Neuronal nitric oxide synthase : a biomarker for Alzheimers disease : interaction of neuronal nitric oxide synthase with beta-amyloid peptides in the brain
- Authors: Padayachee, Eden Rebecca
- Date: 2011 , 2013-07-19
- Subjects: Alzheimer's disease , Nitric-oxide synthase , Biochemical markers , Amyloid beta-protein , Peptide hormones
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4086 , http://hdl.handle.net/10962/d1007677 , Alzheimer's disease , Nitric-oxide synthase , Biochemical markers , Amyloid beta-protein , Peptide hormones
- Description: High levels of the amino acid arginine and low levels of the product citrulline in the cerebrospinal fluid of Alzheimer's patients could mean that there is a decrease in the enzymes that metabolize this amino acid. One such enzyme is neuronal nitric oxide synthase (nNOS). In this study, neuronal nitric oxide synthase (nNOS), sourced from bovine brain was extracted and concentrated using two methods of precipitation: poly (ethylene glycol) 20 000 (PEG) and ammonium sulphate [(NH₄)₂S0₄). These two techniques gave no increase in yield nor fold purification and hence were abandoned in favour of ion exchange chromatography by DEAE-Sepharose. The enzyme was then successfully purified by anion-exchange and after dialysis produced a 38% yield and three fold purification and yielded the highest specific activity of 2.27 U/mg. Neuronal nitric oxide synthase (nNOS) was a heterodimeric protein with a total molecular mass of ± 225 kDa (95 and 130 kDa monomers). The temperature and pH optima of the enzyme were 40⁰C and 6.5, respectively. The kinetic parameters (KM and Vmax) of nNOS were 70 μM and 0.332 μmol.min⁻¹, respectively. Moreover neuronal nitric oxide synthase (nNOS) was relatively stable at 40⁰C (t½ = 3 h). It was also confirmed that β-amyloid peptides inhibited nNOS when bound to the enzyme and that nNOS behaved as a catalyst in fibril formation through association-dissociation between enzyme and β-amyloid peptide. It was further shown that Aβ₁₇₋₂₈ inhibited nNOS the most with a Ki of 1.92 μM and also had the highest Stern-Volmer value (Ksv) of 0.11 μM⁻¹ indicating tight binding affinity to nNOS and easier accessibility to fluor molecules during binding. Congo red, turbidity, thioflavin-T assays and transmission electron microscopy were successfully used to detect and visualize the presence of fibrils by studying the process of fibrillogenesis. Computerized molecular modeling successfully studied protein dynamics and conformational changes of nNOS. These results correlated with resonance energy transfer (FRET) results which revealed the distance of tryptophan residues from the arginine bound at enzyme active site. Both the aforementioned techniques revealed that in the natural state of the enzyme with arginine bound at the active site, the tryptophan residues (TRP₆₂₅ and TRP₇₂₁) were positioned at the surface of the enzyme 28 Å away from the active site. When the amyloid peptide (Aβ₁₇₋₂₈) was bound to the active site, these same two amino acids moved 14 Å closer to the active site. A five residue hydrophobic fragment Aβ₁₇₋₂₁ [Leu₁₇ - Val₁₈ - Phe₁₉ - Phe₂₀ - Ala₁] within Aβ₁₇₋₂₈ was shown by computer modeling to be critical to the binding of the peptide to the active site of nNOS.
- Full Text:
- Date Issued: 2011
- Authors: Padayachee, Eden Rebecca
- Date: 2011 , 2013-07-19
- Subjects: Alzheimer's disease , Nitric-oxide synthase , Biochemical markers , Amyloid beta-protein , Peptide hormones
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4086 , http://hdl.handle.net/10962/d1007677 , Alzheimer's disease , Nitric-oxide synthase , Biochemical markers , Amyloid beta-protein , Peptide hormones
- Description: High levels of the amino acid arginine and low levels of the product citrulline in the cerebrospinal fluid of Alzheimer's patients could mean that there is a decrease in the enzymes that metabolize this amino acid. One such enzyme is neuronal nitric oxide synthase (nNOS). In this study, neuronal nitric oxide synthase (nNOS), sourced from bovine brain was extracted and concentrated using two methods of precipitation: poly (ethylene glycol) 20 000 (PEG) and ammonium sulphate [(NH₄)₂S0₄). These two techniques gave no increase in yield nor fold purification and hence were abandoned in favour of ion exchange chromatography by DEAE-Sepharose. The enzyme was then successfully purified by anion-exchange and after dialysis produced a 38% yield and three fold purification and yielded the highest specific activity of 2.27 U/mg. Neuronal nitric oxide synthase (nNOS) was a heterodimeric protein with a total molecular mass of ± 225 kDa (95 and 130 kDa monomers). The temperature and pH optima of the enzyme were 40⁰C and 6.5, respectively. The kinetic parameters (KM and Vmax) of nNOS were 70 μM and 0.332 μmol.min⁻¹, respectively. Moreover neuronal nitric oxide synthase (nNOS) was relatively stable at 40⁰C (t½ = 3 h). It was also confirmed that β-amyloid peptides inhibited nNOS when bound to the enzyme and that nNOS behaved as a catalyst in fibril formation through association-dissociation between enzyme and β-amyloid peptide. It was further shown that Aβ₁₇₋₂₈ inhibited nNOS the most with a Ki of 1.92 μM and also had the highest Stern-Volmer value (Ksv) of 0.11 μM⁻¹ indicating tight binding affinity to nNOS and easier accessibility to fluor molecules during binding. Congo red, turbidity, thioflavin-T assays and transmission electron microscopy were successfully used to detect and visualize the presence of fibrils by studying the process of fibrillogenesis. Computerized molecular modeling successfully studied protein dynamics and conformational changes of nNOS. These results correlated with resonance energy transfer (FRET) results which revealed the distance of tryptophan residues from the arginine bound at enzyme active site. Both the aforementioned techniques revealed that in the natural state of the enzyme with arginine bound at the active site, the tryptophan residues (TRP₆₂₅ and TRP₇₂₁) were positioned at the surface of the enzyme 28 Å away from the active site. When the amyloid peptide (Aβ₁₇₋₂₈) was bound to the active site, these same two amino acids moved 14 Å closer to the active site. A five residue hydrophobic fragment Aβ₁₇₋₂₁ [Leu₁₇ - Val₁₈ - Phe₁₉ - Phe₂₀ - Ala₁] within Aβ₁₇₋₂₈ was shown by computer modeling to be critical to the binding of the peptide to the active site of nNOS.
- Full Text:
- Date Issued: 2011
Probing the biocompatibility of biomedical interfaces using the Quartz Crystal Microbalance with Dissipation
- Authors: Cromhout, Mary
- Date: 2011
- Subjects: Biomedical materials , Nanostructured materials , Biomedical engineering , Quartz crystal microbalances , Blood proteins , Nanoparticles
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4102 , http://hdl.handle.net/10962/d1010660
- Description: The biomedical application of nanotechnology has come into the spotlight, with the promise of ‘personalised’ therapeutics that couple early diagnosis with targeted therapeutic activity. Due to the rapid growth of the biomedical applications of nanoparticles, along with the lack of understanding concerning their interactions with biomolecules, there is a pressing need for the development of standard methods directed at investigating the effect of introducing these unique particles into the human body. The central aim of this research is to establish a platform directed at assessing the biological fate of pioneering therapeutic particulate agents, such as metallophthalocyanines (MPcs) and multi-walled carbon nanotubes (FMWCNTs). In particular, we proposed, that Quartz Crystal Microbalance with Dissipation (QCM-D) technology may be employed to assess the composition of blood protein corona deposited on the therapeutic surface, and subsequently assess the biocompatibility of such particles. The proposed method of protein detection utilises the nanogram sensitivity of QCM-D technology to monitor highly specific antibody-antigen interactions. In particular those interactions which occur when probe antibodies are used to detect adsorbed blood proteins deposited on target particle-modified sensor surfaces. Protein detection analysis was directed toward identification of surface bound human serum albumin, complement factor C3c, and human plasma fibrinogen. Preliminary analysis of generic biomedical surfaces indicated human serum albumin demonstrates a higher binding affinity towards positively charged surfaces (i.e. cysteamine self-assembled monolayer), followed by hydrophobic surfaces. Detection of complement C3c, corresponded with literature, where lower levels were detected on negatively charged surfaces (i.e. mercapto undecanoic acid self-assembled monolayer), and higher levels of more hydrophobic surfaces (i.e. 11-amino undecane thiol self-assembled monolayer). Human plasma fibrinogen was observed to favour hydrophilic over hydrophobic self-assembled monolayer surfaces, which was in accordance with literature. Application of the proposed protein detection method for biocompatibility analysis of target therapeutic molecules, namely metallophthalocyanines and acid functionalised multi-walled carbon nanotubes, demonstrated a dependence on modified-surface film characteristics, such as surface charge and topography with regards to human serum albumin and human plasma fibrinogen analysis representing new insights into their potential biomolecular interactions The highest levels of detected human serum albumin and complement C3c were detected on the GePcSmix-modified surfaces. AlPcSmix-modified surfaces analysis suggested the highest levels of human plasma fibrinogen. Two methods of acid functionalisation were employed, using both nitric and sulphuric acid, and pure nitric acid. A general increase in detected human serum albumin, corresponding with an increase in functionalisation time, was observed. Complement C3c detection suggested an increase in deposited complement C3c, with increasing functionalisation time, when assessing nitric acid functionalised multi-walled carbon nanotubes, and a decrease, with increasing functionalisation time, when assessing nitric and sulphuric acid functionalised multi-walled carbon nanotubes. Analysis of human plasma fibrinogen was inconclusive, as were cytotoxicity experiments utilising MCF-7 cells in the presence of metallophthalocyanine complexes, raising simultaneously important considerations for their application and study. In the first such detailed examination of its kind it was concluded that the proposed method of protein detection, using QCM-D, allows for the rudimentary but rapid means of analysis of select protein corona deposited on particulate biomedical surfaces.
- Full Text:
- Date Issued: 2011
- Authors: Cromhout, Mary
- Date: 2011
- Subjects: Biomedical materials , Nanostructured materials , Biomedical engineering , Quartz crystal microbalances , Blood proteins , Nanoparticles
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4102 , http://hdl.handle.net/10962/d1010660
- Description: The biomedical application of nanotechnology has come into the spotlight, with the promise of ‘personalised’ therapeutics that couple early diagnosis with targeted therapeutic activity. Due to the rapid growth of the biomedical applications of nanoparticles, along with the lack of understanding concerning their interactions with biomolecules, there is a pressing need for the development of standard methods directed at investigating the effect of introducing these unique particles into the human body. The central aim of this research is to establish a platform directed at assessing the biological fate of pioneering therapeutic particulate agents, such as metallophthalocyanines (MPcs) and multi-walled carbon nanotubes (FMWCNTs). In particular, we proposed, that Quartz Crystal Microbalance with Dissipation (QCM-D) technology may be employed to assess the composition of blood protein corona deposited on the therapeutic surface, and subsequently assess the biocompatibility of such particles. The proposed method of protein detection utilises the nanogram sensitivity of QCM-D technology to monitor highly specific antibody-antigen interactions. In particular those interactions which occur when probe antibodies are used to detect adsorbed blood proteins deposited on target particle-modified sensor surfaces. Protein detection analysis was directed toward identification of surface bound human serum albumin, complement factor C3c, and human plasma fibrinogen. Preliminary analysis of generic biomedical surfaces indicated human serum albumin demonstrates a higher binding affinity towards positively charged surfaces (i.e. cysteamine self-assembled monolayer), followed by hydrophobic surfaces. Detection of complement C3c, corresponded with literature, where lower levels were detected on negatively charged surfaces (i.e. mercapto undecanoic acid self-assembled monolayer), and higher levels of more hydrophobic surfaces (i.e. 11-amino undecane thiol self-assembled monolayer). Human plasma fibrinogen was observed to favour hydrophilic over hydrophobic self-assembled monolayer surfaces, which was in accordance with literature. Application of the proposed protein detection method for biocompatibility analysis of target therapeutic molecules, namely metallophthalocyanines and acid functionalised multi-walled carbon nanotubes, demonstrated a dependence on modified-surface film characteristics, such as surface charge and topography with regards to human serum albumin and human plasma fibrinogen analysis representing new insights into their potential biomolecular interactions The highest levels of detected human serum albumin and complement C3c were detected on the GePcSmix-modified surfaces. AlPcSmix-modified surfaces analysis suggested the highest levels of human plasma fibrinogen. Two methods of acid functionalisation were employed, using both nitric and sulphuric acid, and pure nitric acid. A general increase in detected human serum albumin, corresponding with an increase in functionalisation time, was observed. Complement C3c detection suggested an increase in deposited complement C3c, with increasing functionalisation time, when assessing nitric acid functionalised multi-walled carbon nanotubes, and a decrease, with increasing functionalisation time, when assessing nitric and sulphuric acid functionalised multi-walled carbon nanotubes. Analysis of human plasma fibrinogen was inconclusive, as were cytotoxicity experiments utilising MCF-7 cells in the presence of metallophthalocyanine complexes, raising simultaneously important considerations for their application and study. In the first such detailed examination of its kind it was concluded that the proposed method of protein detection, using QCM-D, allows for the rudimentary but rapid means of analysis of select protein corona deposited on particulate biomedical surfaces.
- Full Text:
- Date Issued: 2011
SphereZyme (TM) technology for enhanced enzyme immobilisation application in biosensors
- Authors: Molawa, Letshego Gloria
- Date: 2011
- Subjects: Immobilized enzymes , Hydrolases , Hydrolysis , SphereZyme , Biosensors , Proteolytic enzymes
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3989 , http://hdl.handle.net/10962/d1004048 , Immobilized enzymes , Hydrolases , Hydrolysis , SphereZyme , Biosensors , Proteolytic enzymes
- Description: Self-immobilisation enzyme technologies, such as SphereZyme™, suffer from the lack of applicability to hydrolyse large substrates. Solid support immobilisation is usually a method of choice, to produce a stable biocatalyst for large substrates hydrolysis in the industry. In order to investigate this limitation, a commercial protease called Alcalase® was chosen as a model enzyme due to its natural activity (hydrolysis of large substrates-proteins). Prior to immobilising through the SphereZyme™ technology, Alcalase® was partially purified through dialysis followed by CM Sepharose™ FF cation exchanger. Sample contaminants, such as salts and stabilisers can inhibit protein crosslinking by reacting with glutaraldehyde. Alcalase® was successfully separated into 3 proteases with the major peak correlating to a positive control run on native PAGE, indicating that it was likely subtilisin Carlsberg. A 16% alkaline protease activity for azo-casein hydrolysis was retained when 5% v/v PEI: 25% v/v glutaraldehyde solution was used as a crosslinking agent in Alcalase® SphereZyme™ production. An increase in activity was also observed for monomeric substrates (PNPA) where the highest was 55%. The highest % activities maintained when 0.33 M EDA: 25% v/v glutaraldehyde solution was initially used as crosslinking agent were 4.5% and 1.6% for monomeric and polymeric substrates, respectively. PEI is a hydrophilic branched polymer with an abundance of amine groups compared to EDA. A comparison study of immobilisation efficiencies of SphereZyme™, Eupergit® and Dendrispheres was also performed for large substrate biocatalysis. The two latter technologies are solid-support immobilisation methods. Dendrispheres reached its maximum loading capacity in the first 5 minute of the one hour binding time. Twenty minutes was chosen as a maximum binding time since there was constant protein maintained on the solid support and no enzyme loss was observed during the 1 hour binding time. PEI at pH 11.5, its native pH, gave the highest immobilisation yield and specific activity over the PEI pH range of 11.5 to 7. SphereZyme™ had the highest ratio for azocasein hydrolysis followed by Dendrispheres and Eupergit®. The SphereZyme™ was also shown to be applicable to biosensors for phenol detection. Different modifications of glassy carbon electrode (GCE) were evaluated as a benchmark for the fabrication of SphereZyme™ modified phenol biosensor. GCE modified with laccase SphereZyme™ entrapped in cellulose membrane was the best modification due to the broad catechol range (<0.950 mM), high correlation coefficient (R2, 0.995) and relative high sensitivity factor (0.305 μA.mM-1). This type of biosensor was also shown to be electroactive at pH 7.0 for which its control, free laccase, lacked electroactivity. From the catalytic constants calculated, GCE modified with laccase SphereZyme™ entrapped in cellulose membrane also gave the highest effectiveness factor (Imax/Km app) of 1.84 μA.mM-1. The modified GCE with Alcalase® SphereZyme™ was relatively more sensitive than GCE modified with free Alcalase®.
