Using bioinformatics tools to screen for trypanosomal cathepsin B cysteine protease inhibitors from the SANCDB as a novel therapeutic modality against Human African Trypanosomiasis (HAT)
- Authors: Mokhawa, Gaone
- Date: 2016
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/3304 , vital:20470
- Description: Human African Trypanosomiasis (HAT), also known as sleeping sickness, is a fatal chronic disease that is caused by flagellated protozoans, Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense. HAT is spread by a bite from an infected tsetse fly of the Glosina genus. Up to 60 million people in 36 countries in sub-Saharan Africa are at a risk of infection from HAT with up to 30 000 deaths reported every year. Current chemotherapy for HAT is insufficient since the available drugs exhibit unacceptable side effects (toxicity) and parasite resistance. Novel treatments and approaches for development of specific and more potent drugs for HAT are therefore required. One approach is to target vital proteins that are essential to the life cycle of the parasite. The main interest of this study is to explore Trypanosoma brucei cathepsin B-like protease (TbCatB) structural and functional properties with the primary goal of discovering non peptide small molecule inhibitors of TbCatB using bioinformatics approaches. TbCatB is a papain family C1 cysteine protease which belongs to clan CA group and it has emerged as a potential HAT drug target. Papain family cysteine proteases of Clan CA group of Trypanosoma brucei (rhodesain and TbCatB) have demonstrated potential as chemotherapeutic targets using synthetic protease inhibitors like Z-Phe-Ala-CHN2 to kill the parasite in vitro and in vivo. TbCatB has been identified as the essential cysteine protease of T. brucei since mRNA silencing of TbCatB killed the parasite and resulted in a cure in mice infected with T. brucei while mRNA silencing of rhodesain only extended mice life. TbCatB is therefore a promising drug target against HAT and the discovery and development of compounds that can selectively inhibit TbCatB without posing any danger to the human host represent a great therapeutic solution for treatment of HAT. To understand protein-inhibitor interactions, useful information can be obtained from high resolution protease-inhibitor crystal structure complexes. This study aims to use bioinformatics approaches to carry out comparative sequence, structural and functional analysis of TbCatB protease and its homologs from T. congolense, T, cruzi, T. vivax and H. sapien as well as to identify non-peptide small molecule inhibitors of TbCatB cysteine proteases from natural compounds of South African origin. Sequences of TbCatB (PDB ID: 3HHI) homologs were retrieved by a BLAST search. Human cathepsin B (PDB ID: 3CBJ) was selected from a list of templates for homology modelling found by HHpred. MODELLER version 9.10 program was used to generate a hundred models for T. congolense, T, cruzi and T. vivax cathepsin B like proteases using 3HHI and 3CBJ as templates. The best models were chosen based on their low DOPE Z scores before validation using MetaMQAPII, ANOLEA, PROCHECK and QMEAN6. The DOPE Z scores and the RMSD (RMS) values of the calculated models indicate that the models are of acceptable energy (stability) and fold (conformation). Results from the different MQAPs indicate the models are of acceptable quality and they can be used for docking studies. High throughput screening of SANCDB using AutoDock Vina revealed nine compounds, SANC00 478, 479, 480, 481, 482, 488, 489, 490 and 491, having a strong affinity for Trypanosoma spp. cathepsin B proteases than HsCatB. SANC00488 has the strongest binding to Trypanosoma spp. cathepsin B proteases and the weakest binding to HsCatB protease. Molecular dynamics (MD) simulations show that the complexes between SANC00488 and TbCatB, TcCatB, TcrCatB and TvCatB are stable and do not come apart during simulation. The complex between this compound and HsCatB however is unstable and comes apart during simulation. Residues that are important for the stability of SANC00488-TbCatB complex are Gly328 of the S2 subsite, Phe208, and Ala256. In conclusion SANC00488 is a good candidate for development of a drug against HAT.
- Full Text:
- Date Issued: 2016
- Authors: Mokhawa, Gaone
- Date: 2016
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/3304 , vital:20470
- Description: Human African Trypanosomiasis (HAT), also known as sleeping sickness, is a fatal chronic disease that is caused by flagellated protozoans, Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense. HAT is spread by a bite from an infected tsetse fly of the Glosina genus. Up to 60 million people in 36 countries in sub-Saharan Africa are at a risk of infection from HAT with up to 30 000 deaths reported every year. Current chemotherapy for HAT is insufficient since the available drugs exhibit unacceptable side effects (toxicity) and parasite resistance. Novel treatments and approaches for development of specific and more potent drugs for HAT are therefore required. One approach is to target vital proteins that are essential to the life cycle of the parasite. The main interest of this study is to explore Trypanosoma brucei cathepsin B-like protease (TbCatB) structural and functional properties with the primary goal of discovering non peptide small molecule inhibitors of TbCatB using bioinformatics approaches. TbCatB is a papain family C1 cysteine protease which belongs to clan CA group and it has emerged as a potential HAT drug target. Papain family cysteine proteases of Clan CA group of Trypanosoma brucei (rhodesain and TbCatB) have demonstrated potential as chemotherapeutic targets using synthetic protease inhibitors like Z-Phe-Ala-CHN2 to kill the parasite in vitro and in vivo. TbCatB has been identified as the essential cysteine protease of T. brucei since mRNA silencing of TbCatB killed the parasite and resulted in a cure in mice infected with T. brucei while mRNA silencing of rhodesain only extended mice life. TbCatB is therefore a promising drug target against HAT and the discovery and development of compounds that can selectively inhibit TbCatB without posing any danger to the human host represent a great therapeutic solution for treatment of HAT. To understand protein-inhibitor interactions, useful information can be obtained from high resolution protease-inhibitor crystal structure complexes. This study aims to use bioinformatics approaches to carry out comparative sequence, structural and functional analysis of TbCatB protease and its homologs from T. congolense, T, cruzi, T. vivax and H. sapien as well as to identify non-peptide small molecule inhibitors of TbCatB cysteine proteases from natural compounds of South African origin. Sequences of TbCatB (PDB ID: 3HHI) homologs were retrieved by a BLAST search. Human cathepsin B (PDB ID: 3CBJ) was selected from a list of templates for homology modelling found by HHpred. MODELLER version 9.10 program was used to generate a hundred models for T. congolense, T, cruzi and T. vivax cathepsin B like proteases using 3HHI and 3CBJ as templates. The best models were chosen based on their low DOPE Z scores before validation using MetaMQAPII, ANOLEA, PROCHECK and QMEAN6. The DOPE Z scores and the RMSD (RMS) values of the calculated models indicate that the models are of acceptable energy (stability) and fold (conformation). Results from the different MQAPs indicate the models are of acceptable quality and they can be used for docking studies. High throughput screening of SANCDB using AutoDock Vina revealed nine compounds, SANC00 478, 479, 480, 481, 482, 488, 489, 490 and 491, having a strong affinity for Trypanosoma spp. cathepsin B proteases than HsCatB. SANC00488 has the strongest binding to Trypanosoma spp. cathepsin B proteases and the weakest binding to HsCatB protease. Molecular dynamics (MD) simulations show that the complexes between SANC00488 and TbCatB, TcCatB, TcrCatB and TvCatB are stable and do not come apart during simulation. The complex between this compound and HsCatB however is unstable and comes apart during simulation. Residues that are important for the stability of SANC00488-TbCatB complex are Gly328 of the S2 subsite, Phe208, and Ala256. In conclusion SANC00488 is a good candidate for development of a drug against HAT.
