Hsp90 organising protein (Hop) functions as a cochaperone for entry of client proteins into the Hsp90 folding cycle. Hop is upregulated in cancer and regulates a number of cell biology processes via interactions with proteins independently of Hsp90. In a previous study using global whole cell mass spectrometry, emerin was shown to be the most significantly down regulated protein in Hop depleted cell lysates. In this current study, it was postulated that emerin interacts with Hop, and this interaction regulates the stability, and level of emerin in the nucleus which impacts on the structure of the nuclear envelope. We used HEK293T cell lines stably expressing shRNA against Hop, emerin and a non-targeting control alongside the over expression of Hop in HEK293 cells to determine the effect of Hop levels on emerin expression and vice versa via Western blotting. The effect of Hop on the localization of emerin was assessed via subcellullar fractionation and confocal microscopy, while the impact on the structure of the nucleus was determined by transmission electron microscopy (TEM). We established that the depletion of Hop using shRNA and the over expression of Hop both result in the proteasomal and lysosomal degradation of emerin. Co-immunoprecipitation assays confirmed that Hop and emerin are in a common complex, which was not dependent on the presence of Hsp90. Loss of Hop or emerin led to a deformation of nuclear structure and a statistically significant decrease in nuclear size compared to control cells and was associated with an increase in the levels of nuclear protein, lamin A-C. Loss of emerin and Hop resulted in increased long term cell survival, but only after restriction of the nucleus when the cells had migrated across a transwell membrane. Taken together, the results obtained suggest that Hop acts as a scaffold for the stabilization of emerin and that the effects of Hop depletion on the structure of the nucleus and long term survival are mediated via the depletion of emerin. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2017
- Full Text:
- Date Issued: 2017
- Authors: Kituyi, Sarah Naulikha
- Date: 2017
- Subjects: Heat shock proteins , Nuclear structure , Nuclear membranes , Cancer Treatment , Molecular chaperones , Cytoskeleton , Cytoplasm
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/59230 , vital:27485 , DOI 10.21504/10962/59230
- Description: A vital component of the integral nuclear membrane is emerin, a Lamin Emerin and Man1 (LEM) domain protein whose concentration determines the levels of partner proteins that together constitute the structure of the nuclear envelope. Deficiencies in any of these proteins causes the failure of the structure and assembly and disassembly of the nuclear envelope, which disrupts chromosome segregation and nuclear compartmentalization that are both associated with disease. Emerin also localizes in the cytoplasm where it is implicated in the structure of the cytoskeleton via interaction with tubulin and actin and thus its deficiency may equally contribute to the collapse of the cytoskeleton. The Hsp70-Hsp90 organising protein (Hop) functions as a cochaperone for entry of client proteins into the Hsp90 folding cycle. Hop is upregulated in cancer and regulates a number of cell biology processes via interactions with proteins independently of Hsp90. In a previous study using global whole cell mass spectrometry, emerin was shown to be the most significantly down regulated protein in Hop depleted cell lysates. In this current study, it was postulated that emerin interacts with Hop, and this interaction regulates the stability, and level of emerin in the nucleus which impacts on the structure of the nuclear envelope. We used HEK293T cell lines stably expressing shRNA against Hop, emerin and a non-targeting control alongside the over expression of Hop in HEK293 cells to determine the effect of Hop levels on emerin expression and vice versa via Western blotting. The effect of Hop on the localization of emerin was assessed via subcellullar fractionation and confocal microscopy, while the impact on the structure of the nucleus was determined by transmission electron microscopy (TEM). We established that the depletion of Hop using shRNA and the over expression of Hop both result in the proteasomal and lysosomal degradation of emerin. Co-immunoprecipitation assays confirmed that Hop and emerin are in a common complex, which was not dependent on the presence of Hsp90. Loss of Hop or emerin led to a deformation of nuclear structure and a statistically significant decrease in nuclear size compared to control cells and was associated with an increase in the levels of nuclear protein, lamin A-C. Loss of emerin and Hop resulted in increased long term cell survival, but only after restriction of the nucleus when the cells had migrated across a transwell membrane. Taken together, the results obtained suggest that Hop acts as a scaffold for the stabilization of emerin and that the effects of Hop depletion on the structure of the nucleus and long term survival are mediated via the depletion of emerin. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2017
- Full Text:
- Date Issued: 2017
An investigation into the use of anaerobic digestion for the treatment of tannery wastewaters
- Authors: Jackson-Moss, Clive Alan
- Date: 1991
- Subjects: Tanneries -- Waste disposal , Sewage sludge -- South Africa -- Management , Sewage -- Purification -- Biological treatment -- South Africa , Water quality management -- South Africa , Water -- Purification -- Biological treatment -- South Africa
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4059 , http://hdl.handle.net/10962/d1004120 , Tanneries -- Waste disposal , Sewage sludge -- South Africa -- Management , Sewage -- Purification -- Biological treatment -- South Africa , Water quality management -- South Africa , Water -- Purification -- Biological treatment -- South Africa