- Full Text:
- Date Issued: 2011
- Authors: Molawa, Letshego Gloria
- Date: 2011
- Subjects: Immobilized enzymes , Hydrolases , Hydrolysis , SphereZyme , Biosensors , Proteolytic enzymes
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3989 , http://hdl.handle.net/10962/d1004048 , Immobilized enzymes , Hydrolases , Hydrolysis , SphereZyme , Biosensors , Proteolytic enzymes
- Description: Self-immobilisation enzyme technologies, such as SphereZyme™, suffer from the lack of applicability to hydrolyse large substrates. Solid support immobilisation is usually a method of choice, to produce a stable biocatalyst for large substrates hydrolysis in the industry. In order to investigate this limitation, a commercial protease called Alcalase® was chosen as a model enzyme due to its natural activity (hydrolysis of large substrates-proteins). Prior to immobilising through the SphereZyme™ technology, Alcalase® was partially purified through dialysis followed by CM Sepharose™ FF cation exchanger. Sample contaminants, such as salts and stabilisers can inhibit protein crosslinking by reacting with glutaraldehyde. Alcalase® was successfully separated into 3 proteases with the major peak correlating to a positive control run on native PAGE, indicating that it was likely subtilisin Carlsberg. A 16% alkaline protease activity for azo-casein hydrolysis was retained when 5% v/v PEI: 25% v/v glutaraldehyde solution was used as a crosslinking agent in Alcalase® SphereZyme™ production. An increase in activity was also observed for monomeric substrates (PNPA) where the highest was 55%. The highest % activities maintained when 0.33 M EDA: 25% v/v glutaraldehyde solution was initially used as crosslinking agent were 4.5% and 1.6% for monomeric and polymeric substrates, respectively. PEI is a hydrophilic branched polymer with an abundance of amine groups compared to EDA. A comparison study of immobilisation efficiencies of SphereZyme™, Eupergit® and Dendrispheres was also performed for large substrate biocatalysis. The two latter technologies are solid-support immobilisation methods. Dendrispheres reached its maximum loading capacity in the first 5 minute of the one hour binding time. Twenty minutes was chosen as a maximum binding time since there was constant protein maintained on the solid support and no enzyme loss was observed during the 1 hour binding time. PEI at pH 11.5, its native pH, gave the highest immobilisation yield and specific activity over the PEI pH range of 11.5 to 7. SphereZyme™ had the highest ratio for azocasein hydrolysis followed by Dendrispheres and Eupergit®. The SphereZyme™ was also shown to be applicable to biosensors for phenol detection. Different modifications of glassy carbon electrode (GCE) were evaluated as a benchmark for the fabrication of SphereZyme™ modified phenol biosensor. GCE modified with laccase SphereZyme™ entrapped in cellulose membrane was the best modification due to the broad catechol range (<0.950 mM), high correlation coefficient (R2, 0.995) and relative high sensitivity factor (0.305 μA.mM-1). This type of biosensor was also shown to be electroactive at pH 7.0 for which its control, free laccase, lacked electroactivity. From the catalytic constants calculated, GCE modified with laccase SphereZyme™ entrapped in cellulose membrane also gave the highest effectiveness factor (Imax/Km app) of 1.84 μA.mM-1. The modified GCE with Alcalase® SphereZyme™ was relatively more sensitive than GCE modified with free Alcalase®.
- Full Text:
- Date Issued: 2011
The development of an in vitro system for the production of drug metabolites using microsomal enzymes from bovine liver
- Authors: Morrison, Roxanne
- Date: 2011
- Subjects: Drugs -- Metabolism , Xenobiotics -- Metabolism , Metabolites , Drugs -- Testing , Toxicity testing -- In vitro , Doping in horse racing -- Control -- Research
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4087 , http://hdl.handle.net/10962/d1007698 , Drugs -- Metabolism , Xenobiotics -- Metabolism , Metabolites , Drugs -- Testing , Toxicity testing -- In vitro , Doping in horse racing -- Control -- Research
- Description: Drug metabolism is a specialised subset of xenobiotic metabolism, pertaining to the breakdown and elimination of pharmaceutical drugs. The enzymes involved in these pathways are the cytochrome P450 family of isozymes. Metabolism is an important factor in determining the pharmacological effects of drugs. The main aim of this study was to develop a system whereby the major metabolites of drugs can be produced in vitro. An in vitro system was developed and optimised using commercially prepared microsomes from rat liver and coumarin (by monitoring its conversion to 7-hydroxycoumarin) as a model. The optimum running conditions for the incubations were 50 μM coumarin, 50 μg protein/ml microsomes, 1 mM NADP⁺, 5 mM G6P and 1U/ml G6PDH incubated for 30 minutes at 38℃. The HPLC method for the detection of coumarin and 7-hydroxycoumarin was also validated with respect to linearity, reproducibility, precision, accuracy and lower limits of detection and quantification. The system developed was then tested using microsomes prepared from fresh bovine liver on these ten drugs of interest in doping control in horse racing: diazepam, nordiazepam, oxazepam, promazine, acepromazine, chlorpromazine, morphine, codeine, etoricoxib and lumiracoxib. The bovine liver microsomes were prepared using differential centrifugation and had activity on a par with the commercial preparations. This in vitro system metabolised the drugs and produced both phase I and II metabolites, similar to those observed in humans and horses in vivo. For example, the major metabolites of the benzodiazepine drug, diazepam, nordiazepam, temazepam and oxazepam as well as the glucuronidated phase II products were all found after incubations with the bovine liver microsomes. The metabolism of the drugs was also investigated in silico using the computational procedure, MetaSite. MetaSite was able to successfully predict known metabolites for most of the drugs studied. Differences were observed from the in vitro incubations and this is most likely due to MetaSite using only human cytochrome P450s for analysis.
- Full Text:
- Date Issued: 2011
- Authors: Morrison, Roxanne
- Date: 2011
- Subjects: Drugs -- Metabolism , Xenobiotics -- Metabolism , Metabolites , Drugs -- Testing , Toxicity testing -- In vitro , Doping in horse racing -- Control -- Research
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4087 , http://hdl.handle.net/10962/d1007698 , Drugs -- Metabolism , Xenobiotics -- Metabolism , Metabolites , Drugs -- Testing , Toxicity testing -- In vitro , Doping in horse racing -- Control -- Research
- Description: Drug metabolism is a specialised subset of xenobiotic metabolism, pertaining to the breakdown and elimination of pharmaceutical drugs. The enzymes involved in these pathways are the cytochrome P450 family of isozymes. Metabolism is an important factor in determining the pharmacological effects of drugs. The main aim of this study was to develop a system whereby the major metabolites of drugs can be produced in vitro. An in vitro system was developed and optimised using commercially prepared microsomes from rat liver and coumarin (by monitoring its conversion to 7-hydroxycoumarin) as a model. The optimum running conditions for the incubations were 50 μM coumarin, 50 μg protein/ml microsomes, 1 mM NADP⁺, 5 mM G6P and 1U/ml G6PDH incubated for 30 minutes at 38℃. The HPLC method for the detection of coumarin and 7-hydroxycoumarin was also validated with respect to linearity, reproducibility, precision, accuracy and lower limits of detection and quantification. The system developed was then tested using microsomes prepared from fresh bovine liver on these ten drugs of interest in doping control in horse racing: diazepam, nordiazepam, oxazepam, promazine, acepromazine, chlorpromazine, morphine, codeine, etoricoxib and lumiracoxib. The bovine liver microsomes were prepared using differential centrifugation and had activity on a par with the commercial preparations. This in vitro system metabolised the drugs and produced both phase I and II metabolites, similar to those observed in humans and horses in vivo. For example, the major metabolites of the benzodiazepine drug, diazepam, nordiazepam, temazepam and oxazepam as well as the glucuronidated phase II products were all found after incubations with the bovine liver microsomes. The metabolism of the drugs was also investigated in silico using the computational procedure, MetaSite. MetaSite was able to successfully predict known metabolites for most of the drugs studied. Differences were observed from the in vitro incubations and this is most likely due to MetaSite using only human cytochrome P450s for analysis.
- Full Text:
- Date Issued: 2011
The role of Hsp90 in the Wnt pathway of MCF7 breast cancer cells
- Authors: Cooper, Leanne Claire
- Date: 2011
- Subjects: Cancer -- Treatment , Heat shock proteins , Cancer cells , Molecular chaperones
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3985 , http://hdl.handle.net/10962/d1004044 , Cancer -- Treatment , Heat shock proteins , Cancer cells , Molecular chaperones
- Description: Breast cancer is one of the most common forms of cancer in not only South African women, but women all over the world. The molecular chaperone heat shock protein 90 (HSP90) is upregulated in cancer and is almost exclusively associated with proteins involved in intracellular signal transduction, thus it plays an important role in signalling pathways within the cell. In cancer, there is an aberrant activation of the Wnt signaling pathway, which results in stabilized β-catenin being able to translocate to the nucleus where it can trigger the transcription of oncogenes found to be involved in the self-renewal of cells. The level of β-catenin is usually kept in check by a destruction complex comprising glycogen synthase kinase 3-beta (GSK-3β), axin1, adenomatous polyposis coli (APC) which phosphorylate β-catenin, resulting in its ubiquitination and degradation. HSP90 has been found to be associated with GSK-3β, but whether this association is only transient is debatable. Very little is known about the association of HSP90 with other members of the Wnt pathway in breast cancer. In this study, we have attempted to further identify the direct associations between HSP90 and GSK-3β, β-catenin, p-β-catenin and axin1. Immunofluorescence and confocal microscopy co-localization studies suggested a potential association between HSP90 and these proteins. Treatment with HSP90 inhibitors, 17-AAG and novobiocin resulted in a shift of axin1 to what appeared to be the plasma membrane. The associations of HSP90 with GSK-3β, β-catenin, p-β-catenin and axin1 were confirmed biochemically by co-immunoprecipitation and inhibition using 17-AAG, geldanamycin and novobiocin. We showed, for the first time that HSP90 is associated in a possible complex with β-catenin, p-β-catenin and axin1 therefore is potentially involved in the modulation of p-β-catenin in the Wnt pathway through the stabilization of the destruction complex.
- Full Text:
- Date Issued: 2011
- Authors: Cooper, Leanne Claire
- Date: 2011
- Subjects: Cancer -- Treatment , Heat shock proteins , Cancer cells , Molecular chaperones
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3985 , http://hdl.handle.net/10962/d1004044 , Cancer -- Treatment , Heat shock proteins , Cancer cells , Molecular chaperones
- Description: Breast cancer is one of the most common forms of cancer in not only South African women, but women all over the world. The molecular chaperone heat shock protein 90 (HSP90) is upregulated in cancer and is almost exclusively associated with proteins involved in intracellular signal transduction, thus it plays an important role in signalling pathways within the cell. In cancer, there is an aberrant activation of the Wnt signaling pathway, which results in stabilized β-catenin being able to translocate to the nucleus where it can trigger the transcription of oncogenes found to be involved in the self-renewal of cells. The level of β-catenin is usually kept in check by a destruction complex comprising glycogen synthase kinase 3-beta (GSK-3β), axin1, adenomatous polyposis coli (APC) which phosphorylate β-catenin, resulting in its ubiquitination and degradation. HSP90 has been found to be associated with GSK-3β, but whether this association is only transient is debatable. Very little is known about the association of HSP90 with other members of the Wnt pathway in breast cancer. In this study, we have attempted to further identify the direct associations between HSP90 and GSK-3β, β-catenin, p-β-catenin and axin1. Immunofluorescence and confocal microscopy co-localization studies suggested a potential association between HSP90 and these proteins. Treatment with HSP90 inhibitors, 17-AAG and novobiocin resulted in a shift of axin1 to what appeared to be the plasma membrane. The associations of HSP90 with GSK-3β, β-catenin, p-β-catenin and axin1 were confirmed biochemically by co-immunoprecipitation and inhibition using 17-AAG, geldanamycin and novobiocin. We showed, for the first time that HSP90 is associated in a possible complex with β-catenin, p-β-catenin and axin1 therefore is potentially involved in the modulation of p-β-catenin in the Wnt pathway through the stabilization of the destruction complex.
- Full Text:
- Date Issued: 2011
The spatial evolution of the chemotaxis proteins of the Bacillus subtilis group
- Yssel, Anna Elizabeth Johanna
- Authors: Yssel, Anna Elizabeth Johanna
- Date: 2011
- Subjects: Chemotaxis , Bacillus subtilis , Bacillus (Bacteria) , Homology (Biology) , Plants -- Microbiology
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4027 , http://hdl.handle.net/10962/d1004087 , Chemotaxis , Bacillus subtilis , Bacillus (Bacteria) , Homology (Biology) , Plants -- Microbiology
- Description: The aim of this work was to study spatial evolution of the chemotaxis proteins of a group of plant-associated soil-dwelling bacteria vernacularly referred to as the B. subtilis group. This was achieved by creating homology models for the chemotaxis proteins if a suitable template was available, and by analysing the selective forces (positive, purifying or neutral) acting upon the chemotaxis proteins. Chemotaxis is the phenomenon in which bacteria direct their movement towards more favourable conditions, and is critical for processes such as obtaining nutrients, escaping toxic compounds, host colonization and bio-film formation. Members of the B. subtilis group exhibit different preferences for certain host plants, and it is therefore feasible that their chemotactic machinery are fine-tuned to respond optimally to the conditions of the various niches that the strains inhabit. Homology models were inferred for the plant growth promoting B. amyloliquefaciens FZB42 proteins CheB, CheC, CheD, CheR, CheW and CheY. The interactions between: CheC-CheD, the P1 and P2 domains of CheA with CheY and CheB, and the P4 and P5 domains of CheA with CheW were also modelled. The hydrophobic interactions contributing to intra- and inter-protein contacts were analysed. The models of the interactions between CheB and the various domains of CheA are of particular interest, because to date no structures have been solved that show an interaction between a histidine kinase (such as CheA) and a multidomain response regulator (such as CheB). Furthermore, evidence that phospho-CheB may inhibit the formation of phospho-CheY by competitively binding to the P2 domain of CheA is also presented. Proteins were analysed to determine if individual amino acid sites are under positive, neutral or purifying selection. The Methyl Accepting Chemotaxis Proteins (MCPs), CheA and CheV were also analyzed, but due to a lack of suitable templates, no homology models were constructed. Site-specific positive and purifying selection were estimated by comparing the ratios of non-synonymous to synonymous substitutions at each site in the sequences for the chemotaxis proteins as well as for the receptors McpA, McpB, and McpC. Homology models were coloured according to intensity of selective forces. It was found that the chemotaxis proteins of member of the B. subtilis group are under strong evolutionary constraints, hence it is unlikely that positive selection in these proteins are responsible for the differences in habitat preference that these organism exhibit.