- Full Text:
- Date Issued: 2016
Localizing selected endocytosis protein candidates in Plasmodium falciparum using GFP-tagged fusion constructs
- Authors: Basson, Travis
- Date: 2016
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/2680 , vital:20316
- Description: Malaria is a mosquito-borne infectious disease caused by several obligate intracellular protozoan parasites in the Plasmodium genus, with Plasmodium falciparum causing the most widespread cases and malaria deaths. In 2013 there were approximately 190 million cases of the disease and between 584,000 and 855,000 deaths. It is essential to identify novel drug targets and develop novel drug candidates due to the increase in resistance of P. falciparum parasites to the current arsenal of antimalarial drugs. Endocytosis is an essential process in eukaryotic cells in which the external environment is internalized by the cell in order to obtain various particles from the extracellular space. This extracellular cytoplasm is internalized in membrane-bound invaginations at the plasma membrane. During the blood stage of malaria infection, the parasite requires nutrients from the host red blood cell. To obtain these nutrients, the parasite internalizes haemoglobin in large amounts and degrades it in an acidic, lysosome-like organelle, known as the digestive vacuole. Whilst the exact molecular mechanism of malaria parasite endocytosis is not yet fully understood, a number of proteins have been suggested to be involved. The most expedient approach in identifying candidate endocytosis proteins is to investigate parasite homologues of proteins known to be involved in endocytosis in mammalian cells. The three proteins selected for investigation in this study were the P. falciparum homologues of coronin, dynamin 2, and μ4. The coding sequences for the candidate endocytosis proteins were amplified by PCR and cloned into the pARL2-GFP expression vector. P. falciparum 3D7 parasites were transfected with these vectors and the episomal expression of full-length GFP-tagged fusion protein was confirmed by Western blot analysis using commercially available anti-GFP antibodies. Microscopic analysis of live parasites using fluorescence and confocal microscopy was used to determine the localization of the candidate endocytosis proteins. Coronin appeared to display diffuse cytoplasmic GFP localization during the trophozoite stage, arguing against a role in endocytosis. However, distinct localization during the schizont stage at what appears to be the inner membrane complex was observed. Coronin is thus likely required to coordinate the formation of the actin network between the merozoite IMC and the plasma membrane on which the glideosome is dependant for generating the motile forces required for the merozoite motility and invasion of RBCs. Dynamin 2 displayed localization at three potential locii: the parasite periphery (plasma membrane), punctuate regions within the cytoplasm (potentially at membrane bound organelles) and at the parasite food vacuole. The data suggested that dynamin 2 is involved in endocytosis and membrane trafficking in a similar manner to classical dynamins, potentially as a vesicle scission molecule at the plasma membrane, mediating vesicle formation at the food vacuole to recycle membrane to the plasma membrane, and possibly mitochondria organelle division. μ4 displayed transient localization, cycling between cytosolic localization, and localization to distinct regions at the plasma membrane and the food vacuole. Localization of Pfμ4 to the plasma membrane is indicative of a role for μ4 as a part of an adaptor protein (AP) complex which may be responsible for recruitment of clathrin to initiate endocytosis in a manner similar to mammalian AP-2. As was observed with PfDYN2, Pfμ4 localizes to the FV, which suggests that Pfμ4 forms part of a coat complex that mediates the formation of vesicles that recycle membrane from the FV to the parasite plasma membrane. This study showed that expressing proteins as full-length GFP-tagged fusion constructs is an effective approach in the early stages of determining the localization and function of P. falciparum proteins in vitro, and distinguishing between candidates that have a potential role in endocytosis and those that are unlikely to do so.
- Full Text:
- Date Issued: 2016
- Authors: Basson, Travis
- Date: 2016
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/2680 , vital:20316
- Description: Malaria is a mosquito-borne infectious disease caused by several obligate intracellular protozoan parasites in the Plasmodium genus, with Plasmodium falciparum causing the most widespread cases and malaria deaths. In 2013 there were approximately 190 million cases of the disease and between 584,000 and 855,000 deaths. It is essential to identify novel drug targets and develop novel drug candidates due to the increase in resistance of P. falciparum parasites to the current arsenal of antimalarial drugs. Endocytosis is an essential process in eukaryotic cells in which the external environment is internalized by the cell in order to obtain various particles from the extracellular space. This extracellular cytoplasm is internalized in membrane-bound invaginations at the plasma membrane. During the blood stage of malaria infection, the parasite requires nutrients from the host red blood cell. To obtain these nutrients, the parasite internalizes haemoglobin in large amounts and degrades it in an acidic, lysosome-like organelle, known as the digestive vacuole. Whilst the exact molecular mechanism of malaria parasite endocytosis is not yet fully understood, a number of proteins have been suggested to be involved. The most expedient approach in identifying candidate endocytosis proteins is to investigate parasite homologues of proteins known to be involved in endocytosis in mammalian cells. The three proteins selected for investigation in this study were the P. falciparum homologues of coronin, dynamin 2, and μ4. The coding sequences for the candidate endocytosis proteins were amplified by PCR and cloned into the pARL2-GFP expression vector. P. falciparum 3D7 parasites were transfected with these vectors and the episomal expression of full-length GFP-tagged fusion protein was confirmed by Western blot analysis using commercially available anti-GFP antibodies. Microscopic analysis of live parasites using fluorescence and confocal microscopy was used to determine the localization of the candidate endocytosis proteins. Coronin appeared to display diffuse cytoplasmic GFP localization during the trophozoite stage, arguing against a role in endocytosis. However, distinct localization during the schizont stage at what appears to be the inner membrane complex was observed. Coronin is thus likely required to coordinate the formation of the actin network between the merozoite IMC and the plasma membrane on which the glideosome is dependant for generating the motile forces required for the merozoite motility and invasion of RBCs. Dynamin 2 displayed localization at three potential locii: the parasite periphery (plasma membrane), punctuate regions within the cytoplasm (potentially at membrane bound organelles) and at the parasite food vacuole. The data suggested that dynamin 2 is involved in endocytosis and membrane trafficking in a similar manner to classical dynamins, potentially as a vesicle scission molecule at the plasma membrane, mediating vesicle formation at the food vacuole to recycle membrane to the plasma membrane, and possibly mitochondria organelle division. μ4 displayed transient localization, cycling between cytosolic localization, and localization to distinct regions at the plasma membrane and the food vacuole. Localization of Pfμ4 to the plasma membrane is indicative of a role for μ4 as a part of an adaptor protein (AP) complex which may be responsible for recruitment of clathrin to initiate endocytosis in a manner similar to mammalian AP-2. As was observed with PfDYN2, Pfμ4 localizes to the FV, which suggests that Pfμ4 forms part of a coat complex that mediates the formation of vesicles that recycle membrane from the FV to the parasite plasma membrane. This study showed that expressing proteins as full-length GFP-tagged fusion constructs is an effective approach in the early stages of determining the localization and function of P. falciparum proteins in vitro, and distinguishing between candidates that have a potential role in endocytosis and those that are unlikely to do so.
- Full Text:
- Date Issued: 2016
Development of an enzyme-synergy based bioreactor system for the beneficiation of apple pomace lignocellulosic waste
- Authors: Abboo, Sagaran
- Date: 2016
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/315 , vital:19947
- Description: Due to the finite supply of non-renewable fossil fuels, agro-industrial wastes are identified as alternate, renewable sources for energy supply. Large amounts of fruit waste are generated in South Africa due to fruit juice and wine processing from apples, grapes and citrus fruit. Apple pomace is the solid residue that is left over after juice, cider and wine processing and constitutes between 25-30% of the total fruit. On a global scale millions of tonnes of apple pomace are produced; between 2006-2007 over 46 million tonnes were produced. In South Africa a total production of 244 469 tonnes were produced during the 2011- 2012 season. Initially, apple pomace was regarded as a waste by-product used for animal feed and compost in soil, however presently it is considered a source of dietary fiber and natural antioxidants like polyphenols. In addition, apple pomace has a high carbohydrate content and can be enzymatically hydrolysed to produce sugar monomers which, in turn, can be fermented by yeasts to produce bioethanol. The polyphenols present in apple pomace can be used for their health properties, and the bioethanol can be used as a replacement for fossil fuel. Apple pomace is lignocellulosic in nature and consists of hemicellulose, cellulose, lignin and pectin. A combination of enzymes such as cellulases, hemicellulases, pectinases and lignases are required to operate in synergy for the degradation of lignocellulosic biomass. This is due to the recalcitrant nature of lignocellulose. This study investigated the degradation of apple pomace using a combination of commercially obtained enzyme cocktails viz. Viscozyme L , Celluclast 1.5L and Novozyme 188. The commercial enzymes Viscozyme L and Celluclast 1.5L were added in a ratio of 1:1 (50%:50%). The final concentrations of the enzymes were 0.019 mg/ml each. Novozyme 188 was added to provide a final concentration of 0.0024 mg/ml. A novel cost effective 20L bioreactor was designed, constructed and implemented for the degradation of apple pomace to produce value added products. The hydrolysis of the apple pomace was performed initially in 1 L flasks (batch fed) and, once optimized, scaled up to a 20 L bioreactor in batch mode. The bioreactors were operated at room temperature (22 ± 2ºC) and in an unbuffered system. The sugars released were detected and quantified using an optimized validated HPLC method established in this study. The sugars released in the bioreactors were mainly glucose, galactose, arabinose, cellobiose and fructose. The polyphenols released in this study were gallic acid, catechin, epicatechin, chlorogenic acid, rutin and phloridzin, which have a number of health benefits. The simultaneous analyses of the polyphenols were performed using a newly developed and validated HPLC method established in this study. This method was developed to detect nine polyphenols simultaneously. The two HPLC methods developed and validated in this study for the analysis of sugars and polyphenols demonstrated good accuracy, precision, reproducibility, linearity, robustness and sensitivity. Both analytical methods were validated according to the International Convention on Harmonization (ICH). The HPLC parameters for sugar analysis were: refractive index (RI) as the detection mode, the stationary phase was a ligand-exchange sugar column (Shodex SP0810) and an aqueous mobile phase in isocratic mode was used. The HPLC method for polyphenols employed UV diode array detection (DAD) as the detection mode, a reverse phase column as the stationary phase and a mobile phase of consisting of 0.01 M phosphoric acid in water and 100% methanol using gradient elution mode. The highest concentrations of sugars released in the novel 20 L bioreactor with 20% apple pomace (w/v) substrate loading were as follow: glucose (6.5 mg/ml), followed by galactose (2.1 mg/ml), arabinose (1.4 mg/ml), cellobiose (0.7 mg/ml) and fructose (0.5 mg/ml). The amounts of polyphenols released at 20% (w/v) apple pomace substrate were epicatechin (0.01 mg/ml), catechin (0.002 mg/ml), rutin (0.03 mg/ml), chlorogenic acid (0.002 mg/ml) and gallic acid 0.01 (mg/ml). Two mathematical models were developed in this study for kinetic analysis of lignocellulose (apple pomace) hydrolysis in the novel 20 L bioreactor, using the experimental data generated by the above HPLC analyses. The first model, modelling with regression, defines the hydrolysis of the sugars glucose, galactose, cellobiose and arabinose produced in the novel 20 L bioreactor at 5%, 10%, 15% and 20% (w/v) substrate concentrations. The regression model describes the sugars produced in the 20 L bioreactor by minimizing the error of the sugars released by finding a value for K which minimises the function which computes the sum of squares of errors between the solution curves and the data points. The second, more complex, model developed in this study used a system of differential equations model (ODE). This model solved the system by using a numerical method, such as the Runge-Kutta method, then fitted the solution curves to the data. Both models simulated (and had the ability to predict) the production of sugars in the novel 20 L bioreactor for apple pomace hydrolysis. These two models also revealed the time at which the maximum amount of sugars were released, which revealed the optimum time to run the 20 L bioreactor in order to be more cost effective. The optimum time for maximum glucose (the main sugar used in fermentation for biofuel production) release was determined to be around 60 h. The ODE model, in addition, determined the rate at which the substrate became depleted, as well as the rate at which the enzymes became deactivated for the various substrate loadings in the 20 L bioreactor. A third model was developed to determine the optimal running cost of the bioreactor which incorporated the substrate loading and the amount of glucose (g/L) produced. The novel 20 L bioreactor constructed from cost effective materials demonstrated that agro-industrial waste can be converted to value-added products by lignocellolytic enzymes. The sugars released from apple pomace can be used in biofuel production and the polyphenols as food supplements and nutraceuticals for health benefits. This novel study contributes to agro-industrial waste beneficiation via fuel production. In addition, using agro-industrial waste for the generation of value added products (instead of mere disposal) will help prevent environmental pollution.