- Description: The anaerobic digestion of tannery wastewaters was investigated with a view to using this form of treatment in the tanning industry. As these wastewaters are extremely complex and contain high concentrations of both inorganic and organic compounds, the effect of these individual compounds on the anaerobic digestion process was investigated in detail, in order to ascertain the fate of these compounds during the digestion process. The experiments comprising the initial toxicity study were carried out as adaptation experiments using a synthetic wastewater. It was found that the heavy metals such as chrome, aluminium and iron precipitated and accumulated in the sludge bed of the digesters . The soluble ions such as sodium and chloride were not retained and passed through the digesters. Approximately 20 % of the calcium ions were removed through precipitation, with the remainder being present in the digester effluent . Under the anaerobic conditions, ammonification of the organic nitrogen occurred, and influent sulphates were reduced to sulphides . These sulphides were present as either H2S, HS or insoluble sulphides. As these compounds under investigation on caused no inhibition of the anaerobic digestion process at the concentrations found in tannery wastewaters, the anaerobic treatment of these wastewaters appeared to be possible, provided the bacteria were given sufficient time to adapt to the potentially toxic compounds. However, despite the findings of the synthetic study, the successful anaerobic digestion of the tannery effluents could not be achieved. Although the use of acid was found to be essential in order to control the digester pH in the optimum range, the metabolism of the methanogenic bacteria was inhibited by the presence or absence of unknown compounds. Neither the addition of essential trace nutrients, nor the prevention of the competition between the methanogens and the sulphate-reducing bacteria were able to reverse this inhibition. As tannery effluents contain very low concentrations of phosphorous, it is possible that the methanogens were inhibited by a lack of phosphorous, which is essential during methanogenesis. In contrast to the results obtained from the effluent experiments, the anaerobic digestion of tannery sludge was found to be possible. Of the organic solids present in the sludge, 60 % were degraded and converted into biogas, which had a methane content greater than 70 %. The degradation of the organic solids ensured that COD and PV reductions of greater than 90 % were achieved, and the fate of the compounds in the digesters were in agreement with the findings of the v synthetic study. Efforts to improve the efficiency of the digestion process through the addition of trace nutrients and the use of a two-stage process were only successful in bringing about a minor improvement in digester performance. The overall results of this investigation show, therefore, that although the anaerobic treatment of the tannery effluent was not achieved, the successful anaerobic digestion of tannery sludge is possible at low loading rates. As many difficulties still need to be solved, a great deal of further research is necessary if anaerobic digestion is to be used on an industrial scale for the treatment and disposal of tannery wastewaters.
- Full Text:
- Date Issued: 1991
- Authors: Jackson-Moss, Clive Alan
- Date: 1991
- Subjects: Tanneries -- Waste disposal , Sewage sludge -- South Africa -- Management , Sewage -- Purification -- Biological treatment -- South Africa , Water quality management -- South Africa , Water -- Purification -- Biological treatment -- South Africa
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4059 , http://hdl.handle.net/10962/d1004120 , Tanneries -- Waste disposal , Sewage sludge -- South Africa -- Management , Sewage -- Purification -- Biological treatment -- South Africa , Water quality management -- South Africa , Water -- Purification -- Biological treatment -- South Africa
- Description: The anaerobic digestion of tannery wastewaters was investigated with a view to using this form of treatment in the tanning industry. As these wastewaters are extremely complex and contain high concentrations of both inorganic and organic compounds, the effect of these individual compounds on the anaerobic digestion process was investigated in detail, in order to ascertain the fate of these compounds during the digestion process. The experiments comprising the initial toxicity study were carried out as adaptation experiments using a synthetic wastewater. It was found that the heavy metals such as chrome, aluminium and iron precipitated and accumulated in the sludge bed of the digesters . The soluble ions such as sodium and chloride were not retained and passed through the digesters. Approximately 20 % of the calcium ions were removed through precipitation, with the remainder being present in the digester effluent . Under the anaerobic conditions, ammonification of the organic nitrogen occurred, and influent sulphates were reduced to sulphides . These sulphides were present as either H2S, HS or insoluble sulphides. As these compounds under investigation on caused no inhibition of the anaerobic digestion process at the concentrations found in tannery wastewaters, the anaerobic treatment of these wastewaters appeared to be possible, provided the bacteria were given sufficient time to adapt to the potentially toxic compounds. However, despite the findings of the synthetic study, the successful anaerobic digestion of the tannery effluents could not be achieved. Although the use of acid was found to be essential in order to control the digester pH in the optimum range, the metabolism of the methanogenic bacteria was inhibited by the presence or absence of unknown compounds. Neither the addition of essential trace nutrients, nor the prevention of the competition between the methanogens and the sulphate-reducing bacteria were able to reverse this inhibition. As tannery effluents contain very low concentrations of phosphorous, it is possible that the methanogens were inhibited by a lack of phosphorous, which is essential during methanogenesis. In contrast to the results obtained from the effluent experiments, the anaerobic digestion of tannery sludge was found to be possible. Of the organic solids present in the sludge, 60 % were degraded and converted into biogas, which had a methane content greater than 70 %. The degradation of the organic solids ensured that COD and PV reductions of greater than 90 % were achieved, and the fate of the compounds in the digesters were in agreement with the findings of the v synthetic study. Efforts to improve the efficiency of the digestion process through the addition of trace nutrients and the use of a two-stage process were only successful in bringing about a minor improvement in digester performance. The overall results of this investigation show, therefore, that although the anaerobic treatment of the tannery effluent was not achieved, the successful anaerobic digestion of tannery sludge is possible at low loading rates. As many difficulties still need to be solved, a great deal of further research is necessary if anaerobic digestion is to be used on an industrial scale for the treatment and disposal of tannery wastewaters.
- Full Text:
- Date Issued: 1991