- Full Text:
- Date Issued: 2011
- Authors: Yssel, Anna Elizabeth Johanna
- Date: 2011
- Subjects: Chemotaxis , Bacillus subtilis , Bacillus (Bacteria) , Homology (Biology) , Plants -- Microbiology
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4027 , http://hdl.handle.net/10962/d1004087 , Chemotaxis , Bacillus subtilis , Bacillus (Bacteria) , Homology (Biology) , Plants -- Microbiology
- Description: The aim of this work was to study spatial evolution of the chemotaxis proteins of a group of plant-associated soil-dwelling bacteria vernacularly referred to as the B. subtilis group. This was achieved by creating homology models for the chemotaxis proteins if a suitable template was available, and by analysing the selective forces (positive, purifying or neutral) acting upon the chemotaxis proteins. Chemotaxis is the phenomenon in which bacteria direct their movement towards more favourable conditions, and is critical for processes such as obtaining nutrients, escaping toxic compounds, host colonization and bio-film formation. Members of the B. subtilis group exhibit different preferences for certain host plants, and it is therefore feasible that their chemotactic machinery are fine-tuned to respond optimally to the conditions of the various niches that the strains inhabit. Homology models were inferred for the plant growth promoting B. amyloliquefaciens FZB42 proteins CheB, CheC, CheD, CheR, CheW and CheY. The interactions between: CheC-CheD, the P1 and P2 domains of CheA with CheY and CheB, and the P4 and P5 domains of CheA with CheW were also modelled. The hydrophobic interactions contributing to intra- and inter-protein contacts were analysed. The models of the interactions between CheB and the various domains of CheA are of particular interest, because to date no structures have been solved that show an interaction between a histidine kinase (such as CheA) and a multidomain response regulator (such as CheB). Furthermore, evidence that phospho-CheB may inhibit the formation of phospho-CheY by competitively binding to the P2 domain of CheA is also presented. Proteins were analysed to determine if individual amino acid sites are under positive, neutral or purifying selection. The Methyl Accepting Chemotaxis Proteins (MCPs), CheA and CheV were also analyzed, but due to a lack of suitable templates, no homology models were constructed. Site-specific positive and purifying selection were estimated by comparing the ratios of non-synonymous to synonymous substitutions at each site in the sequences for the chemotaxis proteins as well as for the receptors McpA, McpB, and McpC. Homology models were coloured according to intensity of selective forces. It was found that the chemotaxis proteins of member of the B. subtilis group are under strong evolutionary constraints, hence it is unlikely that positive selection in these proteins are responsible for the differences in habitat preference that these organism exhibit.
- Full Text:
- Date Issued: 2011
Analysis of the anti-cancer activity of novel indigenous algal compounds in breast cancer: towards the development of a model for screening anti-cancer stem cell activity
- Authors: Lawson, Jessica Clair
- Date: 2010
- Subjects: Breast -- Cancer , Breast -- Cancer -- Chemotherapy , Breast -- Cancer -- Treatment , Red algae , Brown algae , Algae -- Biotechnology
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3925 , http://hdl.handle.net/10962/d1003984 , Breast -- Cancer , Breast -- Cancer -- Chemotherapy , Breast -- Cancer -- Treatment , Red algae , Brown algae , Algae -- Biotechnology
- Description: Breast cancer, the most common malignancy diagnosed in women, is one of the leading causes of death in women worldwide. In South Africa only 32% of women diagnosed with advanced breast cancer survive more than five years. The search for new chemotherapeutic agents capable of effectively treating breast cancer is therefore essential. Recent evidence supporting the cancer stem cell theory of cancer development for breast cancer challenges the current theories of cancer development and hence treatment. Cancer stem cells are a small subpopulation of tumour cells that possess properties of both cancer cells and stem cells and are believed to be the tumour-initiating population of many cancers. Cancer stem cells are inherently resistant to many chemotherapeutic agents and in this way have been associated with repopulation of tumours after chemotherapy. This phenomenon is proposed as a possible mechanism for cancer relapse after treatment. Cancer stem cells have also been implicated in metastasis, the major cause of mortality in cancer patients. Therefore, any treatment that is capable of targeting and removing breast cancer stem cells may have the theoretical potential to effectively treat breast cancer. However, there are currently no such treatments available for clinical use. We were provided access to a library of novel indigenous small molecules isolated from red and brown algae found off the Eastern Cape of South Africa. The aim of this project was to analyse the anti-cancer and anti-cancer stem cell properties of the compounds in this library and to identify „hit‟ compounds which could form the basis for future development into new anti-cancer drugs. Ten novel compounds of algal origin were tested for cytotoxicity, by determining their ability to inhibit the growth of MCF12A breast epithelial cells and MCF7 breast cancer cells using the colorimetric MTT [(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] cell proliferation assay. All but one of the compounds tested exhibited cytotoxicity towards the MCF7 cancer cell line, with IC50 values (the concentration of the compound that leads to a 50% inhibition in cell growth) of between 3 μM and 90 μM. The chemotherapeutic drug paclitaxel was used as a positive control. Four of the compounds (RUMB-001, RUMB-002, RUMB-007 and RUMB-010/saragaquinoic acid) were significantly more toxic to the MCF7 cancer cell line, than the „normal‟ MCF12A breast cells and were selected as priority compounds for further analyses. In addition, two other compounds were selected as priority compounds, one highly cytotoxic towards both MCF12A and MCF7 cell lines (RUMB-015) and one which was non toxic to either cell line (RUMB-017/018). Preliminary studies into the mechanism of cytotoxicity using Western blot analysis for poly (ADP-ribose) polymerase (PARP) cleavage and Hoechst 33342 immunostaining in MCF-7 cells were largely unsuccessful. The Hoechst 33342 immunostaining assay did provide tentative evidence that selected priority compounds were capable of inducing apoptosis, although these assays will need to be repeated using a less subjective assay to confirm the results. The priority compounds were subsequently investigated for their cytotoxic effect on the cancer stem cell-enriched side population in MCF7 cells. The ability of the priority compounds to selectively target the cancer stem cell containing side population was assessed using two complementary flow cytometry-based techniques – namely the Hoechst 33342-exclusion assay, and fluorescent immunostaining for the expression of the putative cancer stem cell marker, ABCG2+. The ABCG2+ staining assay was a novel technique developed during the course of this study. It remains to be fully validated, but it may provide a new and reliable way to identify and analyse cancer stem cell containing side population cells. The MCF7 cells were treated with the compounds and the proportion of putative cancer stem cells compared with the size of the population in untreated cells was assessed. Three compounds (RUMB-010, RUMB-015 and RUMB-017/018) capable of reducing the proportion of side population cells within the MCF7 cell line were identified. Taking these data together, we identified two potential „hit‟ compounds which should be prioritised for future research. These are compounds RUMB-010/sargaquinoic acid and RUMB-017/018. RUMB-010 is of interest as it was shown to target the putative cancer stem cell population, in addition to the bulk MCF7 tumour line, but was relatively less toxic to the „normal‟ MCF12A cell line. RUMB-017/018 is of interest due to the ability to selectively target the cancer stem cell enriched side population, while having little effect on the normal (MCF12A) or bulk tumour (MCF7) cell lines tested. These compounds will be important as „hit‟ compounds for drug development and as tool compounds to study cancer and cancer stem cell biology.
- Full Text:
- Date Issued: 2010
- Authors: Lawson, Jessica Clair
- Date: 2010
- Subjects: Breast -- Cancer , Breast -- Cancer -- Chemotherapy , Breast -- Cancer -- Treatment , Red algae , Brown algae , Algae -- Biotechnology
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3925 , http://hdl.handle.net/10962/d1003984 , Breast -- Cancer , Breast -- Cancer -- Chemotherapy , Breast -- Cancer -- Treatment , Red algae , Brown algae , Algae -- Biotechnology
- Description: Breast cancer, the most common malignancy diagnosed in women, is one of the leading causes of death in women worldwide. In South Africa only 32% of women diagnosed with advanced breast cancer survive more than five years. The search for new chemotherapeutic agents capable of effectively treating breast cancer is therefore essential. Recent evidence supporting the cancer stem cell theory of cancer development for breast cancer challenges the current theories of cancer development and hence treatment. Cancer stem cells are a small subpopulation of tumour cells that possess properties of both cancer cells and stem cells and are believed to be the tumour-initiating population of many cancers. Cancer stem cells are inherently resistant to many chemotherapeutic agents and in this way have been associated with repopulation of tumours after chemotherapy. This phenomenon is proposed as a possible mechanism for cancer relapse after treatment. Cancer stem cells have also been implicated in metastasis, the major cause of mortality in cancer patients. Therefore, any treatment that is capable of targeting and removing breast cancer stem cells may have the theoretical potential to effectively treat breast cancer. However, there are currently no such treatments available for clinical use. We were provided access to a library of novel indigenous small molecules isolated from red and brown algae found off the Eastern Cape of South Africa. The aim of this project was to analyse the anti-cancer and anti-cancer stem cell properties of the compounds in this library and to identify „hit‟ compounds which could form the basis for future development into new anti-cancer drugs. Ten novel compounds of algal origin were tested for cytotoxicity, by determining their ability to inhibit the growth of MCF12A breast epithelial cells and MCF7 breast cancer cells using the colorimetric MTT [(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] cell proliferation assay. All but one of the compounds tested exhibited cytotoxicity towards the MCF7 cancer cell line, with IC50 values (the concentration of the compound that leads to a 50% inhibition in cell growth) of between 3 μM and 90 μM. The chemotherapeutic drug paclitaxel was used as a positive control. Four of the compounds (RUMB-001, RUMB-002, RUMB-007 and RUMB-010/saragaquinoic acid) were significantly more toxic to the MCF7 cancer cell line, than the „normal‟ MCF12A breast cells and were selected as priority compounds for further analyses. In addition, two other compounds were selected as priority compounds, one highly cytotoxic towards both MCF12A and MCF7 cell lines (RUMB-015) and one which was non toxic to either cell line (RUMB-017/018). Preliminary studies into the mechanism of cytotoxicity using Western blot analysis for poly (ADP-ribose) polymerase (PARP) cleavage and Hoechst 33342 immunostaining in MCF-7 cells were largely unsuccessful. The Hoechst 33342 immunostaining assay did provide tentative evidence that selected priority compounds were capable of inducing apoptosis, although these assays will need to be repeated using a less subjective assay to confirm the results. The priority compounds were subsequently investigated for their cytotoxic effect on the cancer stem cell-enriched side population in MCF7 cells. The ability of the priority compounds to selectively target the cancer stem cell containing side population was assessed using two complementary flow cytometry-based techniques – namely the Hoechst 33342-exclusion assay, and fluorescent immunostaining for the expression of the putative cancer stem cell marker, ABCG2+. The ABCG2+ staining assay was a novel technique developed during the course of this study. It remains to be fully validated, but it may provide a new and reliable way to identify and analyse cancer stem cell containing side population cells. The MCF7 cells were treated with the compounds and the proportion of putative cancer stem cells compared with the size of the population in untreated cells was assessed. Three compounds (RUMB-010, RUMB-015 and RUMB-017/018) capable of reducing the proportion of side population cells within the MCF7 cell line were identified. Taking these data together, we identified two potential „hit‟ compounds which should be prioritised for future research. These are compounds RUMB-010/sargaquinoic acid and RUMB-017/018. RUMB-010 is of interest as it was shown to target the putative cancer stem cell population, in addition to the bulk MCF7 tumour line, but was relatively less toxic to the „normal‟ MCF12A cell line. RUMB-017/018 is of interest due to the ability to selectively target the cancer stem cell enriched side population, while having little effect on the normal (MCF12A) or bulk tumour (MCF7) cell lines tested. These compounds will be important as „hit‟ compounds for drug development and as tool compounds to study cancer and cancer stem cell biology.
- Full Text:
- Date Issued: 2010
Enhancing the saccharolytic phase of sugar beet pulp via hemicellulase synergy
- Authors: Dredge, Roselyn Ann
- Date: 2010
- Subjects: Sugar plantations , Sugar plantations -- South Africa , Sugar beet industry -- South Africa , Saccharomyces cerevisiae -- Biotechnology , Biomass energy industries -- South Africa
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3955 , http://hdl.handle.net/10962/d1004014 , Sugar plantations , Sugar plantations -- South Africa , Sugar beet industry -- South Africa , Saccharomyces cerevisiae -- Biotechnology , Biomass energy industries -- South Africa
- Description: The sugar beet (Beta vulgaris) plant has in recent years been added to the Biofuel Industrial Strategy (Department of Minerals and Energy, 2007) by the South African government as a crop grown for the production of bio-ethanol. Sugar beet is commonly grown in Europe for the production of sucrose and has recently been cultivated in Cradock and the surrounding areas (Engineering News, 2008). The biofuel industry usually ferments the sucrose with Saccharomyces cerevisiae to yield bio-ethanol. However, researchers are presented with a critical role to increase current yields as there are concerns over the process costs from industrial biotechnologists. The beet factories produce a pulp by-product removed of all sucrose. The hemicellulose-rich pulp can be degraded by microbial enzymes to simple sugars that can be subsequently fermented to bio-ethanol. Thus, the pulp represents a potential source for second generation biofuel. The process of utilising microbial hemicellulases requires an initial chemical pre-treatment step to delignify the sugar beet pulp (SBP). An alkaline pre-treatment with ‘slake lime’ (calcium hydroxide) was investigated using a 23 factorial design and the factors examined were: lime load; temperature and time. The analysed results showed the highest release of reducing sugars at the pre-treatment conditions of: 0.4 g lime / g SBP; 40°C and 36 hours. A partial characterisation of the Clostridium cellulovorans hemicellulases was carried out to verify the optimal activity conditions stated in literature. The highest release of reducing sugars was measured at pH 6.5 – 7.0 and at 45°C for arabinofuranosidase A (ArfA); at pH 5.5 and 40°C for mannanase A (ManA) and pH 5.0 – 6.0 and 45°C for xylanase A (XynA). Temperature studies showed that a complete loss of enzymatic activity occurred after 11 hours for ManA; and 84-96 hours for ArfA. XynA was still active after 120 hours. The optimised lime pre-treated SBP was subsequently degraded using various combinations and percentages of C. cellulovorans ArfA, ManA and XynA to determine the maximal release of reducing sugars. Synergistically, the highest synergy was observed at 75% ArfA and 25% ManA, with a specific activity of 2.9 μmol/min/g protein. However, the highest release of sugars was observed at 4.2 μmol/min/g protein at 100% ArfA. This study has initiated the research within South Africa on SBP and its degradation by C. cellulovorans. Preliminary studies show that SBP has the potential to be utilised as a second generation biofuel source.