- Full Text:
- Date Issued: 2016
- Authors: Abboo, Sagaran
- Date: 2016
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/315 , vital:19947
- Description: Due to the finite supply of non-renewable fossil fuels, agro-industrial wastes are identified as alternate, renewable sources for energy supply. Large amounts of fruit waste are generated in South Africa due to fruit juice and wine processing from apples, grapes and citrus fruit. Apple pomace is the solid residue that is left over after juice, cider and wine processing and constitutes between 25-30% of the total fruit. On a global scale millions of tonnes of apple pomace are produced; between 2006-2007 over 46 million tonnes were produced. In South Africa a total production of 244 469 tonnes were produced during the 2011- 2012 season. Initially, apple pomace was regarded as a waste by-product used for animal feed and compost in soil, however presently it is considered a source of dietary fiber and natural antioxidants like polyphenols. In addition, apple pomace has a high carbohydrate content and can be enzymatically hydrolysed to produce sugar monomers which, in turn, can be fermented by yeasts to produce bioethanol. The polyphenols present in apple pomace can be used for their health properties, and the bioethanol can be used as a replacement for fossil fuel. Apple pomace is lignocellulosic in nature and consists of hemicellulose, cellulose, lignin and pectin. A combination of enzymes such as cellulases, hemicellulases, pectinases and lignases are required to operate in synergy for the degradation of lignocellulosic biomass. This is due to the recalcitrant nature of lignocellulose. This study investigated the degradation of apple pomace using a combination of commercially obtained enzyme cocktails viz. Viscozyme L , Celluclast 1.5L and Novozyme 188. The commercial enzymes Viscozyme L and Celluclast 1.5L were added in a ratio of 1:1 (50%:50%). The final concentrations of the enzymes were 0.019 mg/ml each. Novozyme 188 was added to provide a final concentration of 0.0024 mg/ml. A novel cost effective 20L bioreactor was designed, constructed and implemented for the degradation of apple pomace to produce value added products. The hydrolysis of the apple pomace was performed initially in 1 L flasks (batch fed) and, once optimized, scaled up to a 20 L bioreactor in batch mode. The bioreactors were operated at room temperature (22 ± 2ºC) and in an unbuffered system. The sugars released were detected and quantified using an optimized validated HPLC method established in this study. The sugars released in the bioreactors were mainly glucose, galactose, arabinose, cellobiose and fructose. The polyphenols released in this study were gallic acid, catechin, epicatechin, chlorogenic acid, rutin and phloridzin, which have a number of health benefits. The simultaneous analyses of the polyphenols were performed using a newly developed and validated HPLC method established in this study. This method was developed to detect nine polyphenols simultaneously. The two HPLC methods developed and validated in this study for the analysis of sugars and polyphenols demonstrated good accuracy, precision, reproducibility, linearity, robustness and sensitivity. Both analytical methods were validated according to the International Convention on Harmonization (ICH). The HPLC parameters for sugar analysis were: refractive index (RI) as the detection mode, the stationary phase was a ligand-exchange sugar column (Shodex SP0810) and an aqueous mobile phase in isocratic mode was used. The HPLC method for polyphenols employed UV diode array detection (DAD) as the detection mode, a reverse phase column as the stationary phase and a mobile phase of consisting of 0.01 M phosphoric acid in water and 100% methanol using gradient elution mode. The highest concentrations of sugars released in the novel 20 L bioreactor with 20% apple pomace (w/v) substrate loading were as follow: glucose (6.5 mg/ml), followed by galactose (2.1 mg/ml), arabinose (1.4 mg/ml), cellobiose (0.7 mg/ml) and fructose (0.5 mg/ml). The amounts of polyphenols released at 20% (w/v) apple pomace substrate were epicatechin (0.01 mg/ml), catechin (0.002 mg/ml), rutin (0.03 mg/ml), chlorogenic acid (0.002 mg/ml) and gallic acid 0.01 (mg/ml). Two mathematical models were developed in this study for kinetic analysis of lignocellulose (apple pomace) hydrolysis in the novel 20 L bioreactor, using the experimental data generated by the above HPLC analyses. The first model, modelling with regression, defines the hydrolysis of the sugars glucose, galactose, cellobiose and arabinose produced in the novel 20 L bioreactor at 5%, 10%, 15% and 20% (w/v) substrate concentrations. The regression model describes the sugars produced in the 20 L bioreactor by minimizing the error of the sugars released by finding a value for K which minimises the function which computes the sum of squares of errors between the solution curves and the data points. The second, more complex, model developed in this study used a system of differential equations model (ODE). This model solved the system by using a numerical method, such as the Runge-Kutta method, then fitted the solution curves to the data. Both models simulated (and had the ability to predict) the production of sugars in the novel 20 L bioreactor for apple pomace hydrolysis. These two models also revealed the time at which the maximum amount of sugars were released, which revealed the optimum time to run the 20 L bioreactor in order to be more cost effective. The optimum time for maximum glucose (the main sugar used in fermentation for biofuel production) release was determined to be around 60 h. The ODE model, in addition, determined the rate at which the substrate became depleted, as well as the rate at which the enzymes became deactivated for the various substrate loadings in the 20 L bioreactor. A third model was developed to determine the optimal running cost of the bioreactor which incorporated the substrate loading and the amount of glucose (g/L) produced. The novel 20 L bioreactor constructed from cost effective materials demonstrated that agro-industrial waste can be converted to value-added products by lignocellolytic enzymes. The sugars released from apple pomace can be used in biofuel production and the polyphenols as food supplements and nutraceuticals for health benefits. This novel study contributes to agro-industrial waste beneficiation via fuel production. In addition, using agro-industrial waste for the generation of value added products (instead of mere disposal) will help prevent environmental pollution.
- Full Text:
- Date Issued: 2016
Investigating soil microbial interactions of Portulacaria afra
- Authors: Fulmaka, Aviwe
- Date: 2016
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/54598 , vital:26592
- Description: Portulacaria afra commonly known as Spekboom contributes significantly to carbon sequestration and has been widely planted in degraded areas of the Eastern Cape. Approximately 50% of planted cuttings do not survive although the cause of this decline is unknown. Like many indigenous plants, Spekboom forms a symbiotic relationship with mycorrhizal fungi and the interaction with rhizobacteria may enhance and improve plant growth and establishment. This study aims to investigate these relationships which will include a survey of the arbuscular mycorrhizal (AM) fungal populations associated with Spekboom, determination of the causal agent of Spekboom decline, isolation and identification of the associated rhizobacteria and investigation of their plant growth promotion properties and assessing the ability of arbuscular mycorrhizal fungi and selected rhizobacteria to enhance establishment and growth of Spekboom. Soil and root samples from selected trial sites were used to assess AM fungal spore abundance and colonisation; isolation, characterization, and identification of rhizobacteria and determine the interaction of the microbes on Spekboom growth and tolerance to Fusarium. AM spore abundance and percentage root colonisation did not differ between the three Spekboom plots. Molecular analyses of the SSU region from the plots showed 4 families of AM fungi and were identified as Ambisporaceae, Glomeraceae, Claroideoglomeraceae and Paraglomeraceae. A suspected Fusarium pathogen was isolated and molecularly identified. Pathogenicity tests indicated reduced Spekboom growth with poor root development. Thirty four rhizobacterial isolates were tested for various plant growth promoting abilities. Of these, 6 were able to produce IAA which may promote plant root growth, 27 siderophores and 23 were phosphate solubilisers. Bacterial isolates were molecularly identified to be from various species of Bacillus, with some Arthrobacter, Enterobacter, Pseudomonas and Microbacterium. Inoculation of Spekboom cuttings with mycorrhizal fungi and selected rhizobacterial isolates significantly improved shoot height. Spekboom cuttings challenged with Fusarium and inoculated with mycorrhizal fungi and two rhizobacterial isolates significantly improved growth. The inoculation of cuttings in the nursery with mycorrhizal fungi and selected rhizobacteria is recommended prior to establishing Spekboom in the field.
- Full Text:
- Date Issued: 2016
- Authors: Fulmaka, Aviwe
- Date: 2016
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/54598 , vital:26592
- Description: Portulacaria afra commonly known as Spekboom contributes significantly to carbon sequestration and has been widely planted in degraded areas of the Eastern Cape. Approximately 50% of planted cuttings do not survive although the cause of this decline is unknown. Like many indigenous plants, Spekboom forms a symbiotic relationship with mycorrhizal fungi and the interaction with rhizobacteria may enhance and improve plant growth and establishment. This study aims to investigate these relationships which will include a survey of the arbuscular mycorrhizal (AM) fungal populations associated with Spekboom, determination of the causal agent of Spekboom decline, isolation and identification of the associated rhizobacteria and investigation of their plant growth promotion properties and assessing the ability of arbuscular mycorrhizal fungi and selected rhizobacteria to enhance establishment and growth of Spekboom. Soil and root samples from selected trial sites were used to assess AM fungal spore abundance and colonisation; isolation, characterization, and identification of rhizobacteria and determine the interaction of the microbes on Spekboom growth and tolerance to Fusarium. AM spore abundance and percentage root colonisation did not differ between the three Spekboom plots. Molecular analyses of the SSU region from the plots showed 4 families of AM fungi and were identified as Ambisporaceae, Glomeraceae, Claroideoglomeraceae and Paraglomeraceae. A suspected Fusarium pathogen was isolated and molecularly identified. Pathogenicity tests indicated reduced Spekboom growth with poor root development. Thirty four rhizobacterial isolates were tested for various plant growth promoting abilities. Of these, 6 were able to produce IAA which may promote plant root growth, 27 siderophores and 23 were phosphate solubilisers. Bacterial isolates were molecularly identified to be from various species of Bacillus, with some Arthrobacter, Enterobacter, Pseudomonas and Microbacterium. Inoculation of Spekboom cuttings with mycorrhizal fungi and selected rhizobacterial isolates significantly improved shoot height. Spekboom cuttings challenged with Fusarium and inoculated with mycorrhizal fungi and two rhizobacterial isolates significantly improved growth. The inoculation of cuttings in the nursery with mycorrhizal fungi and selected rhizobacteria is recommended prior to establishing Spekboom in the field.
- Full Text:
- Date Issued: 2016
Identification of potential novel roles for Hsp70/Hsp90 organising protein (Hop) using proteomic analysis in human cells
- Authors: Wingate, Ianthe
- Date: 2016
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/64758 , vital:28598
- Description: Expected release date-May 2018
- Full Text:
- Date Issued: 2016
- Authors: Wingate, Ianthe
- Date: 2016
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/64758 , vital:28598
- Description: Expected release date-May 2018
- Full Text:
- Date Issued: 2016
In silico analysis of the effects of non-synonymous single nucleotide polymorphisms on the human macrophage migration inhibitory factor gene and their possible role in human African trypanosomiasis susceptibility
- Authors: Kimuda, Magambo Philip
- Date: 2016
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/3047 , vital:20355
- Description: Human African trypanosomiasis (HAT) is a public health problem in sub-Saharan Africa, with approximately 10,000 cases being reported per year. The Macrophage Migration Inhibitory Factor (MIF) which is encoded by a functionally polymorphic gene is important in both innate andadaptive immune responses, and has been implicated in affecting the outcome and processes of several inflammatory conditions. A recent study in mice to that effect showed that MIF deficient and anti-MIF antibody treated mice showed lowered inflammatory responses, liver damage and anaemia than the wild type mice when experimentally challenged with Trypanosomes. These findings could mean that the transcript levels and/or polymorphisms in this gene can possibly affect individual risk to trypanosomiasis. This is especially of interest because there have been reports of spontaneous recovery i.e self-cure/resistance in some HAT cases in West Africa. Prior to this discovery the general paradigm was that trypanosomiasis is fatal if left untreated. The aim of this study was to gain insights into how human genetic variation in forms of nonsynonymous SNPs affects the MIF structure and function and possibly HAT susceptibility. NsSNPs in the mif gene were obtained from dbSNP. Through homology modeling, SNP prediction tools, protein interface analysis, alanine scanning, changes in free energy of folding, protein interactions calculator (PIC), and molecular dynamics simulations, SNP effects on the protein structure and function were studied. The study cohort comprised of human genome sequence data from 50 North Western Uganda Lugbara endemic individuals of whom 20 were cases (previous HAT patients) and 30 were controls (HAT free individuals). None of the 26 nsSNPs retrieved from dbSNP (July 2015) were present in the mif gene region in the study cohort. Out of the eight variants called in the mif coding region there was only one missense variant rs36065127 whose clinical significance is unknown. It was not possible to test for association of this variant with HAT due to its low global MAF that was less than 0.05. Alanine scanning provided a fast and computationally cheap means of quickly assessing nsSNPs of importance. NsSNPs that were interface residues were more likely to be hotspots (important in protein stability). Assessment of possible compensatory mutations using PIC analysis showed that some nsSNP sites were interacting with others, but this requires further experimentation. Analysis of changes in free energy using FOLDX was not enough to predict which nsSNPs would adversely affect protein structure, function and kinetics. The MD simulations were unfortunately too short to glean any meaningful inferences. This was the first genetic study carried out on the people of Lugbara ethnicity from North Western Uganda.
- Full Text:
- Date Issued: 2016
- Authors: Kimuda, Magambo Philip
- Date: 2016
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/3047 , vital:20355
- Description: Human African trypanosomiasis (HAT) is a public health problem in sub-Saharan Africa, with approximately 10,000 cases being reported per year. The Macrophage Migration Inhibitory Factor (MIF) which is encoded by a functionally polymorphic gene is important in both innate andadaptive immune responses, and has been implicated in affecting the outcome and processes of several inflammatory conditions. A recent study in mice to that effect showed that MIF deficient and anti-MIF antibody treated mice showed lowered inflammatory responses, liver damage and anaemia than the wild type mice when experimentally challenged with Trypanosomes. These findings could mean that the transcript levels and/or polymorphisms in this gene can possibly affect individual risk to trypanosomiasis. This is especially of interest because there have been reports of spontaneous recovery i.e self-cure/resistance in some HAT cases in West Africa. Prior to this discovery the general paradigm was that trypanosomiasis is fatal if left untreated. The aim of this study was to gain insights into how human genetic variation in forms of nonsynonymous SNPs affects the MIF structure and function and possibly HAT susceptibility. NsSNPs in the mif gene were obtained from dbSNP. Through homology modeling, SNP prediction tools, protein interface analysis, alanine scanning, changes in free energy of folding, protein interactions calculator (PIC), and molecular dynamics simulations, SNP effects on the protein structure and function were studied. The study cohort comprised of human genome sequence data from 50 North Western Uganda Lugbara endemic individuals of whom 20 were cases (previous HAT patients) and 30 were controls (HAT free individuals). None of the 26 nsSNPs retrieved from dbSNP (July 2015) were present in the mif gene region in the study cohort. Out of the eight variants called in the mif coding region there was only one missense variant rs36065127 whose clinical significance is unknown. It was not possible to test for association of this variant with HAT due to its low global MAF that was less than 0.05. Alanine scanning provided a fast and computationally cheap means of quickly assessing nsSNPs of importance. NsSNPs that were interface residues were more likely to be hotspots (important in protein stability). Assessment of possible compensatory mutations using PIC analysis showed that some nsSNP sites were interacting with others, but this requires further experimentation. Analysis of changes in free energy using FOLDX was not enough to predict which nsSNPs would adversely affect protein structure, function and kinetics. The MD simulations were unfortunately too short to glean any meaningful inferences. This was the first genetic study carried out on the people of Lugbara ethnicity from North Western Uganda.