- Full Text:
- Date Issued: 2010
- Authors: Dredge, Roselyn Ann
- Date: 2010
- Subjects: Sugar plantations , Sugar plantations -- South Africa , Sugar beet industry -- South Africa , Saccharomyces cerevisiae -- Biotechnology , Biomass energy industries -- South Africa
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3955 , http://hdl.handle.net/10962/d1004014 , Sugar plantations , Sugar plantations -- South Africa , Sugar beet industry -- South Africa , Saccharomyces cerevisiae -- Biotechnology , Biomass energy industries -- South Africa
- Description: The sugar beet (Beta vulgaris) plant has in recent years been added to the Biofuel Industrial Strategy (Department of Minerals and Energy, 2007) by the South African government as a crop grown for the production of bio-ethanol. Sugar beet is commonly grown in Europe for the production of sucrose and has recently been cultivated in Cradock and the surrounding areas (Engineering News, 2008). The biofuel industry usually ferments the sucrose with Saccharomyces cerevisiae to yield bio-ethanol. However, researchers are presented with a critical role to increase current yields as there are concerns over the process costs from industrial biotechnologists. The beet factories produce a pulp by-product removed of all sucrose. The hemicellulose-rich pulp can be degraded by microbial enzymes to simple sugars that can be subsequently fermented to bio-ethanol. Thus, the pulp represents a potential source for second generation biofuel. The process of utilising microbial hemicellulases requires an initial chemical pre-treatment step to delignify the sugar beet pulp (SBP). An alkaline pre-treatment with ‘slake lime’ (calcium hydroxide) was investigated using a 23 factorial design and the factors examined were: lime load; temperature and time. The analysed results showed the highest release of reducing sugars at the pre-treatment conditions of: 0.4 g lime / g SBP; 40°C and 36 hours. A partial characterisation of the Clostridium cellulovorans hemicellulases was carried out to verify the optimal activity conditions stated in literature. The highest release of reducing sugars was measured at pH 6.5 – 7.0 and at 45°C for arabinofuranosidase A (ArfA); at pH 5.5 and 40°C for mannanase A (ManA) and pH 5.0 – 6.0 and 45°C for xylanase A (XynA). Temperature studies showed that a complete loss of enzymatic activity occurred after 11 hours for ManA; and 84-96 hours for ArfA. XynA was still active after 120 hours. The optimised lime pre-treated SBP was subsequently degraded using various combinations and percentages of C. cellulovorans ArfA, ManA and XynA to determine the maximal release of reducing sugars. Synergistically, the highest synergy was observed at 75% ArfA and 25% ManA, with a specific activity of 2.9 μmol/min/g protein. However, the highest release of sugars was observed at 4.2 μmol/min/g protein at 100% ArfA. This study has initiated the research within South Africa on SBP and its degradation by C. cellulovorans. Preliminary studies show that SBP has the potential to be utilised as a second generation biofuel source.
- Full Text:
- Date Issued: 2010
Generation of polyclonal antibodies against Theiler's Murine Encephalomyelitis virus protein 2C, and their use in investigating localisation of the protein in infected cells
- Authors: Jauka, Tembisa Innocencia
- Date: 2010
- Subjects: Picornaviruses , RNA viruses , Immunoglobulins , Encephalomyelitis
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3959 , http://hdl.handle.net/10962/d1004018 , Picornaviruses , RNA viruses , Immunoglobulins , Encephalomyelitis
- Description: The Picornavirus family of positive sense RNA viruses includes some significant human and animal pathogens including Poliovirus (PV), Foot-and-Mouth disease virus (FMDV) and Human Rhinovirus (HRV). The genome is translated within the host cell into a polyprotein that is proteolytically cleaved into the structural and nonstructural proteins. The highly conserved, non-structural protein 2C has numerous roles during the virus life cycle and is essential for virus replication. Although the protein has been well studied in the case of PV, its interactions with the host cell during picornavirus infection is poorly understood. Theiler’s Encephalomyelitis virus (TMEV) is a picornavirus that infects mice, and is being used in our laboratory as a model in which to study the 2C protein. In this study, polyclonal antibodies against the TMEV 2C protein were generated and used to localise the protein in infected cells by indirect immunofluorescence. To produce antigen for immunisation purposes, the TMEV-2C protein sequence was analysed to identify hydrophilic and antigenic regions. An internal region of the 2C representing amino acid residues 31-210 was selected, expressed in bacteria and purified by nickel NTA affinity chromatography. Time course analysis of 2C (31-210) showed that the peptide was maximally expressed at 5 hours post induction. The peptide was solubilised using a mild detergent and 1.5 mg of purified antigen was used for immunisation of rabbits. Western blot analysis confirmed that the antibodies could detect both bacteriallyexpressed antigen, and virally-expressed 2C. Examination of virus-infected baby hamster kidney cells by immunofluorescence and confocal microscopy using the antiserum (anti-TMEV 2C antibodies) showed that the protein had a diffuse distribution upon early infection and at later stages it was located in a large perinuclear structure representing the viral replication complex. Furthermore, 2C localised to the Golgi apparatus as revealed by dual-label immunofluorescence using anti-TMEV 2C antibodies and wheat germ agglutinin (WGA). Furthermore, it was shown that TMEV infection results in changes in cell morphology and a redistribution of the cytoskeletal protein, β-actin. The successful production of antibodies that recognise TMEV 2C opens the way for further studies to investigate interactions between 2C and hostencoded factors.
- Full Text:
- Date Issued: 2010
- Authors: Jauka, Tembisa Innocencia
- Date: 2010
- Subjects: Picornaviruses , RNA viruses , Immunoglobulins , Encephalomyelitis
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3959 , http://hdl.handle.net/10962/d1004018 , Picornaviruses , RNA viruses , Immunoglobulins , Encephalomyelitis
- Description: The Picornavirus family of positive sense RNA viruses includes some significant human and animal pathogens including Poliovirus (PV), Foot-and-Mouth disease virus (FMDV) and Human Rhinovirus (HRV). The genome is translated within the host cell into a polyprotein that is proteolytically cleaved into the structural and nonstructural proteins. The highly conserved, non-structural protein 2C has numerous roles during the virus life cycle and is essential for virus replication. Although the protein has been well studied in the case of PV, its interactions with the host cell during picornavirus infection is poorly understood. Theiler’s Encephalomyelitis virus (TMEV) is a picornavirus that infects mice, and is being used in our laboratory as a model in which to study the 2C protein. In this study, polyclonal antibodies against the TMEV 2C protein were generated and used to localise the protein in infected cells by indirect immunofluorescence. To produce antigen for immunisation purposes, the TMEV-2C protein sequence was analysed to identify hydrophilic and antigenic regions. An internal region of the 2C representing amino acid residues 31-210 was selected, expressed in bacteria and purified by nickel NTA affinity chromatography. Time course analysis of 2C (31-210) showed that the peptide was maximally expressed at 5 hours post induction. The peptide was solubilised using a mild detergent and 1.5 mg of purified antigen was used for immunisation of rabbits. Western blot analysis confirmed that the antibodies could detect both bacteriallyexpressed antigen, and virally-expressed 2C. Examination of virus-infected baby hamster kidney cells by immunofluorescence and confocal microscopy using the antiserum (anti-TMEV 2C antibodies) showed that the protein had a diffuse distribution upon early infection and at later stages it was located in a large perinuclear structure representing the viral replication complex. Furthermore, 2C localised to the Golgi apparatus as revealed by dual-label immunofluorescence using anti-TMEV 2C antibodies and wheat germ agglutinin (WGA). Furthermore, it was shown that TMEV infection results in changes in cell morphology and a redistribution of the cytoskeletal protein, β-actin. The successful production of antibodies that recognise TMEV 2C opens the way for further studies to investigate interactions between 2C and hostencoded factors.
- Full Text:
- Date Issued: 2010
Isolation of xylanolytic multi-enzyme complexes from Bacillus subtilis SJ01
- Authors: Jones, Sarah Melissa Jane
- Date: 2010
- Subjects: Bacillus subtilis , Xylans , Multienzyme complexes , Botanical chemistry , Cellulose , Hemicellulose , Polysaccharides
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3974 , http://hdl.handle.net/10962/d1004033 , Bacillus subtilis , Xylans , Multienzyme complexes , Botanical chemistry , Cellulose , Hemicellulose , Polysaccharides
- Description: Cellulose and hemicellulose account for a large portion of the world‘s plant biomass. In nature, these polysaccharides are intertwined forming complex materials that require multiple enzymes to degrade them. Multi-enzyme complexes (MECs) consist of a number of enzymes working in close proximity and synergistically to degrade complex substrates with higher efficiency than individual enzymes. The cellulosome is a cellulolytic MEC produced by anaerobic bacteria that has been studied extensively since its discovery in 1983. The aim of this study was to purify a cellulolytic and/or hemicellulolytic MEC from an aerobic bacterium of the Bacillus genus. Several bacterial isolates were identified using morphological characteristics and 16S rDNA sequencing, and screened for their ability to degrade cellulose and xylan using a MEC. The isolate that produced a high molecular weight protein fraction with the greatest ability to degrade Avicel®, carboxymethyl cellulose (CMC) and birchwood xylan was identified as Bacillus subtilis SJ01. An optimised growth medium, consisting of vitamins, trace elements, birchwood xylan (as the carbon source), and yeast and ammonium sulphate (as the nitrogen sources), increased the production of CMCase and xylanase enzymes from this bacterium. The removal of a competing bacterial strain from the culture and the inhibition of proteases also increased enzyme activities. A growth curve of B. subtilis SJ01 indicated that xylanase production was highest in early stationary growth phase and thus 84 hours was chosen as the best cell harvesting time. To purify the MECs produced by B. subtilis SJ01 size-exclusion chromatography on a Sephacryl S-400 column was used. It was concluded that (for the purposes of this study) the best method of concentrating the culture supernatant prior to loading onto Sephacryl S-400 was the use of ultrafiltration with a 50 kDa cut-off membrane. Two MECs, named C1 and C2 of 371 and 267 kDa, respectively, were purified from the culture supernatant of B. subtilis SJ01. Electrophoretic analysis revealed that these MECs consisted of 16 and 18 subunits, respectively, 4 of which degraded birchwood xylan and 5 of which degraded oat spelt xylan. The MECs degraded xylan substrates (C1: 0.24 U/mg, C2: 0.14 U/mg birchwood xylan) with higher efficiency than cellulose substrates (C1: 0.002 U/mg, C2: 0.01 U/mg CMC), and could therefore be considered xylanosomes. Interestingly, the MECs did not bind to insoluble birchwood xylan or Avicel® and did not contain glycosylated proteins, which are common features of cellulosomes. This study is, therefore, important in revealing the presence of MECs that differ from the cellulosome and that may have particular application in industries requiring high xylanase activity, such as the paper and pulp industry. The abundant genetic information available on B. subtilis means that this organism could also be used for genetic engineering of cellulolytic/hemicellulolytic MECs.
- Full Text:
- Date Issued: 2010
- Authors: Jones, Sarah Melissa Jane
- Date: 2010
- Subjects: Bacillus subtilis , Xylans , Multienzyme complexes , Botanical chemistry , Cellulose , Hemicellulose , Polysaccharides
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3974 , http://hdl.handle.net/10962/d1004033 , Bacillus subtilis , Xylans , Multienzyme complexes , Botanical chemistry , Cellulose , Hemicellulose , Polysaccharides
- Description: Cellulose and hemicellulose account for a large portion of the world‘s plant biomass. In nature, these polysaccharides are intertwined forming complex materials that require multiple enzymes to degrade them. Multi-enzyme complexes (MECs) consist of a number of enzymes working in close proximity and synergistically to degrade complex substrates with higher efficiency than individual enzymes. The cellulosome is a cellulolytic MEC produced by anaerobic bacteria that has been studied extensively since its discovery in 1983. The aim of this study was to purify a cellulolytic and/or hemicellulolytic MEC from an aerobic bacterium of the Bacillus genus. Several bacterial isolates were identified using morphological characteristics and 16S rDNA sequencing, and screened for their ability to degrade cellulose and xylan using a MEC. The isolate that produced a high molecular weight protein fraction with the greatest ability to degrade Avicel®, carboxymethyl cellulose (CMC) and birchwood xylan was identified as Bacillus subtilis SJ01. An optimised growth medium, consisting of vitamins, trace elements, birchwood xylan (as the carbon source), and yeast and ammonium sulphate (as the nitrogen sources), increased the production of CMCase and xylanase enzymes from this bacterium. The removal of a competing bacterial strain from the culture and the inhibition of proteases also increased enzyme activities. A growth curve of B. subtilis SJ01 indicated that xylanase production was highest in early stationary growth phase and thus 84 hours was chosen as the best cell harvesting time. To purify the MECs produced by B. subtilis SJ01 size-exclusion chromatography on a Sephacryl S-400 column was used. It was concluded that (for the purposes of this study) the best method of concentrating the culture supernatant prior to loading onto Sephacryl S-400 was the use of ultrafiltration with a 50 kDa cut-off membrane. Two MECs, named C1 and C2 of 371 and 267 kDa, respectively, were purified from the culture supernatant of B. subtilis SJ01. Electrophoretic analysis revealed that these MECs consisted of 16 and 18 subunits, respectively, 4 of which degraded birchwood xylan and 5 of which degraded oat spelt xylan. The MECs degraded xylan substrates (C1: 0.24 U/mg, C2: 0.14 U/mg birchwood xylan) with higher efficiency than cellulose substrates (C1: 0.002 U/mg, C2: 0.01 U/mg CMC), and could therefore be considered xylanosomes. Interestingly, the MECs did not bind to insoluble birchwood xylan or Avicel® and did not contain glycosylated proteins, which are common features of cellulosomes. This study is, therefore, important in revealing the presence of MECs that differ from the cellulosome and that may have particular application in industries requiring high xylanase activity, such as the paper and pulp industry. The abundant genetic information available on B. subtilis means that this organism could also be used for genetic engineering of cellulolytic/hemicellulolytic MECs.