- Full Text:
- Date Issued: 2016
Comparative analysis of existing pipelines for assessment of arbuscular mycorrhizal fungal biodiversity in natural and commercial rooibos (aspalathus linearis) and honeybush (cyclopia intermedia) soil samples
- Authors: De Wit, Hermina Johanna
- Date: 2016
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/2915 , vital:20342
- Full Text:
- Date Issued: 2016
- Authors: De Wit, Hermina Johanna
- Date: 2016
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/2915 , vital:20342
- Full Text:
- Date Issued: 2016
Structural bioinformatics studies and tool development related to drug discovery
- Authors: Hatherley, Rowan
- Date: 2016
- Subjects: Structural bioinformatics , Drug development , Natural products -- Databases , Natural products -- Biotechnology , Sequence alignment (Bioinformatics) , Malaria -- Chemotherapy , Heat shock proteins , Plasmodium falciparum
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4164 , http://hdl.handle.net/10962/d1020021
- Description: This thesis is divided into two distinct sections which can be combined under the broad umbrella of structural bioinformatics studies related to drug discovery. The first section involves the establishment of an online South African natural products database. Natural products (NPs) are chemical entities synthesised in nature and are unrivalled in their structural complexity, chemical diversity, and biological specificity, which has long made them crucial to the drug discovery process. South Africa is rich in both plant and marine biodiversity and a great deal of research has gone into isolating compounds from organisms found in this country. However, there is no official database containing this information, making it difficult to access for research purposes. This information was extracted manually from literature to create a database of South African natural products. In order to make the information accessible to the general research community, a website, named “SANCDB”, was built to enable compounds to be quickly and easily searched for and downloaded in a number of different chemical formats. The content of the database was assessed and compared to other established natural product databases. Currently, SANCDB is the only database of natural products in Africa with an online interface. The second section of the thesis was aimed at performing structural characterisation of proteins with the potential to be targeted for antimalarial drug therapy. This looked specifically at 1) The interactions between an exported heat shock protein (Hsp) from Plasmodium falciparum (P. falciparum), PfHsp70-x and various host and exported parasite J proteins, as well as 2) The interface between PfHsp90 and the heat shock organising protein (PfHop). The PfHsp70-x:J protein study provided additional insight into how these two proteins potentially interact. Analysis of the PfHsp90:PfHop also provided a structural insight into the interaction interface between these two proteins and identified residues that could be targeted due to their contribution to the stability of the Hsp90:Hop binding complex and differences between parasite and human proteins. These studies inspired the development of a homology modelling tool, which can be used to assist researchers with homology modelling, while providing them with step-by-step control over the entire process. This thesis presents the establishment of a South African NP database and the development of a homology modelling tool, inspired by protein structural studies. When combined, these two applications have the potential to contribute greatly towards in silico drug discovery research.
- Full Text:
- Date Issued: 2016
- Authors: Hatherley, Rowan
- Date: 2016
- Subjects: Structural bioinformatics , Drug development , Natural products -- Databases , Natural products -- Biotechnology , Sequence alignment (Bioinformatics) , Malaria -- Chemotherapy , Heat shock proteins , Plasmodium falciparum
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4164 , http://hdl.handle.net/10962/d1020021
- Description: This thesis is divided into two distinct sections which can be combined under the broad umbrella of structural bioinformatics studies related to drug discovery. The first section involves the establishment of an online South African natural products database. Natural products (NPs) are chemical entities synthesised in nature and are unrivalled in their structural complexity, chemical diversity, and biological specificity, which has long made them crucial to the drug discovery process. South Africa is rich in both plant and marine biodiversity and a great deal of research has gone into isolating compounds from organisms found in this country. However, there is no official database containing this information, making it difficult to access for research purposes. This information was extracted manually from literature to create a database of South African natural products. In order to make the information accessible to the general research community, a website, named “SANCDB”, was built to enable compounds to be quickly and easily searched for and downloaded in a number of different chemical formats. The content of the database was assessed and compared to other established natural product databases. Currently, SANCDB is the only database of natural products in Africa with an online interface. The second section of the thesis was aimed at performing structural characterisation of proteins with the potential to be targeted for antimalarial drug therapy. This looked specifically at 1) The interactions between an exported heat shock protein (Hsp) from Plasmodium falciparum (P. falciparum), PfHsp70-x and various host and exported parasite J proteins, as well as 2) The interface between PfHsp90 and the heat shock organising protein (PfHop). The PfHsp70-x:J protein study provided additional insight into how these two proteins potentially interact. Analysis of the PfHsp90:PfHop also provided a structural insight into the interaction interface between these two proteins and identified residues that could be targeted due to their contribution to the stability of the Hsp90:Hop binding complex and differences between parasite and human proteins. These studies inspired the development of a homology modelling tool, which can be used to assist researchers with homology modelling, while providing them with step-by-step control over the entire process. This thesis presents the establishment of a South African NP database and the development of a homology modelling tool, inspired by protein structural studies. When combined, these two applications have the potential to contribute greatly towards in silico drug discovery research.
- Full Text:
- Date Issued: 2016
Genetic and biological characterisation of a novel South African Cydia pomonella granulovirus (CpGV-SA) isolate
- Motsoeneng, Boitumelo Madika
- Authors: Motsoeneng, Boitumelo Madika
- Date: 2016
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:20503 , http://hdl.handle.net/10962/d1021266
- Description: The codling moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae), is the primary pest of pome fruit cultivated worldwide. The control of this insect pest has been dependent on the frequent use of broad-spectrum chemical pesticides, which has led to the development of resistance in pest populations and negative effects on human health and the environment. The Betabaculovirus of C. pomonella has successfully been applied as a biological control agent in integrated pest management (IPM) programmes for the suppression of pest populations worldwide. Previously, all Cydia pomonella granulovirus (CpGV) biopesticides were based on a Mexican isolate (CpGV-M) and although these products are highly efficient at controlling C. pomonella, resistance cases have been reported across Europe. The identification of novel CpGV isolates as additional or alternative control agents to manage resistance is therefore necessary. This study aimed to genetically and biologically characterise a novel South African C. pomonella granulovirus isolate and to test its virulence against neonate larvae. Based on the morphology of the occlusion bodies observed using transmission electron microscopy, granuloviruses were recovered from diseased and dead larvae collected from an orchard in South Africa where no virus applications had been made. DNA was extracted and the identification of the isolated granulovirus was achieved through the PCR amplification and sequencing of the lef-8, lef-9, granulin and egt genes. Submission of the gene sequences to BLAST revealed high percentage identities to sequences from various CpGV isolates, resulting in the naming of the isolate in this study as the South African Cydia pomonella granulovirus (CpGV-SA) isolate. Phylogenetic analysis based on the single nucleotide polymorphisms (SNPs) detected in the lef-8, lef-9 and granulin nucleotide sequences grouped the South African isolate with CpGV-E2 (genome type B) and CpGV-S (genome type E). The CpGV-SA isolate was further genetically characterised by restriction endonuclease analysis and complete sequencing of the genomic DNA. Differences were observed for the BamHI, EcoRI, PstI and XhoI profiles of CpGV-SA in comparison to the respective profiles generated for CpGV-M extracted from a biopesticide, Carpovirusine® (Arysta Lifescience, France). Several genetic variations between the complete genome sequence of CpGV-SA and the reference isolate, CpGV-M1, as well as a recent genome submission of CpGV-M, both representing genome type A were observed. The complete genome analysis confirmed that CpGV-SA is genetically different from the Mexican CpGV isolate, used in thedevelopment of most biopesticides. In silico restriction profiles of the genome sequence obtained for CpGV-SA and genome sequences of genetically different CpGV isolates originating from Mexico (M1 and M), England (E2), Canada (S) and Iran (I12 and I07), available on the NCBI’s GenBank database confirmed that CpGV-SA is of mixed genotypes. Furthermore, the South African isolate shared the single common difference found in the pe38 gene of resistance overcoming isolates, which was the absence of an internal 24 nucleotide repeat present in CpGV-M1. In addition to the common difference, SNPs detected in the pe38 gene grouped the isolate with the CpGV-S isolate, suggesting that the CpGV-SA isolate is predominantly of genome type E. To determine the biological activity of CpGV-SA against neonate C. pomonella larvae, surface bioassays were conducted alongside CpGV-M (Carpovirusine®) bioassays. The LC50 and LC90 values for the South African isolate were 1.6 × 103 and 1.2 × 105 OBs/ml respectively. The LT50 was determined to be 135 hours. These values were similar to the values obtained for CpGV-M (Carpovirusine®). The results in this study suggest that a novel South African CpGV isolate of mixed genotypes, potentially able to overcome resistance in C. pomonella, with biological activity similar to CpGV-M (Carpovirusine®) and important for the control of C. pomonella was recovered. The CpGV-SA isolate could therefore potentially be developed into a biopesticide for use in resistance management strategies against C. pomonella populations in South Africa.