- Full Text:
- Date Issued: 2010
The E.coli RNA degradosome analysis of molecular chaperones and enolase
- Authors: Burger, Adélle
- Date: 2010
- Subjects: Molecular chaperones , Escherichia coli -- Biotechnology , Polyphosphates , Polyphosphates -- Biotechnology , RNA-protein interactions
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3950 , http://hdl.handle.net/10962/d1004009 , Molecular chaperones , Escherichia coli -- Biotechnology , Polyphosphates , Polyphosphates -- Biotechnology , RNA-protein interactions
- Description: Normal mRNA turnover is essential for genetic regulation within cells. The E. coli RNA degradosome, a large multi-component protein complex which originates through specific protein interactions, has been referred to as the “RNA decay machine” and is responsible for mRNA turnover. The degradosome functions to process RNA and its key components have been identified. The scaffold protein is RNase E and it tethers the degradosome to the cytoplasmic membrane. Polynucleotide phosphorylase (PNPase), ATP-dependent RNA helicase (RhlB helicase) and the glycolytic enzyme enolase associate with RNase E to form the degradosome. Polyphosphate kinase associates with the degradosome in substoichiometric amounts, as do the molecular chaperones DnaK and GroEL. The role of DnaK as well as that of enolase in the RNA degradosome is unknown. Very limited research has been conducted on the components of the RNA degradosome under conditions of stress. The aim of this study was to understand the role played by enolase in the assembly of the degradosome under conditions of stress, as well as investigating the protein levels of molecular chaperones under these conditions. The RNA degradosome was successfully purified through its scaffold protein using nickel-affinity chromatography. In vivo studies were performed to investigate the protein levels of DnaK and GroEL present in the degradosome under conditions of heat stress, and whether GroEL could functionally replace DnaK in the degradosome. To investigate the recruitment of enolase to the degradosome under heat stress, a subcellular fractionation was performed to determine the localization of enolase upon heat shock in vivo. The elevated temperature resulted in an increased concentration of enolase in the membrane fraction. To determine whether there is an interaction between enolase and DnaK, enolase activity assays were conducted in vitro. The effect of DnaK on enolase activity was measured upon quantifying DnaK and adding it to the enolase assays. For the first time it was observed that the activity of enolase increased with the addition of substoichiometric amounts of DnaK. This indicates that DnaK may be interacting with the RNA degradosome via enolase.
- Full Text:
- Date Issued: 2010
- Authors: Burger, Adélle
- Date: 2010
- Subjects: Molecular chaperones , Escherichia coli -- Biotechnology , Polyphosphates , Polyphosphates -- Biotechnology , RNA-protein interactions
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3950 , http://hdl.handle.net/10962/d1004009 , Molecular chaperones , Escherichia coli -- Biotechnology , Polyphosphates , Polyphosphates -- Biotechnology , RNA-protein interactions
- Description: Normal mRNA turnover is essential for genetic regulation within cells. The E. coli RNA degradosome, a large multi-component protein complex which originates through specific protein interactions, has been referred to as the “RNA decay machine” and is responsible for mRNA turnover. The degradosome functions to process RNA and its key components have been identified. The scaffold protein is RNase E and it tethers the degradosome to the cytoplasmic membrane. Polynucleotide phosphorylase (PNPase), ATP-dependent RNA helicase (RhlB helicase) and the glycolytic enzyme enolase associate with RNase E to form the degradosome. Polyphosphate kinase associates with the degradosome in substoichiometric amounts, as do the molecular chaperones DnaK and GroEL. The role of DnaK as well as that of enolase in the RNA degradosome is unknown. Very limited research has been conducted on the components of the RNA degradosome under conditions of stress. The aim of this study was to understand the role played by enolase in the assembly of the degradosome under conditions of stress, as well as investigating the protein levels of molecular chaperones under these conditions. The RNA degradosome was successfully purified through its scaffold protein using nickel-affinity chromatography. In vivo studies were performed to investigate the protein levels of DnaK and GroEL present in the degradosome under conditions of heat stress, and whether GroEL could functionally replace DnaK in the degradosome. To investigate the recruitment of enolase to the degradosome under heat stress, a subcellular fractionation was performed to determine the localization of enolase upon heat shock in vivo. The elevated temperature resulted in an increased concentration of enolase in the membrane fraction. To determine whether there is an interaction between enolase and DnaK, enolase activity assays were conducted in vitro. The effect of DnaK on enolase activity was measured upon quantifying DnaK and adding it to the enolase assays. For the first time it was observed that the activity of enolase increased with the addition of substoichiometric amounts of DnaK. This indicates that DnaK may be interacting with the RNA degradosome via enolase.
- Full Text:
- Date Issued: 2010
Investigating the enzymatic mechanism of platinum nanoparticle synthesis in sulfate-reducing bacteria
- Authors: Riddin, Tamsyn Louise
- Date: 2009
- Subjects: Platinum , Nanoparticles , Sulfate-reducing bacteria
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3965 , http://hdl.handle.net/10962/d1004024 , Platinum , Nanoparticles , Sulfate-reducing bacteria
- Description: Efforts to discover an efficient yet environmentally friendly mode of metal nanoparticle (NP) synthesis are increasing rapidly. A ‘green’ route that avoids the high costs, toxic wastes and complicated protocols associated with chemical synthesis methods is therefore highly sought after. A biologically based protocol will provide the possibility of gaining control over the mechanism merely by manipulating the experimental conditions of the system. Given that the properties of nanoparticles are highly dependant on the morphology of the particles themselves, this mechanistic control will provide significant industrial advantages with regards to tailoring specific properties of the nanoparticles produced. The key objectives of this study were to: a) determine whether a consortium of sulfate-reducing bacteria was capable of platinum nanoparticle synthesis, b) elucidate the bioreductive, enzymatic mechanism responsible, and c) attempt to control the morphologies of the particles produced. A consortium of sulfate-reducing bacteria (SRB), isolated from sewage sludge, was used in these investigations due to the advantages a consortium provides in comparison to pure cultures. The syntrophic relationships established within the constituent species not only prevent the growth of contaminant microbes, but increases the oxygen-tolerance of the system as a whole. The sulfate-reducing consortium was shown to possess an aerobic mechanism for Pt(IV) reduction which, though different from the anaerobic bioreductive mechanism previously identified in literature, did not require an exogenous electron donor. It was demonstrated that the Pt(IV) ion becomes reduced to Pt(0) via a two-cycle mechanism involving Pt(II) as the intermediate. Further investigation elucidated the reduction of Pt(IV) to Pt(II) to be dependant on a novel Pt(IV) reductase which becomes upregulated in the presence of Cu(II), while the reduction of Pt(II) to Pt(0) occurred by means of a periplasmic hydrogenase. To our knowledge, this is the first time a coupled mechanism for Pt(IV) reduction by micro-organisms has been proposed. A cell-free, crude protein solution from the consortium produced both geometric and irregular platinum nanoparticles. The wavelength of 334 nm was chosen as a nonquantitative indicator of Pt(0) nanoparticle formation over time. The optimum conditions for nanoparticle synthesis were pH 9.0, 65 ˚C and 0.75 mM Pt(IV) as H2PtCl6 salt. In the absence of a buffer a Pt(IV) concentration > 1 mM resulted in the precipitation of protein-nanoparticle bioconjugates, due to unfavourable acidic conditions. This demonstrated that the nanoparticles were binding to and becoming stabilised by general protein in the cell-free solution. Upon addition of a sodium-bicarbonate buffer, a general increase in Pt(IV) reduction to Pt(II) was observed. The addition of the buffer also resulted in an unexplained change in particle morphology and for this reason was not used in subsequent investigations. Polyvinylpyrrolidone (PVP) was shown to compromise the reduction rate of the Pt(IV) ion by SRB cells. The presence of extracellular NP’s was suggested by the colour of the supernatant turning brown and the A334 increasing over time. Attempts to visualise the particles by transmission electron microscopy (TEM) resulted in an unexpected phenomenon where nanoparticles could be observed to form dynamically upon irradiation by the electron beam. Extended irradiation by the electron beam also resulted in structural changes of the particles occurring during observation. An increase in temperature was shown to increase the reduction rate which in turn resulted in particles decreasing in size. The starting pH was shown to have a significant effect on the reduction rate and particle morphology although specific trends could not be identified. In conclusion, the cell-soluble extract from the sulfate-reducing consortium investigated, is capable of Pt(0) nanoparticle synthesis. Precise control over the particle morphology was not attained although the mechanism was further clarified and optimal conditions for nanoparticle synthesis were determined.
- Full Text:
- Date Issued: 2009
- Authors: Riddin, Tamsyn Louise
- Date: 2009
- Subjects: Platinum , Nanoparticles , Sulfate-reducing bacteria
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3965 , http://hdl.handle.net/10962/d1004024 , Platinum , Nanoparticles , Sulfate-reducing bacteria
- Description: Efforts to discover an efficient yet environmentally friendly mode of metal nanoparticle (NP) synthesis are increasing rapidly. A ‘green’ route that avoids the high costs, toxic wastes and complicated protocols associated with chemical synthesis methods is therefore highly sought after. A biologically based protocol will provide the possibility of gaining control over the mechanism merely by manipulating the experimental conditions of the system. Given that the properties of nanoparticles are highly dependant on the morphology of the particles themselves, this mechanistic control will provide significant industrial advantages with regards to tailoring specific properties of the nanoparticles produced. The key objectives of this study were to: a) determine whether a consortium of sulfate-reducing bacteria was capable of platinum nanoparticle synthesis, b) elucidate the bioreductive, enzymatic mechanism responsible, and c) attempt to control the morphologies of the particles produced. A consortium of sulfate-reducing bacteria (SRB), isolated from sewage sludge, was used in these investigations due to the advantages a consortium provides in comparison to pure cultures. The syntrophic relationships established within the constituent species not only prevent the growth of contaminant microbes, but increases the oxygen-tolerance of the system as a whole. The sulfate-reducing consortium was shown to possess an aerobic mechanism for Pt(IV) reduction which, though different from the anaerobic bioreductive mechanism previously identified in literature, did not require an exogenous electron donor. It was demonstrated that the Pt(IV) ion becomes reduced to Pt(0) via a two-cycle mechanism involving Pt(II) as the intermediate. Further investigation elucidated the reduction of Pt(IV) to Pt(II) to be dependant on a novel Pt(IV) reductase which becomes upregulated in the presence of Cu(II), while the reduction of Pt(II) to Pt(0) occurred by means of a periplasmic hydrogenase. To our knowledge, this is the first time a coupled mechanism for Pt(IV) reduction by micro-organisms has been proposed. A cell-free, crude protein solution from the consortium produced both geometric and irregular platinum nanoparticles. The wavelength of 334 nm was chosen as a nonquantitative indicator of Pt(0) nanoparticle formation over time. The optimum conditions for nanoparticle synthesis were pH 9.0, 65 ˚C and 0.75 mM Pt(IV) as H2PtCl6 salt. In the absence of a buffer a Pt(IV) concentration > 1 mM resulted in the precipitation of protein-nanoparticle bioconjugates, due to unfavourable acidic conditions. This demonstrated that the nanoparticles were binding to and becoming stabilised by general protein in the cell-free solution. Upon addition of a sodium-bicarbonate buffer, a general increase in Pt(IV) reduction to Pt(II) was observed. The addition of the buffer also resulted in an unexplained change in particle morphology and for this reason was not used in subsequent investigations. Polyvinylpyrrolidone (PVP) was shown to compromise the reduction rate of the Pt(IV) ion by SRB cells. The presence of extracellular NP’s was suggested by the colour of the supernatant turning brown and the A334 increasing over time. Attempts to visualise the particles by transmission electron microscopy (TEM) resulted in an unexpected phenomenon where nanoparticles could be observed to form dynamically upon irradiation by the electron beam. Extended irradiation by the electron beam also resulted in structural changes of the particles occurring during observation. An increase in temperature was shown to increase the reduction rate which in turn resulted in particles decreasing in size. The starting pH was shown to have a significant effect on the reduction rate and particle morphology although specific trends could not be identified. In conclusion, the cell-soluble extract from the sulfate-reducing consortium investigated, is capable of Pt(0) nanoparticle synthesis. Precise control over the particle morphology was not attained although the mechanism was further clarified and optimal conditions for nanoparticle synthesis were determined.
- Full Text:
- Date Issued: 2009
Molecular and biochemical analysis of the diet of the black rhinoceros
- Authors: Kgopa, Ananias Hodi
- Date: 2009 , 2013-07-15
- Subjects: Black rhinoceros -- South Africa -- Eastern Cape , Browse (Animal food) -- South Africa -- Eastern Cape -- Analysis , Black rhinoceros -- Manure -- Analysis , Phenols , Antioxidants , Plant ecology -- South Africa -- Eastern Cape
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4064 , http://hdl.handle.net/10962/d1004721 , Black rhinoceros -- South Africa -- Eastern Cape , Browse (Animal food) -- South Africa -- Eastern Cape -- Analysis , Black rhinoceros -- Manure -- Analysis , Phenols , Antioxidants , Plant ecology -- South Africa -- Eastern Cape
- Description: The black rhinoceros, Diceros bicornis, is listed as critically endangered. The black rhinoceros population in the Great Fish River Reserve (GFRR) has increased steadily to a current estimate of one hundred animals since the re-introduction of four animals in 1986. In an effort to contribute to the effective conservation and management of this species, dietary composition was studied in the medium Portulcaria thicket vegetation of the GFRR. This study used a molecular approach to determine the diet of the black rhinoceros of the GFRR by sequencing the ribulose bisphosphate carboxylase large subunit (rbcL) gene in plants and dung. Twenty-three plant species were collected from the reserve, and 802 bp of the rbcL gene were sequenced. These plant sequences were used as a reference database for the identification of plant sequences generated from black rhinoceros dung. Initial studies investigated the amplification, cloning and sequencing of DNA extracted from the dung samples which indicated the viability of the molecular approach. Thereafter, dung generated rbcL DNA was analyzed by GS FLX sequencing. Of the plant sequences identified by comparison to the GenBank database, Carissa bispinosa was the most prevalent. The study further characterized the antioxidant activities and phenolic content of plants eaten by the black rhinoceros using four different assays. Phyllanthus verrucosus, Putterlickia pyracantha, Maytenus capitata, Euclea undulata and Ozoroa mucrunata consistently had high antioxidant activities when assayed against 2,2-azinobis (3-ethyl benzothiazolium-6-sulfonic acid) (ABTSʹ⁺), 2,2-diphenyl-1-picrylhydrazyl (DPPHʹ), and ferric reducing antioxidant potentials (FRAP) and phenolic content when evaluated using the Folin-Ciocalteu assay. The majority of plants investigated showed low antioxidant potentials and low phenolic content. The extent to which antioxidants influenced the browse selection by the black rhinoceros remains inconclusive.