- Full Text:
- Date Issued: 2016
- Authors: Motsoeneng, Boitumelo Madika
- Date: 2016
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:20503 , http://hdl.handle.net/10962/d1021266
- Description: The codling moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae), is the primary pest of pome fruit cultivated worldwide. The control of this insect pest has been dependent on the frequent use of broad-spectrum chemical pesticides, which has led to the development of resistance in pest populations and negative effects on human health and the environment. The Betabaculovirus of C. pomonella has successfully been applied as a biological control agent in integrated pest management (IPM) programmes for the suppression of pest populations worldwide. Previously, all Cydia pomonella granulovirus (CpGV) biopesticides were based on a Mexican isolate (CpGV-M) and although these products are highly efficient at controlling C. pomonella, resistance cases have been reported across Europe. The identification of novel CpGV isolates as additional or alternative control agents to manage resistance is therefore necessary. This study aimed to genetically and biologically characterise a novel South African C. pomonella granulovirus isolate and to test its virulence against neonate larvae. Based on the morphology of the occlusion bodies observed using transmission electron microscopy, granuloviruses were recovered from diseased and dead larvae collected from an orchard in South Africa where no virus applications had been made. DNA was extracted and the identification of the isolated granulovirus was achieved through the PCR amplification and sequencing of the lef-8, lef-9, granulin and egt genes. Submission of the gene sequences to BLAST revealed high percentage identities to sequences from various CpGV isolates, resulting in the naming of the isolate in this study as the South African Cydia pomonella granulovirus (CpGV-SA) isolate. Phylogenetic analysis based on the single nucleotide polymorphisms (SNPs) detected in the lef-8, lef-9 and granulin nucleotide sequences grouped the South African isolate with CpGV-E2 (genome type B) and CpGV-S (genome type E). The CpGV-SA isolate was further genetically characterised by restriction endonuclease analysis and complete sequencing of the genomic DNA. Differences were observed for the BamHI, EcoRI, PstI and XhoI profiles of CpGV-SA in comparison to the respective profiles generated for CpGV-M extracted from a biopesticide, Carpovirusine® (Arysta Lifescience, France). Several genetic variations between the complete genome sequence of CpGV-SA and the reference isolate, CpGV-M1, as well as a recent genome submission of CpGV-M, both representing genome type A were observed. The complete genome analysis confirmed that CpGV-SA is genetically different from the Mexican CpGV isolate, used in thedevelopment of most biopesticides. In silico restriction profiles of the genome sequence obtained for CpGV-SA and genome sequences of genetically different CpGV isolates originating from Mexico (M1 and M), England (E2), Canada (S) and Iran (I12 and I07), available on the NCBI’s GenBank database confirmed that CpGV-SA is of mixed genotypes. Furthermore, the South African isolate shared the single common difference found in the pe38 gene of resistance overcoming isolates, which was the absence of an internal 24 nucleotide repeat present in CpGV-M1. In addition to the common difference, SNPs detected in the pe38 gene grouped the isolate with the CpGV-S isolate, suggesting that the CpGV-SA isolate is predominantly of genome type E. To determine the biological activity of CpGV-SA against neonate C. pomonella larvae, surface bioassays were conducted alongside CpGV-M (Carpovirusine®) bioassays. The LC50 and LC90 values for the South African isolate were 1.6 × 103 and 1.2 × 105 OBs/ml respectively. The LT50 was determined to be 135 hours. These values were similar to the values obtained for CpGV-M (Carpovirusine®). The results in this study suggest that a novel South African CpGV isolate of mixed genotypes, potentially able to overcome resistance in C. pomonella, with biological activity similar to CpGV-M (Carpovirusine®) and important for the control of C. pomonella was recovered. The CpGV-SA isolate could therefore potentially be developed into a biopesticide for use in resistance management strategies against C. pomonella populations in South Africa.
- Full Text:
- Date Issued: 2016
Bio-prospecting a Soil Metagenomic Library for Carbohydrate Active Esterases
- Authors: Shezi, Ntombifuthi
- Date: 2016
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4172 , http://hdl.handle.net/10962/d1021266
- Description: Lignocellulosic biomass is a promising renewable resource on earth. Plant biomass contains fermentable sugars and other moieties that can be converted to biofuels or other chemicals. Enzymatic hydrolysis of these biopolymers is significant in the liberation of sugars for fermentation into desired products. Owing to its complex structure, synergistic action of enzymes is required for its degradation. Enzymes that are involved in biomass degradation include cellulases, hemicellulases and the accessory enzymes acetyl xylan esterases and ferulic acid esterases. Ferulic acid esterases (FAEs, EC 3.1.1.73), represent a subclass of carboxylester hydrolases (EC 3.1.1.-) that catalyse the release of hydroxycinnamic acids (such as ferulic acid, p-coumaric, ferulic, sinapic and caffeic acid) that are generally found esterified to polysaccharides, such as arabinoxylans. Hydroxycinnamic acids have widespread potential applications due to their antimicrobial, photoprotectant and antioxidant properties, as well as their use as flavour precursors. Therefore, this interesting group of FAEs has a potentially wide variety of applications in agriculture, food and pharmaceutical industries. In the search for novel biocatalysts, metagenomics is considered as an alternative approach to conventional microbe screening, therefore, searching for novel biocatalysts from a soil metagenome that harbours a unique diversity of biocatalyst is significant. The aim of this study was to extract DNA from soil associated with cattle manure and construct a soil metagenomic library using a fosmid based plasmid vector and subsequently functionally screen for ferulic acid esterases using ethyl ferulate as a model substrate. A total of 59 recombinant fosmids conferring ferulic acid esterase phenotypes were identified (Hit rate 1:3122) and the two fosmids that consistently showed high FAE activities were selected for further study. Following nucleotide sequencing and translational analysis, two fae encoding open reading frames (FAE9 and FAE27) of approximately 274 and 322 aa, respectively, were identified. The amino acid sequence of the two ORFs contained a classical conserved esterase/lipase G-x-S-x-G sequence motif. The two genes (fae9 and fae27) were successfully expressed in Escherichia coli BL21 (DE3) and the purified enzymes exhibited respective temperature optima of 50 °C and 40 °C, and respective pH optima of 6.0 and 7.0. Further biochemical characterisation showed that FAE9 and FAE27 have high substrate specificity, following the fact that EFA is the preferred substrate for FAE9 (kcat/Km value of 128 s−1.mM-1) and also the preferred substrate for FAE27 (kcat/Km value of 137 s−1.mM-1). This work proves that soil is a valuable environmental source for novel esterase screening through functional based metagenomic approach. Therefore, this method may be used to screen for other valuable enzymes from environmental sources using inexpensive natural sources to encourage the screening of specific enzymes. Biochemistry of the two isolated enzymes makes these enzymes to be useful in industrial applications due to broad substrate activity that could replace the specialised enzymes to complete plant biomass degradation.
- Full Text:
- Date Issued: 2016
- Authors: Shezi, Ntombifuthi
- Date: 2016
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4172 , http://hdl.handle.net/10962/d1021266
- Description: Lignocellulosic biomass is a promising renewable resource on earth. Plant biomass contains fermentable sugars and other moieties that can be converted to biofuels or other chemicals. Enzymatic hydrolysis of these biopolymers is significant in the liberation of sugars for fermentation into desired products. Owing to its complex structure, synergistic action of enzymes is required for its degradation. Enzymes that are involved in biomass degradation include cellulases, hemicellulases and the accessory enzymes acetyl xylan esterases and ferulic acid esterases. Ferulic acid esterases (FAEs, EC 3.1.1.73), represent a subclass of carboxylester hydrolases (EC 3.1.1.-) that catalyse the release of hydroxycinnamic acids (such as ferulic acid, p-coumaric, ferulic, sinapic and caffeic acid) that are generally found esterified to polysaccharides, such as arabinoxylans. Hydroxycinnamic acids have widespread potential applications due to their antimicrobial, photoprotectant and antioxidant properties, as well as their use as flavour precursors. Therefore, this interesting group of FAEs has a potentially wide variety of applications in agriculture, food and pharmaceutical industries. In the search for novel biocatalysts, metagenomics is considered as an alternative approach to conventional microbe screening, therefore, searching for novel biocatalysts from a soil metagenome that harbours a unique diversity of biocatalyst is significant. The aim of this study was to extract DNA from soil associated with cattle manure and construct a soil metagenomic library using a fosmid based plasmid vector and subsequently functionally screen for ferulic acid esterases using ethyl ferulate as a model substrate. A total of 59 recombinant fosmids conferring ferulic acid esterase phenotypes were identified (Hit rate 1:3122) and the two fosmids that consistently showed high FAE activities were selected for further study. Following nucleotide sequencing and translational analysis, two fae encoding open reading frames (FAE9 and FAE27) of approximately 274 and 322 aa, respectively, were identified. The amino acid sequence of the two ORFs contained a classical conserved esterase/lipase G-x-S-x-G sequence motif. The two genes (fae9 and fae27) were successfully expressed in Escherichia coli BL21 (DE3) and the purified enzymes exhibited respective temperature optima of 50 °C and 40 °C, and respective pH optima of 6.0 and 7.0. Further biochemical characterisation showed that FAE9 and FAE27 have high substrate specificity, following the fact that EFA is the preferred substrate for FAE9 (kcat/Km value of 128 s−1.mM-1) and also the preferred substrate for FAE27 (kcat/Km value of 137 s−1.mM-1). This work proves that soil is a valuable environmental source for novel esterase screening through functional based metagenomic approach. Therefore, this method may be used to screen for other valuable enzymes from environmental sources using inexpensive natural sources to encourage the screening of specific enzymes. Biochemistry of the two isolated enzymes makes these enzymes to be useful in industrial applications due to broad substrate activity that could replace the specialised enzymes to complete plant biomass degradation.