- Full Text:
- Date Issued: 2009
- Authors: Kgopa, Ananias Hodi
- Date: 2009 , 2013-07-15
- Subjects: Black rhinoceros -- South Africa -- Eastern Cape , Browse (Animal food) -- South Africa -- Eastern Cape -- Analysis , Black rhinoceros -- Manure -- Analysis , Phenols , Antioxidants , Plant ecology -- South Africa -- Eastern Cape
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4064 , http://hdl.handle.net/10962/d1004721 , Black rhinoceros -- South Africa -- Eastern Cape , Browse (Animal food) -- South Africa -- Eastern Cape -- Analysis , Black rhinoceros -- Manure -- Analysis , Phenols , Antioxidants , Plant ecology -- South Africa -- Eastern Cape
- Description: The black rhinoceros, Diceros bicornis, is listed as critically endangered. The black rhinoceros population in the Great Fish River Reserve (GFRR) has increased steadily to a current estimate of one hundred animals since the re-introduction of four animals in 1986. In an effort to contribute to the effective conservation and management of this species, dietary composition was studied in the medium Portulcaria thicket vegetation of the GFRR. This study used a molecular approach to determine the diet of the black rhinoceros of the GFRR by sequencing the ribulose bisphosphate carboxylase large subunit (rbcL) gene in plants and dung. Twenty-three plant species were collected from the reserve, and 802 bp of the rbcL gene were sequenced. These plant sequences were used as a reference database for the identification of plant sequences generated from black rhinoceros dung. Initial studies investigated the amplification, cloning and sequencing of DNA extracted from the dung samples which indicated the viability of the molecular approach. Thereafter, dung generated rbcL DNA was analyzed by GS FLX sequencing. Of the plant sequences identified by comparison to the GenBank database, Carissa bispinosa was the most prevalent. The study further characterized the antioxidant activities and phenolic content of plants eaten by the black rhinoceros using four different assays. Phyllanthus verrucosus, Putterlickia pyracantha, Maytenus capitata, Euclea undulata and Ozoroa mucrunata consistently had high antioxidant activities when assayed against 2,2-azinobis (3-ethyl benzothiazolium-6-sulfonic acid) (ABTSʹ⁺), 2,2-diphenyl-1-picrylhydrazyl (DPPHʹ), and ferric reducing antioxidant potentials (FRAP) and phenolic content when evaluated using the Folin-Ciocalteu assay. The majority of plants investigated showed low antioxidant potentials and low phenolic content. The extent to which antioxidants influenced the browse selection by the black rhinoceros remains inconclusive.
- Full Text:
- Date Issued: 2009
Phenolic compounds in water and the implications for rapid detection of indicator micro-organisms using ß-D-Galactosidase and ß-D-Glucuronidase
- Authors: Abboo, Sagaran
- Date: 2009
- Subjects: Water -- Purification -- Biological treatment , Pollutants -- Biodegradation , Phenol , Organic water pollutants , Water quality biological assessment , Water -- Pollution
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3978 , http://hdl.handle.net/10962/d1004037 , Water -- Purification -- Biological treatment , Pollutants -- Biodegradation , Phenol , Organic water pollutants , Water quality biological assessment , Water -- Pollution
- Description: Faecal contamination in water is detected using appropriate microbial models such as total coliforms, faecal coliforms and E. coli. Βeta-D-Galactosidase (β-GAL) and Beta-D-glucuronidase (β-GUD) are two marker enzymes that are used to test for the presence of total coliforms and E. coli in water samples, respectively. Various assay methods have been developed using chromogenic and fluorogenic substrates. In this study, the chromogenic substrates chlorophenol red β-D-galactopyranoside (CPRG) for β-GAL and p-nitrophenyl-β-D-galactopyranoside (PNPG) for β-GUD were used. Potential problems associated with this approach include interference from other organisms present in the environment (e.g. plants, algae and other bacteria), as well as the presence of certain chemicals, such as phenolic compounds in water. Phenolic compounds are present in the aquatic environment due to their extensive industrial applications. The USA Enviromental Protection Agency (EPA) lists 11 Priority Pollutant Phenols (PPP) due to their high level of toxicity. This study investigated the interfering effects of the eleven PPP found in water on the enzyme activities of both the β-GAL and β-GUD enzyme assays. The presence of these PPP in the β-GAL and β-GUD enzyme assays showed that over and underestimation of activity may occur due to inhibition or activation of these enzymes. Three types of inhibition to enzyme activities were identified from double reciprocal Lineweaver-Burk plots. The inhibition constants (Ki) were determined for all inhibitory phenolic compounds from appropriate secondary plots. Furthermore, this study presented a validated reverse phase high performance liquid chromatography (RP-HPLC) method, developed for the simultaneous detection, separation and determination of all eleven phenolic compounds found in the environment. This method demonstrated good linearity, reproducibility, accuracy and sensitivity. Environmental water samples were collected from rivers, streams, industrial sites and wastewater treatment plant effluent. These samples were extracted and concentrated using a solid phase extraction (SPE) procedure prior to analysis employing the newly developed HPLC method in this study. Seasonal variations on the presence of the PPP in the environment were observed at certain collection sites. The concentrations found were between 0.033 μg/ml for 2,4-dinitrophenol in a running stream to 0.890 mg/ml for pentachlorophenol from an tannery industrial site. These concentrations of phenolic compounds found in these environments were able to interfere with the β-GAL and β-GUD enzyme assays.
- Full Text:
- Date Issued: 2009
- Authors: Abboo, Sagaran
- Date: 2009
- Subjects: Water -- Purification -- Biological treatment , Pollutants -- Biodegradation , Phenol , Organic water pollutants , Water quality biological assessment , Water -- Pollution
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3978 , http://hdl.handle.net/10962/d1004037 , Water -- Purification -- Biological treatment , Pollutants -- Biodegradation , Phenol , Organic water pollutants , Water quality biological assessment , Water -- Pollution
- Description: Faecal contamination in water is detected using appropriate microbial models such as total coliforms, faecal coliforms and E. coli. Βeta-D-Galactosidase (β-GAL) and Beta-D-glucuronidase (β-GUD) are two marker enzymes that are used to test for the presence of total coliforms and E. coli in water samples, respectively. Various assay methods have been developed using chromogenic and fluorogenic substrates. In this study, the chromogenic substrates chlorophenol red β-D-galactopyranoside (CPRG) for β-GAL and p-nitrophenyl-β-D-galactopyranoside (PNPG) for β-GUD were used. Potential problems associated with this approach include interference from other organisms present in the environment (e.g. plants, algae and other bacteria), as well as the presence of certain chemicals, such as phenolic compounds in water. Phenolic compounds are present in the aquatic environment due to their extensive industrial applications. The USA Enviromental Protection Agency (EPA) lists 11 Priority Pollutant Phenols (PPP) due to their high level of toxicity. This study investigated the interfering effects of the eleven PPP found in water on the enzyme activities of both the β-GAL and β-GUD enzyme assays. The presence of these PPP in the β-GAL and β-GUD enzyme assays showed that over and underestimation of activity may occur due to inhibition or activation of these enzymes. Three types of inhibition to enzyme activities were identified from double reciprocal Lineweaver-Burk plots. The inhibition constants (Ki) were determined for all inhibitory phenolic compounds from appropriate secondary plots. Furthermore, this study presented a validated reverse phase high performance liquid chromatography (RP-HPLC) method, developed for the simultaneous detection, separation and determination of all eleven phenolic compounds found in the environment. This method demonstrated good linearity, reproducibility, accuracy and sensitivity. Environmental water samples were collected from rivers, streams, industrial sites and wastewater treatment plant effluent. These samples were extracted and concentrated using a solid phase extraction (SPE) procedure prior to analysis employing the newly developed HPLC method in this study. Seasonal variations on the presence of the PPP in the environment were observed at certain collection sites. The concentrations found were between 0.033 μg/ml for 2,4-dinitrophenol in a running stream to 0.890 mg/ml for pentachlorophenol from an tannery industrial site. These concentrations of phenolic compounds found in these environments were able to interfere with the β-GAL and β-GUD enzyme assays.
- Full Text:
- Date Issued: 2009
Polymers, catalysts and nanostructures a hybrid approach to biomolecule detection
- Authors: Frith, Kelly-Anne
- Date: 2009
- Subjects: Polymers , Nanostructured materials , Biomolecules , Tryptophan , Melatonin , Electrodes , Electrochemistry , Tryptophan oxygenase
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3980 , http://hdl.handle.net/10962/d1004039 , Polymers , Nanostructured materials , Biomolecules , Tryptophan , Melatonin , Electrodes , Electrochemistry , Tryptophan oxygenase
- Description: The main goals in electroanalytical sensing are towards improved sensitivity and selectivity, or specificity, of an analyte. There are several approaches to achieving these goals with the main approach being modification of an electrode surface with synthetic or natural catalysts (enzymes), polymers and also utilisation of nanostructured materials. At present, there is a strong movement towards hybrid sensing which couple different properties of two or more surface modification approaches. In this thesis, a range of these surface modifications were explored for analysis and detection of two main analytes: the amino acid, tryptophan (Trp); and, the neurotransmitter, dopamine (DA). Specifically, this thesis aimed to utilise these methods to enhance the sensitivity and selectivity for Trp over an interferent, the indoleamine, melatonin (Mel); and, DA over the vitamin, ascorbic acid (AA). For Trp detection, immobilisation of an enzyme, Tryptophanase (Trpase) resulted in poor selectivity for the analyte. However, enhanced sensitivity and selectivity was achieved through pH manipulation of the electrolyte medium at a Nafion®-modified electrode surface for both Trp and Mel. At pH 3.0, the Mel and Trp anodic peak potentials were sufficiently resolved allowing for an LOD of 1.60 and 1.62 nM,respectively, and permitting the accurate analysis of Trp in a dietary supplement containing Mel. Multi-walled carbon nanotubes (MWCNTs) suspended in Nafion® exhibited further increases in the signal responses of these analytes at pH 3.0 and 7.4 with minimal change in the resolution of the anodic peaks. A lower sensitivity was, therefore, observed at the Nafion® and MWCNT modified electrode compared to the Nafion®-modified electrode at pH 3.0 with LODs of 0.59 and 0.80 nM exhibited for Trp and Mel, respectively. Enhanced selectivity for Trp in the presence of Mel can be achieved with MWCNTs in the presence of metallotetrasulphonated phthalocyanines (MTSPcs) particularly at pH 3.0, owing to cation exchange effects. However, the lack of sensitivity towards Trp, and even Mel, at this CoTSPc and MWCNT modified electrode remains a drawback. For DA, detection at the MWCNT and Nafion® surface resulted in improved sensitivity over that of both the bare electrode (613.0 nM) and the Nafion® modified electrode (1045.1 nM) with a calculated LOD of 133.9 nM at this layer. Furthermore, improvements in the selectivity of DA were achieved at the Nafion® and MWCNT modified electrode as exclusion of AA (150 μM) was achieved. At the MWCNT and CoTSPc surface, AA was excluded up to 130 μM with sensitivity for DA extending as low as 14.3 nM, far greater than observed for Trp and Mel. These concentrations are well within physiological concentration ranges and represent the most significant solution yet in terms of AA exclusion and enhanced sensitivity for DA. An examination of the surface layering by impedance spectroscopy and atomic force microscopy indicates that the success of the hybrid sensor utilising CoTSPc and MWCNTs lay in improved dispersion of MWCNTs and improved electron transfer kinetics, facilitated by the net charge of the materials present. This thesis, thus, showed the utility of a judicious selection of synthetic and biological catalysts, polymers and carbon nanomaterials towards a hybrid approach to the electrochemical sensing of Trp, Mel, DA and AA with focus on sensitivity and selectivity of these analytes.
- Full Text:
- Date Issued: 2009
- Authors: Frith, Kelly-Anne
- Date: 2009
- Subjects: Polymers , Nanostructured materials , Biomolecules , Tryptophan , Melatonin , Electrodes , Electrochemistry , Tryptophan oxygenase
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3980 , http://hdl.handle.net/10962/d1004039 , Polymers , Nanostructured materials , Biomolecules , Tryptophan , Melatonin , Electrodes , Electrochemistry , Tryptophan oxygenase
- Description: The main goals in electroanalytical sensing are towards improved sensitivity and selectivity, or specificity, of an analyte. There are several approaches to achieving these goals with the main approach being modification of an electrode surface with synthetic or natural catalysts (enzymes), polymers and also utilisation of nanostructured materials. At present, there is a strong movement towards hybrid sensing which couple different properties of two or more surface modification approaches. In this thesis, a range of these surface modifications were explored for analysis and detection of two main analytes: the amino acid, tryptophan (Trp); and, the neurotransmitter, dopamine (DA). Specifically, this thesis aimed to utilise these methods to enhance the sensitivity and selectivity for Trp over an interferent, the indoleamine, melatonin (Mel); and, DA over the vitamin, ascorbic acid (AA). For Trp detection, immobilisation of an enzyme, Tryptophanase (Trpase) resulted in poor selectivity for the analyte. However, enhanced sensitivity and selectivity was achieved through pH manipulation of the electrolyte medium at a Nafion®-modified electrode surface for both Trp and Mel. At pH 3.0, the Mel and Trp anodic peak potentials were sufficiently resolved allowing for an LOD of 1.60 and 1.62 nM,respectively, and permitting the accurate analysis of Trp in a dietary supplement containing Mel. Multi-walled carbon nanotubes (MWCNTs) suspended in Nafion® exhibited further increases in the signal responses of these analytes at pH 3.0 and 7.4 with minimal change in the resolution of the anodic peaks. A lower sensitivity was, therefore, observed at the Nafion® and MWCNT modified electrode compared to the Nafion®-modified electrode at pH 3.0 with LODs of 0.59 and 0.80 nM exhibited for Trp and Mel, respectively. Enhanced selectivity for Trp in the presence of Mel can be achieved with MWCNTs in the presence of metallotetrasulphonated phthalocyanines (MTSPcs) particularly at pH 3.0, owing to cation exchange effects. However, the lack of sensitivity towards Trp, and even Mel, at this CoTSPc and MWCNT modified electrode remains a drawback. For DA, detection at the MWCNT and Nafion® surface resulted in improved sensitivity over that of both the bare electrode (613.0 nM) and the Nafion® modified electrode (1045.1 nM) with a calculated LOD of 133.9 nM at this layer. Furthermore, improvements in the selectivity of DA were achieved at the Nafion® and MWCNT modified electrode as exclusion of AA (150 μM) was achieved. At the MWCNT and CoTSPc surface, AA was excluded up to 130 μM with sensitivity for DA extending as low as 14.3 nM, far greater than observed for Trp and Mel. These concentrations are well within physiological concentration ranges and represent the most significant solution yet in terms of AA exclusion and enhanced sensitivity for DA. An examination of the surface layering by impedance spectroscopy and atomic force microscopy indicates that the success of the hybrid sensor utilising CoTSPc and MWCNTs lay in improved dispersion of MWCNTs and improved electron transfer kinetics, facilitated by the net charge of the materials present. This thesis, thus, showed the utility of a judicious selection of synthetic and biological catalysts, polymers and carbon nanomaterials towards a hybrid approach to the electrochemical sensing of Trp, Mel, DA and AA with focus on sensitivity and selectivity of these analytes.