- Full Text:
- Date Issued: 2016
Investigating the use of Arbuscular Mycorrhizas and Plant Growth Promoting Bacteria to improve the drought tolerance of maize (Zea mays L.)
- Authors: Moore, Nicolle Maureen
- Date: 2016
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/54587 , vital:26591
- Description: Maize (Zea mays L.) is a direct staple food crop in Africa and remains an essential component of global food security, with maize crops accounting for over 60% of the total harvested area of annual food crops. Stress caused by drought and high soil salinity limits crop growth and productivity more than any other single environmental factor, with grain yield reductions up to 76% depending on the severity of the drought and the plant growth stage. Arbuscular mycorrhizal (AM) fungi and Plant Growth Promotion Rhizobacteria (PGPR) have previously been shown to improve tolerance of plants to drought stress through a number of chemical and physiological processes. The aim of this investigation was to determine whether mycorrhizal fungi and rhizobacteria adapted to drought and saline conditions and possessing plant growth promoting (PGP) traits were able to stimulate plant growth responses when applied to Zea mays seeds growing under greenhouse conditions Bacterial isolates selected were tolerant to concentrations of NaCl up to 600 mM and maintained 50% growth at low water potentials (-1.44 MPa). They were positive for Indole Acetic Acid (IAA) production, phosphate solubilisation and secretion of siderophores. Bacterial isolates showing plant growth promoting potential were identified using 16S rDNA gene sequencing as Achromobacter xylosoxidans strains A8 and C54 and Klebsiella oxytoca strain M1. Mixed inoculum was prepared from indigenous communities of mycorrhizas in soils sampled from the Cerebos Salt Pan and the Kalahari Desert. Mycorrhizal diversity was investigated using 454-Pyrosequencing which revealed that the community composition was dominated by species in the Ambispora, Glomus and Paraglomus genera with a rare component represented by species in the Redeckera, Archaeospora and Geosiphon genera. Microscopic examination of plant roots at the end of the trial revealed the presence of diagnostic mycorrhizal structures within the root cells, confirming that colonization was successful. Plant growth response to microbial inoculation was assessed by monitoring changes in plant photosynthetic capacity over the duration of a 7 week pot trial. A significant difference in photosynthetic and biomass data was observed between drought and well-watered groups but no mycorrhizal or bacterial treatment effect was evident within the groups, despite the high levels of colonization by mycorrhizas. These results suggest that the beneficial effects of mycorrhizal colonization may be primarily attributed to improved nutrient and mineral uptake in conditions where nutrients are limiting, resulting in improved growth. The improved growth may then have secondary effects on the plant‟s ability to withstand drought. Having controlled for nutrient deficiency, it was not evident in this study that mycorrhizal fungi were able to stimulate a change in plant physiology and confer drought tolerance under the conditions imposed.
- Full Text:
- Date Issued: 2016
- Authors: Moore, Nicolle Maureen
- Date: 2016
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/54587 , vital:26591
- Description: Maize (Zea mays L.) is a direct staple food crop in Africa and remains an essential component of global food security, with maize crops accounting for over 60% of the total harvested area of annual food crops. Stress caused by drought and high soil salinity limits crop growth and productivity more than any other single environmental factor, with grain yield reductions up to 76% depending on the severity of the drought and the plant growth stage. Arbuscular mycorrhizal (AM) fungi and Plant Growth Promotion Rhizobacteria (PGPR) have previously been shown to improve tolerance of plants to drought stress through a number of chemical and physiological processes. The aim of this investigation was to determine whether mycorrhizal fungi and rhizobacteria adapted to drought and saline conditions and possessing plant growth promoting (PGP) traits were able to stimulate plant growth responses when applied to Zea mays seeds growing under greenhouse conditions Bacterial isolates selected were tolerant to concentrations of NaCl up to 600 mM and maintained 50% growth at low water potentials (-1.44 MPa). They were positive for Indole Acetic Acid (IAA) production, phosphate solubilisation and secretion of siderophores. Bacterial isolates showing plant growth promoting potential were identified using 16S rDNA gene sequencing as Achromobacter xylosoxidans strains A8 and C54 and Klebsiella oxytoca strain M1. Mixed inoculum was prepared from indigenous communities of mycorrhizas in soils sampled from the Cerebos Salt Pan and the Kalahari Desert. Mycorrhizal diversity was investigated using 454-Pyrosequencing which revealed that the community composition was dominated by species in the Ambispora, Glomus and Paraglomus genera with a rare component represented by species in the Redeckera, Archaeospora and Geosiphon genera. Microscopic examination of plant roots at the end of the trial revealed the presence of diagnostic mycorrhizal structures within the root cells, confirming that colonization was successful. Plant growth response to microbial inoculation was assessed by monitoring changes in plant photosynthetic capacity over the duration of a 7 week pot trial. A significant difference in photosynthetic and biomass data was observed between drought and well-watered groups but no mycorrhizal or bacterial treatment effect was evident within the groups, despite the high levels of colonization by mycorrhizas. These results suggest that the beneficial effects of mycorrhizal colonization may be primarily attributed to improved nutrient and mineral uptake in conditions where nutrients are limiting, resulting in improved growth. The improved growth may then have secondary effects on the plant‟s ability to withstand drought. Having controlled for nutrient deficiency, it was not evident in this study that mycorrhizal fungi were able to stimulate a change in plant physiology and confer drought tolerance under the conditions imposed.
- Full Text:
- Date Issued: 2016
Development of a high-throughput bioassay to determine the rate of antimalarial drug action using fluorescent vitality probes
- Authors: Laming, Dustin
- Date: 2016
- Subjects: Malaria -- Africa , Plasmodium falciparum , Drug development , Fluorescence
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/64434 , vital:28542
- Description: Malaria is one of the most prevalent diseases in Africa and the Plasmodium falciparum species is widely accepted as the most virulent, with a fatality rate of 15 – 20 % of reported cases of infection. While various treatments have been accepted into early stage clinical trials there has been little progress towards a proven vaccine. Pending a long term solution, endemic countries rely heavily on the development of innovative drugs with acute efficacy coupled with rapids mode of action. Until recently the rate of drug action has been measured by light microscopic examination of parasite morphology using blood slides of drug treated parasite cultures at regular time intervals. This technique is tedious and, most importantly, subject to interpretation with regards to distinguishing between viable and comprised parasite cells, thus making it impossible to objectively quantitate the rate of drug action. This study aimed to develop a series of bioassays using the calcein-acetoxymethyl and propidium iodide vitality probes which would allow the rate of drug action on Plasmodium falciparum malaria parasites to be assessed and ranked in relation to each other. A novel bioassay using these fluorescent vitality probes coupled with fluorescence microscopy was developed and optimized and allowed the rate of drug action on malaria parasites to be assessed i) rapidly (in relation to current assay techniques) and ii) in a semi-quantitative manner. Extrapolation to flow cytometry for improved quantification provided favourable rankings of drug killing rates in the pilot study, however, requires further development to increase throughput and approach the ultimate goal of producing a medium-throughput assay for rapidly assessing the rate of action of antimalarial drugs. Attempts to adapt the assay for use in a multiwell plate reader, as well as using ATP measurements as an indication of parasite vitality after drug treatment, was met with erratic results. The viability probes assay as it stands represents an improvement on other assay formats in terms of rapidity and quantification of live/compromised parasites in cultures.
- Full Text:
- Date Issued: 2016
- Authors: Laming, Dustin
- Date: 2016
- Subjects: Malaria -- Africa , Plasmodium falciparum , Drug development , Fluorescence
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/64434 , vital:28542
- Description: Malaria is one of the most prevalent diseases in Africa and the Plasmodium falciparum species is widely accepted as the most virulent, with a fatality rate of 15 – 20 % of reported cases of infection. While various treatments have been accepted into early stage clinical trials there has been little progress towards a proven vaccine. Pending a long term solution, endemic countries rely heavily on the development of innovative drugs with acute efficacy coupled with rapids mode of action. Until recently the rate of drug action has been measured by light microscopic examination of parasite morphology using blood slides of drug treated parasite cultures at regular time intervals. This technique is tedious and, most importantly, subject to interpretation with regards to distinguishing between viable and comprised parasite cells, thus making it impossible to objectively quantitate the rate of drug action. This study aimed to develop a series of bioassays using the calcein-acetoxymethyl and propidium iodide vitality probes which would allow the rate of drug action on Plasmodium falciparum malaria parasites to be assessed and ranked in relation to each other. A novel bioassay using these fluorescent vitality probes coupled with fluorescence microscopy was developed and optimized and allowed the rate of drug action on malaria parasites to be assessed i) rapidly (in relation to current assay techniques) and ii) in a semi-quantitative manner. Extrapolation to flow cytometry for improved quantification provided favourable rankings of drug killing rates in the pilot study, however, requires further development to increase throughput and approach the ultimate goal of producing a medium-throughput assay for rapidly assessing the rate of action of antimalarial drugs. Attempts to adapt the assay for use in a multiwell plate reader, as well as using ATP measurements as an indication of parasite vitality after drug treatment, was met with erratic results. The viability probes assay as it stands represents an improvement on other assay formats in terms of rapidity and quantification of live/compromised parasites in cultures.
- Full Text:
- Date Issued: 2016
Soil microbial properties and apple tree performance under conventional and organic management
- Authors: Meyer, André Harold
- Date: 2016
- Subjects: Soil management South Africa Western Cape , Agricultural chemicals Environmental aspects , Soil microbiology South Africa Western Cape , Vesicular-arbuscular mycorrhizas , Enzymes Biotechnology , Apples Organic farming South Africa Western Cape
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/64545 , vital:28557
- Description: Conventional (CON) soil management that permits the use of agrochemicals is currently the most common form of management in Western Cape deciduous fruit orchards. There is increasing pressure to minimise or eliminate synthetic agrochemical usage due to its potentially harmful effect on the environment, particularly to non-target soil microorganisms, and to the functions and processes they perform or mediate. In apple orchards, organic (ORG) practices exclude the use of synthetic pesticides and herbicides making use instead of organic fertilisers and naturally derived products as defined by organic certification programs. ORG practices aim to improve nutrient availability, yield, and long-term orchard sustainability relative to CON orchard management practices. If ORG and CON orchard floor management practices affect orchard ecosystems differently, such differences should be measurable in terms of differences in microbiological parameters. In this thesis it is hypothesised that ORG practices would induce positive soil microbiological responses in Western Cape apple orchards relative to CON practices, and by inference general soil health and apple tree performance. To test this hypothesis a polyphasic approach was adopted. This involved measurement of soil microbial activities and functional diversities, by enzyme activity (using colorimetric assays) and carbon-substrate utilisation (using the BIOLOG™ system), respectively. With reference to the enzyme analyses, the performance of a literature-validated, enzyme-based soil health index was also tested. The analyses were supported by coarse-level comparisons of the magnitude of bacteria, fungi, actinobacteria and total heterotroph populations using traditional culturing techniques (dilution plating on growth media). The extent to which the microbial status differed between the applied ORG and CON treatments was thought likely to reflect such treatment-induced variables as soil nutrient status, apple tree nutritional response, tree growth and yield, all of which were determined. Because the root systems of deciduous fruit trees commonly extend to depths >60 cm in well-prepared soils, microbial enzyme activities in the soil depth intervals corresponding to the lower rootzone, were also investigated. This research was carried out in a randomized field trial. Finally, to gain a broader understanding of the effects of contrasting soil management systems on soil microbiology under a greater variety of environmental conditions, arbuscular mycorrhizal (AM) fungal dynamics were explored in a survey of commercial apple orchards. These orchards were selected to span the range of environmental conditions that occur in the apple production areas of the Western Cape. Orchard soils under ORG management promoted richer microbial ecosystems, and appeared to be better able to sustain community metabolic diversity and, by inference, the functions mediated by soil microbial communities, than those under CON management. This implies that ORG approaches possibly afford a better option to sustain critical ecosystem functions than CON management. This possibly explains why use of straw mulches and compost in accordance with ORG practices, compared with CON practices, promoted β-glucosidase, acid phosphatase and urease activities rather than affecting the abundance of the micro-organisms that produce these enzymes. Enzyme activities in the 0–30 cm soil intervals were also more effectively promoted by ORG than CON practices, although no differences were observed at lower depth intervals. ORG practices promoted functional AM associations more effectively than CON practices, but the abundance of glomalin, a beneficial by-product of AM fungi, was unaffected. The greater enzyme activities and higher root colonisation levels in the ORG treatments probably contributed to improved nutritional effects that caused greater vegetative growth, but lower yields, in the ORG treatments. Yield suppression was conceivably due to excessive vegetative growth induced by oversupply of compost and the mineral nutrients contained therein. The survey of Western Cape apple orchards suggested that neither glomalin nor root colonisation bore any specific relationship to production area, cultivation practice, scion x rootstock combination, or, in the case of root colonisation, with any chemical parameters. However, the effect of season on glomalin was conclusively shown, being higher in summer than in spring, as was the lack of any effect of year on glomalin and root colonisation. Collectively, these results showed that ORG soil management promote soil microbiology, soil nutrient status, and apple tree performance compared to CON management. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2016
- Full Text:
- Date Issued: 2016
- Authors: Meyer, André Harold
- Date: 2016
- Subjects: Soil management South Africa Western Cape , Agricultural chemicals Environmental aspects , Soil microbiology South Africa Western Cape , Vesicular-arbuscular mycorrhizas , Enzymes Biotechnology , Apples Organic farming South Africa Western Cape
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/64545 , vital:28557
- Description: Conventional (CON) soil management that permits the use of agrochemicals is currently the most common form of management in Western Cape deciduous fruit orchards. There is increasing pressure to minimise or eliminate synthetic agrochemical usage due to its potentially harmful effect on the environment, particularly to non-target soil microorganisms, and to the functions and processes they perform or mediate. In apple orchards, organic (ORG) practices exclude the use of synthetic pesticides and herbicides making use instead of organic fertilisers and naturally derived products as defined by organic certification programs. ORG practices aim to improve nutrient availability, yield, and long-term orchard sustainability relative to CON orchard management practices. If ORG and CON orchard floor management practices affect orchard ecosystems differently, such differences should be measurable in terms of differences in microbiological parameters. In this thesis it is hypothesised that ORG practices would induce positive soil microbiological responses in Western Cape apple orchards relative to CON practices, and by inference general soil health and apple tree performance. To test this hypothesis a polyphasic approach was adopted. This involved measurement of soil microbial activities and functional diversities, by enzyme activity (using colorimetric assays) and carbon-substrate utilisation (using the BIOLOG™ system), respectively. With reference to the enzyme analyses, the performance of a literature-validated, enzyme-based soil health index was also tested. The analyses were supported by coarse-level comparisons of the magnitude of bacteria, fungi, actinobacteria and total heterotroph populations using traditional culturing techniques (dilution plating on growth media). The extent to which the microbial status differed between the applied ORG and CON treatments was thought likely to reflect such treatment-induced variables as soil nutrient status, apple tree nutritional response, tree growth and yield, all of which were determined. Because the root systems of deciduous fruit trees commonly extend to depths >60 cm in well-prepared soils, microbial enzyme activities in the soil depth intervals corresponding to the lower rootzone, were also investigated. This research was carried out in a randomized field trial. Finally, to gain a broader understanding of the effects of contrasting soil management systems on soil microbiology under a greater variety of environmental conditions, arbuscular mycorrhizal (AM) fungal dynamics were explored in a survey of commercial apple orchards. These orchards were selected to span the range of environmental conditions that occur in the apple production areas of the Western Cape. Orchard soils under ORG management promoted richer microbial ecosystems, and appeared to be better able to sustain community metabolic diversity and, by inference, the functions mediated by soil microbial communities, than those under CON management. This implies that ORG approaches possibly afford a better option to sustain critical ecosystem functions than CON management. This possibly explains why use of straw mulches and compost in accordance with ORG practices, compared with CON practices, promoted β-glucosidase, acid phosphatase and urease activities rather than affecting the abundance of the micro-organisms that produce these enzymes. Enzyme activities in the 0–30 cm soil intervals were also more effectively promoted by ORG than CON practices, although no differences were observed at lower depth intervals. ORG practices promoted functional AM associations more effectively than CON practices, but the abundance of glomalin, a beneficial by-product of AM fungi, was unaffected. The greater enzyme activities and higher root colonisation levels in the ORG treatments probably contributed to improved nutritional effects that caused greater vegetative growth, but lower yields, in the ORG treatments. Yield suppression was conceivably due to excessive vegetative growth induced by oversupply of compost and the mineral nutrients contained therein. The survey of Western Cape apple orchards suggested that neither glomalin nor root colonisation bore any specific relationship to production area, cultivation practice, scion x rootstock combination, or, in the case of root colonisation, with any chemical parameters. However, the effect of season on glomalin was conclusively shown, being higher in summer than in spring, as was the lack of any effect of year on glomalin and root colonisation. Collectively, these results showed that ORG soil management promote soil microbiology, soil nutrient status, and apple tree performance compared to CON management. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2016
- Full Text:
- Date Issued: 2016
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