- Full Text:
- Date Issued: 2009
Purification and characterization of fructosyltransferase for the synthesis of short-chain fructo-oligosaccharides and investigation into thier anti-carcinogenic properties
- Authors: Nemukula, Aluwani
- Date: 2009
- Subjects: Oligosaccharides , Polygalacturonase , Aspergillus , Fructose , Inulin , Cancer -- Prevention , Cancer -- Research , Carcinogens , High performance liquid chromatography
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3927 , http://hdl.handle.net/10962/d1003986 , Oligosaccharides , Polygalacturonase , Aspergillus , Fructose , Inulin , Cancer -- Prevention , Cancer -- Research , Carcinogens , High performance liquid chromatography
- Description: There is a growing attention in the synthesis of fructo-oligosaccharides (FOS) due to their excellent bio-functional and health-promoting properties. The current production processes are limited to chemical hydrolysis reactions of plant extracts, which are often associated with several drawbacks. In this study, fructosyltransferase (FTase) and polygalacturonase (PGase) activities, present in a commercial enzyme preparation (Pectinex® Ultra SP-L) sourced from Aspergillus aculeatus, have been separated and fully purified by anion-exchange and sizeexclusion chromatography. The FTase possesses fructosyl transfer activity for FOS synthesis and the PGase has pectin hydrolytic activity. Fructosyltransferase is a single-band protein with a molecular weight of 85 kDa, whereas PGase is a distinct protein of 40 kDa. The temperature and pH optima of FTase were 60 ºC and 6.0, with a half-life of 8 h; while that for PGase were 40 ºC and 6.0, respectively. FTase was slightly inhibited in the presence of Ni²⁺, Mg²⁺ and urea; but PGase was more susceptible to divalent ions such as Ca²⁺, Mg²⁺ and Mn²⁺. The kinetic parameters (Km and Vmax) of FTase for the hydrolysis of β-(2→1) linkages from sucrose were 752.3 mM and 120.5 μmol.min⁻¹.mL⁻¹, respectively; whereas the same parameters for pectin hydrolysis by PGase were 13.0 mg.mL⁻¹ and 263 μmol.min-1.mL⁻¹, respectively. The purified FTase was able to transfer fructosyl residues from sucrose, synthesizing the corresponding chains of FOS. PGase was relatively stable at 40 ºC (t½ > 3 h), depolymerizing the pectin backbone while releasing the inulins from within the chicory roots. Analysis of various mixtures of FOS by mass spectrometry, HPLC and ¹H-NMR was undertaken. Results indicated that MS with electrospray ionization and ¹H-NMR are capable of providing relative quantitative data of the FOS present in the mixtures. The pharmaceutical effects of various sc-FOS (0.5%, v/v) and SCFA (0.3%, v/v) on certain bacterial enzymes (β-glucuronidase, urease and β-glucosidase) associated with the formation of carcinogens were also studied. These enzyme activities were not directly influenced by the sc-FOS, but were found to be remarkably decreased by SCFA, pointing toward the prebiotic effect of FOS in intestinal microflora modulation.
- Full Text:
- Date Issued: 2009
- Authors: Nemukula, Aluwani
- Date: 2009
- Subjects: Oligosaccharides , Polygalacturonase , Aspergillus , Fructose , Inulin , Cancer -- Prevention , Cancer -- Research , Carcinogens , High performance liquid chromatography
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3927 , http://hdl.handle.net/10962/d1003986 , Oligosaccharides , Polygalacturonase , Aspergillus , Fructose , Inulin , Cancer -- Prevention , Cancer -- Research , Carcinogens , High performance liquid chromatography
- Description: There is a growing attention in the synthesis of fructo-oligosaccharides (FOS) due to their excellent bio-functional and health-promoting properties. The current production processes are limited to chemical hydrolysis reactions of plant extracts, which are often associated with several drawbacks. In this study, fructosyltransferase (FTase) and polygalacturonase (PGase) activities, present in a commercial enzyme preparation (Pectinex® Ultra SP-L) sourced from Aspergillus aculeatus, have been separated and fully purified by anion-exchange and sizeexclusion chromatography. The FTase possesses fructosyl transfer activity for FOS synthesis and the PGase has pectin hydrolytic activity. Fructosyltransferase is a single-band protein with a molecular weight of 85 kDa, whereas PGase is a distinct protein of 40 kDa. The temperature and pH optima of FTase were 60 ºC and 6.0, with a half-life of 8 h; while that for PGase were 40 ºC and 6.0, respectively. FTase was slightly inhibited in the presence of Ni²⁺, Mg²⁺ and urea; but PGase was more susceptible to divalent ions such as Ca²⁺, Mg²⁺ and Mn²⁺. The kinetic parameters (Km and Vmax) of FTase for the hydrolysis of β-(2→1) linkages from sucrose were 752.3 mM and 120.5 μmol.min⁻¹.mL⁻¹, respectively; whereas the same parameters for pectin hydrolysis by PGase were 13.0 mg.mL⁻¹ and 263 μmol.min-1.mL⁻¹, respectively. The purified FTase was able to transfer fructosyl residues from sucrose, synthesizing the corresponding chains of FOS. PGase was relatively stable at 40 ºC (t½ > 3 h), depolymerizing the pectin backbone while releasing the inulins from within the chicory roots. Analysis of various mixtures of FOS by mass spectrometry, HPLC and ¹H-NMR was undertaken. Results indicated that MS with electrospray ionization and ¹H-NMR are capable of providing relative quantitative data of the FOS present in the mixtures. The pharmaceutical effects of various sc-FOS (0.5%, v/v) and SCFA (0.3%, v/v) on certain bacterial enzymes (β-glucuronidase, urease and β-glucosidase) associated with the formation of carcinogens were also studied. These enzyme activities were not directly influenced by the sc-FOS, but were found to be remarkably decreased by SCFA, pointing toward the prebiotic effect of FOS in intestinal microflora modulation.
- Full Text:
- Date Issued: 2009
Understanding the complexity of metabolic regulatory systems an investigation into the regulation of hydantoin-hydrolysis in Pseudomonas putida RU-KM3s
- Authors: De la Mare, Jo-Anne
- Date: 2009
- Subjects: Pseudomonas , Hydantoin , Hydrolysis , Enzymes -- Regulation
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3993 , http://hdl.handle.net/10962/d1004053 , Pseudomonas , Hydantoin , Hydrolysis , Enzymes -- Regulation
- Description: It has been well-established that Pseudomonas species possess extremely versatile metabolic systems allowing them to utilise a wide range of nutrient sources and, furthermore, that the regulation of these enzyme systems involves highly evolved and sophisticated regulatory machinery. This study examined the complexity of metabolic regulation in Pseudomonas using the hydantoin-hydrolysing system of the environmental isolate, Pseudomonas putida RU-KM3s. In this system, the genes encoding dihydropyrimidinase and β-ureidopropionase (dhp and bup) are arranged divergently on the chromosome, separated by a 616 bp intergenic region involved in the transcriptional regulation of these genes. The focus was on the transcriptional regulation of dhp expression. DHP activity was found to be sensitive to several environmental signals including growth phase, carbon catabolite repression (CCR), substrate induction and quorum sensing (QS). Bioinformatic analysis of the intergenic region upstream of dhp revealed a number of putative binding sites for transcriptional regulators, including recognition sequences for the alternate sigma factors σ54 and σ38, as well as for the global regulators Anr (for anaerobic regulator) and Vfr (for virulence factor regulator). The targeted disruption of the genes encoding the transcriptional regulators, Vfr and the major CCR protein, Crc, resulted in a partial relief from repression for the vfr- mutant under quorum sensing conditions and a general decrease in activity in the crc- mutant. This data suggested that both Vfr and Crc were involved in regulating DHP activity. Mutational analysis of the dhp promoter revealed that at least two sites were involved in regulating transcriptional activity, one which mediated activation and the other repression. These sites were designated as a putative Anr box, situated 232 bp from the start codon of dhp, and a CRP-like binding site, at a position 213 bp upstream of dhp. Taken together, this data shows the involvement of several global regulatory factors in controlling the expression of dhp. A complex synergistic model was proposed for the transcriptional regulation of dhp, involving alternate sigma factors in addition to both global and specific regulators and responding to a number of environmental signals associated with growth phase, including nutrient availability, cell density and oxygen status.
- Full Text:
- Date Issued: 2009
- Authors: De la Mare, Jo-Anne
- Date: 2009
- Subjects: Pseudomonas , Hydantoin , Hydrolysis , Enzymes -- Regulation
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3993 , http://hdl.handle.net/10962/d1004053 , Pseudomonas , Hydantoin , Hydrolysis , Enzymes -- Regulation
- Description: It has been well-established that Pseudomonas species possess extremely versatile metabolic systems allowing them to utilise a wide range of nutrient sources and, furthermore, that the regulation of these enzyme systems involves highly evolved and sophisticated regulatory machinery. This study examined the complexity of metabolic regulation in Pseudomonas using the hydantoin-hydrolysing system of the environmental isolate, Pseudomonas putida RU-KM3s. In this system, the genes encoding dihydropyrimidinase and β-ureidopropionase (dhp and bup) are arranged divergently on the chromosome, separated by a 616 bp intergenic region involved in the transcriptional regulation of these genes. The focus was on the transcriptional regulation of dhp expression. DHP activity was found to be sensitive to several environmental signals including growth phase, carbon catabolite repression (CCR), substrate induction and quorum sensing (QS). Bioinformatic analysis of the intergenic region upstream of dhp revealed a number of putative binding sites for transcriptional regulators, including recognition sequences for the alternate sigma factors σ54 and σ38, as well as for the global regulators Anr (for anaerobic regulator) and Vfr (for virulence factor regulator). The targeted disruption of the genes encoding the transcriptional regulators, Vfr and the major CCR protein, Crc, resulted in a partial relief from repression for the vfr- mutant under quorum sensing conditions and a general decrease in activity in the crc- mutant. This data suggested that both Vfr and Crc were involved in regulating DHP activity. Mutational analysis of the dhp promoter revealed that at least two sites were involved in regulating transcriptional activity, one which mediated activation and the other repression. These sites were designated as a putative Anr box, situated 232 bp from the start codon of dhp, and a CRP-like binding site, at a position 213 bp upstream of dhp. Taken together, this data shows the involvement of several global regulatory factors in controlling the expression of dhp. A complex synergistic model was proposed for the transcriptional regulation of dhp, involving alternate sigma factors in addition to both global and specific regulators and responding to a number of environmental signals associated with growth phase, including nutrient availability, cell density and oxygen status.
- Full Text:
- Date Issued: 2009
Metal bioaccumulation and precious metal refinery wastewater treatment by phoma glomerata
- Authors: Moore, Bronwyn Ann
- Date: 2008-03-18
- Subjects: Metals -- Bioaccumulation , Water purification -- South Africa , Metal ions , Water -- Purification -- Biological treatment -- South Africa , Water quality management -- South Africa , Factory and trade waste -- Purification -- South Africa , Metals -- Refining , Hyperaccumulator plants
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4097 , http://hdl.handle.net/10962/d1009441 , Metals -- Bioaccumulation , Water purification -- South Africa , Metal ions , Water -- Purification -- Biological treatment -- South Africa , Water quality management -- South Africa , Factory and trade waste -- Purification -- South Africa , Metals -- Refining , Hyperaccumulator plants
- Description: The biosorption of copper, nickel, gold and platinum from single metal aqueous solutions by the nickel hyperaccumulator Berkheya coddii plant biomass was investigated. Potentiometric titrations of the biomass and determination of optimal sorption pH for each metal showed that nickel ions were released from the biomass into solution. The presence of free nickel ions interfered with the uptake of the other three metals and further biosorption investigations were discontinued. Three fungal isolates found colonising metal solutions were cultured and screened for their ability to remove 50 mg.l⁻¹ of copper, nickel, gold and platinum from solution and to survive and grow in precious metal refinery wastewaters. One isolate was selected for further studies based on its superior metal uptake capabilities (35 and 39 mg.l⁻¹ of gold and platinum, respectively) and was identified as Phoma glomerata. Copper, nickel, gold and platinum uptake studies revealed that nickel and gold were the most toxic metal ions, however, toxicity was dependent on pH. At pH 6 more biomass growth was achieved than at lower pH values and metal uptake increased by 51 and 17 % for copper and nickel, respectively. In addition, the production of extracellular polymeric substances played a role in base metal interaction. Precious metals were observed to be preferentially removed from solution, complete removal of gold and platinum was observed at all initial pH values, 89 % of copper was bioaccumulated at an initial metal concentration of 55 mg.l⁻¹ (pH 6) and only 23 % of nickel was removed from solution under the same conditions. Metal bioaccumulation was confirmed through transmission electron microscopy and micro particle induced X-ray emission. The effect of P. glomerata immobilised in a packed bed reactor on precious metal refinery wastewaters was investigated. It was found that the fungal isolate was not able to remove the high salt and chemical oxygen demand concentrations found in the wastewaters, however due to its ability to survive and grow in undiluted wastewater and remove metal ions from solution it may be utilised as a metal detoxification step in the treatment process train. , PDFCreator Version 0.9.0 , AFPL Ghostscript 8.53
- Full Text:
- Authors: Moore, Bronwyn Ann
- Date: 2008-03-18
- Subjects: Metals -- Bioaccumulation , Water purification -- South Africa , Metal ions , Water -- Purification -- Biological treatment -- South Africa , Water quality management -- South Africa , Factory and trade waste -- Purification -- South Africa , Metals -- Refining , Hyperaccumulator plants
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4097 , http://hdl.handle.net/10962/d1009441 , Metals -- Bioaccumulation , Water purification -- South Africa , Metal ions , Water -- Purification -- Biological treatment -- South Africa , Water quality management -- South Africa , Factory and trade waste -- Purification -- South Africa , Metals -- Refining , Hyperaccumulator plants
- Description: The biosorption of copper, nickel, gold and platinum from single metal aqueous solutions by the nickel hyperaccumulator Berkheya coddii plant biomass was investigated. Potentiometric titrations of the biomass and determination of optimal sorption pH for each metal showed that nickel ions were released from the biomass into solution. The presence of free nickel ions interfered with the uptake of the other three metals and further biosorption investigations were discontinued. Three fungal isolates found colonising metal solutions were cultured and screened for their ability to remove 50 mg.l⁻¹ of copper, nickel, gold and platinum from solution and to survive and grow in precious metal refinery wastewaters. One isolate was selected for further studies based on its superior metal uptake capabilities (35 and 39 mg.l⁻¹ of gold and platinum, respectively) and was identified as Phoma glomerata. Copper, nickel, gold and platinum uptake studies revealed that nickel and gold were the most toxic metal ions, however, toxicity was dependent on pH. At pH 6 more biomass growth was achieved than at lower pH values and metal uptake increased by 51 and 17 % for copper and nickel, respectively. In addition, the production of extracellular polymeric substances played a role in base metal interaction. Precious metals were observed to be preferentially removed from solution, complete removal of gold and platinum was observed at all initial pH values, 89 % of copper was bioaccumulated at an initial metal concentration of 55 mg.l⁻¹ (pH 6) and only 23 % of nickel was removed from solution under the same conditions. Metal bioaccumulation was confirmed through transmission electron microscopy and micro particle induced X-ray emission. The effect of P. glomerata immobilised in a packed bed reactor on precious metal refinery wastewaters was investigated. It was found that the fungal isolate was not able to remove the high salt and chemical oxygen demand concentrations found in the wastewaters, however due to its ability to survive and grow in undiluted wastewater and remove metal ions from solution it may be utilised as a metal detoxification step in the treatment process train. , PDFCreator Version 0.9.0 , AFPL Ghostscript 8.53
- Full Text:
An investigation into the synergistic association between the major Clostridium cellulovorans cellulosomal endoglucanase and two hemicellulases on plant cell wall degradation
- Authors: Beukes, Natasha
- Date: 2008
- Subjects: Clostridium , Cellulose , Hemicellulose , Cellulase , Biomass conversion , Biomass energy -- South Africa , Energy crops -- South Africa , Bagasse -- Biodegradation , Pineapple -- Biodegradation
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3968 , http://hdl.handle.net/10962/d1004027 , Clostridium , Cellulose , Hemicellulose , Cellulase , Biomass conversion , Biomass energy -- South Africa , Energy crops -- South Africa , Bagasse -- Biodegradation , Pineapple -- Biodegradation
- Description: The cellulosome is a multimeric enzyme complex that has the ability to metabolise a wide variety of carbonaceous compounds. Cellulosomal composition may vary according to the microbe’s nutritional requirement and allows for the anaerobic degradation of complex substrates. The complex substrates of interest in this research study were sugarcane bagasse and pineapple fibre waste, as they represent two important lignocellulosic, South African agricultural crops. The effective degradation of complex plant biomass wastes may present a valuable source of renewable compounds for the production of a variety of biofuels, for example bioethanol, and a variety of biocomposites of industrial importance. The identification of renewable energy sources for the production of biofuels is becoming increasingly important, as a result of the rapid depletion of the fossil fuels that are traditionally used as energy sources. An effective means of completely degrading lignocellulose biomass still remains elusive due to the complex heterogeneity of the substrate structure, and the fact that the effective degradation of the substrate requires a consortium of enzymes. The cellulosome not only provides a variety of enzymes with varying specificities, but also promote a close proximity between the catalytic components (enzymes). The close proximity between the enzymes promotes the synergistic degradation of complex plant biomass for the production of valuable energy products. Previous synergy studies have focused predominantly on the synergistic associations between cellulases; however, the synergy between hemicellulases has occasionally been documented. This research project established the synergistic associations between two Clostridium cellulovorans hemicellulases that may be incorporated into the cellulosome and a cellulosomal endoglucanase that is conserved in all cellulosomes. This research study indicated that there was indeed a synergistic degradation of the complex plant biomass (sugarcane bagasse and pineapple fibre). The degrees of synergy and the ratio of the enzymes varied between the two complex substrates. The initial degradation of the bagasse required the presence of all the enzymes and proceeded at an enhanced rate under sulphidogenic conditions; however, there was a low production of fermentable sugars. The low quantity of fermentable sugars produced by the degradation of the bagasse may be related to the chemical composition of the substrate. The sugarcane contains a high percentage of lignin forming a protective layer around the holocellulose, thus the glycosidic bonds are shielded extensively from enzymatic attack. In comparison, the initial degradation of the pineapple fibre required the action of hemicellulases, and proceeded at an enhanced rate under sulphidogenic conditions. The initial degradation of the pineapple fibre produced a substantially larger quantity of fermentable sugars in comparison to the bagasse. The higher production of fermentable sugars from the degradation of the pineapple fibre may be explained by the fact that this substrate may have a lower percentage of lignin than the bagasse, thus allowing a larger percentage of the glycosidic bonds to be exposed to enzymatic attack. The data obtained also indicated that the glycosidic bonds from the hemicellulosic components of the pineapple fibre shielded the glycosidic bonds of the cellulose component. The identification of the chemical components of the different substrates may allow for the initial development of an ideal enzyme complex (designer cellulosome) with enzymes in an ideal ratio with optimal synergy that will effectively degrade the complex plant biomass substrate.
- Full Text:
- Date Issued: 2008
- Authors: Beukes, Natasha
- Date: 2008
- Subjects: Clostridium , Cellulose , Hemicellulose , Cellulase , Biomass conversion , Biomass energy -- South Africa , Energy crops -- South Africa , Bagasse -- Biodegradation , Pineapple -- Biodegradation
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3968 , http://hdl.handle.net/10962/d1004027 , Clostridium , Cellulose , Hemicellulose , Cellulase , Biomass conversion , Biomass energy -- South Africa , Energy crops -- South Africa , Bagasse -- Biodegradation , Pineapple -- Biodegradation
- Description: The cellulosome is a multimeric enzyme complex that has the ability to metabolise a wide variety of carbonaceous compounds. Cellulosomal composition may vary according to the microbe’s nutritional requirement and allows for the anaerobic degradation of complex substrates. The complex substrates of interest in this research study were sugarcane bagasse and pineapple fibre waste, as they represent two important lignocellulosic, South African agricultural crops. The effective degradation of complex plant biomass wastes may present a valuable source of renewable compounds for the production of a variety of biofuels, for example bioethanol, and a variety of biocomposites of industrial importance. The identification of renewable energy sources for the production of biofuels is becoming increasingly important, as a result of the rapid depletion of the fossil fuels that are traditionally used as energy sources. An effective means of completely degrading lignocellulose biomass still remains elusive due to the complex heterogeneity of the substrate structure, and the fact that the effective degradation of the substrate requires a consortium of enzymes. The cellulosome not only provides a variety of enzymes with varying specificities, but also promote a close proximity between the catalytic components (enzymes). The close proximity between the enzymes promotes the synergistic degradation of complex plant biomass for the production of valuable energy products. Previous synergy studies have focused predominantly on the synergistic associations between cellulases; however, the synergy between hemicellulases has occasionally been documented. This research project established the synergistic associations between two Clostridium cellulovorans hemicellulases that may be incorporated into the cellulosome and a cellulosomal endoglucanase that is conserved in all cellulosomes. This research study indicated that there was indeed a synergistic degradation of the complex plant biomass (sugarcane bagasse and pineapple fibre). The degrees of synergy and the ratio of the enzymes varied between the two complex substrates. The initial degradation of the bagasse required the presence of all the enzymes and proceeded at an enhanced rate under sulphidogenic conditions; however, there was a low production of fermentable sugars. The low quantity of fermentable sugars produced by the degradation of the bagasse may be related to the chemical composition of the substrate. The sugarcane contains a high percentage of lignin forming a protective layer around the holocellulose, thus the glycosidic bonds are shielded extensively from enzymatic attack. In comparison, the initial degradation of the pineapple fibre required the action of hemicellulases, and proceeded at an enhanced rate under sulphidogenic conditions. The initial degradation of the pineapple fibre produced a substantially larger quantity of fermentable sugars in comparison to the bagasse. The higher production of fermentable sugars from the degradation of the pineapple fibre may be explained by the fact that this substrate may have a lower percentage of lignin than the bagasse, thus allowing a larger percentage of the glycosidic bonds to be exposed to enzymatic attack. The data obtained also indicated that the glycosidic bonds from the hemicellulosic components of the pineapple fibre shielded the glycosidic bonds of the cellulose component. The identification of the chemical components of the different substrates may allow for the initial development of an ideal enzyme complex (designer cellulosome) with enzymes in an ideal ratio with optimal synergy that will effectively degrade the complex plant biomass substrate.
- Full Text:
- Date Issued: 2008
Interaction between arbuscular mycorrhizal fungi and soil microbial populations in the rhizosphere
- Authors: Ike-Izundu, Nnenna Esther
- Date: 2008
- Subjects: Mycorrhizas , Mycorrhizal fungi , Vesicular-arbuscular mycorrhizas , Soil microbiology , Rhizosphere , Revegetation , Restoration ecology
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3962 , http://hdl.handle.net/10962/d1004021 , Mycorrhizas , Mycorrhizal fungi , Vesicular-arbuscular mycorrhizas , Soil microbiology , Rhizosphere , Revegetation , Restoration ecology
- Description: This study examined the rehabilitation potential of AM fungi with organic and inorganic fertilisers under pot and field trial conditions as well as their interaction with rhizospheric organisms and specific functional groups. In addition, the study highlighted the effects of land-use management on AM fungal populations in soil and the mycorrhizal status of some selected plants from one of the study sites. The study focussed on two sites that differ in operational activities and these included a mined area that was to be rehabilitated and a commercial farming site. A pot trial was conducted using an overburdened soil resulting from kaolin clay mining. Pots were seeded with Cynodon dactylon and treated with either Organic Tea or NPK (3:1:5) fertiliser, with or without AM fungal inoculum. The compatibility of these fertilisers with AM fungi was assessed by plant growth and percentage root colonisation. Maximum shoot height and plant biomass were observed at the 28th week with NPK (3:1:5) fertiliser supporting mycorrhizal colonisation by 80%. The result indicated the potential of AM fungi to be used in rehabilitation with minimal phosphate fertiliser. Similarly, a field trial was set-up using 17 x 17 m[superscript 2] plots in the mining site that were treated with the same organic and inorganic fertilisers as well as with AM fungal inoculum in different combinations. The interaction between AM fungi and soil microbial population was determined using culture dependent and culture independent techniques. The culture dependent technique involved the use of soil dilution and plating on general purpose and selective media. The result showed that there was no change in the total culturable bacterial number in the untreated and AM fungal treated plots, while a change in species composition was observed in the functional groups. Different functional groups identified included nitrogen fixing bacteria, pseudomonads, actinomycetes, phosphate solubilisers and the fungal counterparts. Gram-positive bacteria were observed as the predominant phenotypic type, while nitrogen fixers and actinomycetes were the predominant functional groups. Species identified from each functional group were Pseudomonas fulva, Bacillus megaterium, Streptomyces and actinomycetales bacteria. Meanwhile, fungi such as Ampelomyces, Fusarium, Penicillium, Aspergillus, Cephalosporium and Exserohilium were identified morphologically and molecularly. Furthermore, the mining site had a significantly higher bacterial number than the farming site thereby indicating the effects of land-use management on culturable bacterial numbers. The culture independent technique was carried out by cloning of the bacterial 16S rDNA and sequencing. Identified clones were Bradyrhizobium, Propionibacterium and Sporichthya. A cladogram constructed with the nucleotides sequences of identified functional species, clones and closely related nucleotide sequences from the Genbank indicated that nucleotide sequences differed in terms of the method used. The activity and establishment of the introduced AM fungal population was determined by spore enumeration, infectivity assay, percentage root colonisation and assessment of glomalin concentrations. The results indicated that the two land use types affected AM fungal populations. However, the establishment of AM fungi in the farming site was more successful than in the mining site as indicated by the higher infectivity pontential. Selected host plants, which were collected around the mine area, were observed to be mainly colonised by AM fungi and these were identified as Pentzia incana, Elytropappus rhinocerotis, Euphorbia meloformis, Selago corymbosa, Albuca canadensis and Helichrysum rosum. These plant species were able to thrive under harsh environmental conditions, thereby indicating their potential use as rehabilitation host plants. Generally, the findings of this study has provided an insight into the interaction between arbuscular mycorrhizal fungi and other soil microorganisms in two fields with differing land use management practices.
- Full Text:
- Date Issued: 2008
- Authors: Ike-Izundu, Nnenna Esther
- Date: 2008
- Subjects: Mycorrhizas , Mycorrhizal fungi , Vesicular-arbuscular mycorrhizas , Soil microbiology , Rhizosphere , Revegetation , Restoration ecology
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3962 , http://hdl.handle.net/10962/d1004021 , Mycorrhizas , Mycorrhizal fungi , Vesicular-arbuscular mycorrhizas , Soil microbiology , Rhizosphere , Revegetation , Restoration ecology
- Description: This study examined the rehabilitation potential of AM fungi with organic and inorganic fertilisers under pot and field trial conditions as well as their interaction with rhizospheric organisms and specific functional groups. In addition, the study highlighted the effects of land-use management on AM fungal populations in soil and the mycorrhizal status of some selected plants from one of the study sites. The study focussed on two sites that differ in operational activities and these included a mined area that was to be rehabilitated and a commercial farming site. A pot trial was conducted using an overburdened soil resulting from kaolin clay mining. Pots were seeded with Cynodon dactylon and treated with either Organic Tea or NPK (3:1:5) fertiliser, with or without AM fungal inoculum. The compatibility of these fertilisers with AM fungi was assessed by plant growth and percentage root colonisation. Maximum shoot height and plant biomass were observed at the 28th week with NPK (3:1:5) fertiliser supporting mycorrhizal colonisation by 80%. The result indicated the potential of AM fungi to be used in rehabilitation with minimal phosphate fertiliser. Similarly, a field trial was set-up using 17 x 17 m[superscript 2] plots in the mining site that were treated with the same organic and inorganic fertilisers as well as with AM fungal inoculum in different combinations. The interaction between AM fungi and soil microbial population was determined using culture dependent and culture independent techniques. The culture dependent technique involved the use of soil dilution and plating on general purpose and selective media. The result showed that there was no change in the total culturable bacterial number in the untreated and AM fungal treated plots, while a change in species composition was observed in the functional groups. Different functional groups identified included nitrogen fixing bacteria, pseudomonads, actinomycetes, phosphate solubilisers and the fungal counterparts. Gram-positive bacteria were observed as the predominant phenotypic type, while nitrogen fixers and actinomycetes were the predominant functional groups. Species identified from each functional group were Pseudomonas fulva, Bacillus megaterium, Streptomyces and actinomycetales bacteria. Meanwhile, fungi such as Ampelomyces, Fusarium, Penicillium, Aspergillus, Cephalosporium and Exserohilium were identified morphologically and molecularly. Furthermore, the mining site had a significantly higher bacterial number than the farming site thereby indicating the effects of land-use management on culturable bacterial numbers. The culture independent technique was carried out by cloning of the bacterial 16S rDNA and sequencing. Identified clones were Bradyrhizobium, Propionibacterium and Sporichthya. A cladogram constructed with the nucleotides sequences of identified functional species, clones and closely related nucleotide sequences from the Genbank indicated that nucleotide sequences differed in terms of the method used. The activity and establishment of the introduced AM fungal population was determined by spore enumeration, infectivity assay, percentage root colonisation and assessment of glomalin concentrations. The results indicated that the two land use types affected AM fungal populations. However, the establishment of AM fungi in the farming site was more successful than in the mining site as indicated by the higher infectivity pontential. Selected host plants, which were collected around the mine area, were observed to be mainly colonised by AM fungi and these were identified as Pentzia incana, Elytropappus rhinocerotis, Euphorbia meloformis, Selago corymbosa, Albuca canadensis and Helichrysum rosum. These plant species were able to thrive under harsh environmental conditions, thereby indicating their potential use as rehabilitation host plants. Generally, the findings of this study has provided an insight into the interaction between arbuscular mycorrhizal fungi and other soil microorganisms in two fields with differing land use management practices.
- Full Text:
- Date Issued: 2008