Genetic characterisation of a range of geographically distinct Helicoverpa armigera nucleopolyhedrovirus (HearNPV) isolates and evaluation of biological activity against South African populations of the African bollworm, Helicoverpa armigera (Hu bner) (Lepidoptera: Noctuidae)
- Mtambanengwe, Kudzai Tapiwanashe Esau
- Authors: Mtambanengwe, Kudzai Tapiwanashe Esau
- Date: 2019
- Subjects: Helicoverpa armigera -- Biological control -- South Africa , Baculoviruses -- Genetics , Agricultural pests -- Biological control -- South Africa
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/97334 , vital:31426
- Description: The African bollworm, Helicoverpa armigera Hübner (Lepidoptera: Noctuidae) is a pest of economic and agricultural importance globally. It is a polyphagous pest that feeds on a wide range of host plants including economically important crops. The impact it has on agricultural systems makes its control a priority. The most common method of control is using chemical pesticides; however, continuous application of the pesticides has resulted in the development of resistance. The use of biological control has been investigated and established as an effective method of control as a standalone or part of an integrated pest management (IPM) system. The use of the baculovirus Helicoverpa armigera nucleopolyhedrovirus (HearNPV), has shown promise in the control of H. armigera. Commercial formulations based on the virus are available in many global markets. However, the identification of novel HearNPV isolates will aid in the control of H. armigera as well as provide alternative isolates that may have better virulence. Three new HearNPV isolates were purified and identified from three distinct geographical South African locations H. armigera cadavers and named HearNPV-Albany, HearNPV-KZN and HearNPV-Haygrove. The genomes of two of the HearNPV isolates, namely HearNPV-Albany and HearNPV-KZN were genetically characterised and compared to other geographically distinct HearNPV isolates. Virulence studies were performed comparing the new HearNPV isolates against established commercial HearNPV formulations, Helicovir™ and Helicovex® and other geographically distinct isolated HearNPV, HearNPV-G4 and HearNPV-SP1. Two laboratory colonies were established using H. armigera collected from South African fields in the Belmont Valley near Grahamstown labelled as Albany colony and a colony provided from Haygrove Eden farm near George labelled as Haygrove colony. Biological studies were carried out using the Albany H. armigera colony comparing the rate of development, survival and fertility on bell green peppers, cabbage leaves and on artificial diet. From the biological studies, it was recorded that development and survivorship was best on artificial diet. Regular quality control was required for the maintenance of the colony and continuous generations of healthy larvae were eventually established. Diseased cadavers with signs of baculovirus infection were collected after bioprospecting from the Kwa-Zulu Natal Province in South Africa and were labelled KZN isolate; Belmont Valley near Grahamstown and were labelled Albany isolate; and Haygrove Eden farm near George and were labelled Haygrove isolate for the study. A fourth isolate made up of a crude extract of occlusion bodies (OBs) first described by Whitlock was also analysed and labelled Whitlock isolate. Occlusion bodies were extracted, purified and morphologically identified from the KZN, Albany, Haygrove and Whitlock isolates using TEM. Genomic DNA, which was extracted from the purified OBs. Using PCR, the identity of the OBs as HearNPV was confirmed. Genomic analyses were performed on HearNPV-Albany and HearNPV-KZN through genetic characterisation and comparison with other geographically distinct HearNPV genomes to confirm novelty and establish potential genetic relationships between the isolates through evolutionary distances. Full genomic sequencing of the isolated HearNPV and comparison with other geographically distinct HearNPV isolates identified genomic differences that showed that the HearNPV isolates were novel. HearNPV-Albany and HearNPV-KZN were successfully sequenced and identified as novel isolates with unique fragment patterns and unique gene sequences through deletions or insertions when compared to other geographically distinct HearNPV. This raised the potential for differences in biological activity against H. armigera larvae when tested through biological assays. HearNPV-Whit genome assembly had low quality data which resulted in many gaps and failed assembly. The biological activity of HearNPV isolates from Spain, China, South Africa and two commercial formulations were studied against the laboratory established H. armigera South African colony. The LC50 values of the different South African HearNPV isolates were established to be between 7.7 × 101 OBs.ml-1 for the most effective and 3.2 × 102 OBs.ml-1 for the least effective. The Spanish and Chinese HearNPV isolates resulted in LC50 values of 2.0 × 102 OBs.ml-1 and 1.2 × 101 OBs.ml-1 respectively. The commercial formulations resulted in the least virulence observed with an LC50 of 5.84× 102 OBs.ml-1 and 9.0 × 102 OBs.ml-1 for Helicovex® and Helicovir™ respectively. In this study, novel South African HearNPV isolates were isolated and identified. Through characterisation and bioassays against South African H. armigera populations the HearNPV isolates were shown to have different virulence in comparison to geographically distinct isolates. From this research, there is potential for development of new H. armigera biopesticides based on the novel isolates after field trial testing.
- Full Text:
- Date Issued: 2019
- Authors: Mtambanengwe, Kudzai Tapiwanashe Esau
- Date: 2019
- Subjects: Helicoverpa armigera -- Biological control -- South Africa , Baculoviruses -- Genetics , Agricultural pests -- Biological control -- South Africa
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/97334 , vital:31426
- Description: The African bollworm, Helicoverpa armigera Hübner (Lepidoptera: Noctuidae) is a pest of economic and agricultural importance globally. It is a polyphagous pest that feeds on a wide range of host plants including economically important crops. The impact it has on agricultural systems makes its control a priority. The most common method of control is using chemical pesticides; however, continuous application of the pesticides has resulted in the development of resistance. The use of biological control has been investigated and established as an effective method of control as a standalone or part of an integrated pest management (IPM) system. The use of the baculovirus Helicoverpa armigera nucleopolyhedrovirus (HearNPV), has shown promise in the control of H. armigera. Commercial formulations based on the virus are available in many global markets. However, the identification of novel HearNPV isolates will aid in the control of H. armigera as well as provide alternative isolates that may have better virulence. Three new HearNPV isolates were purified and identified from three distinct geographical South African locations H. armigera cadavers and named HearNPV-Albany, HearNPV-KZN and HearNPV-Haygrove. The genomes of two of the HearNPV isolates, namely HearNPV-Albany and HearNPV-KZN were genetically characterised and compared to other geographically distinct HearNPV isolates. Virulence studies were performed comparing the new HearNPV isolates against established commercial HearNPV formulations, Helicovir™ and Helicovex® and other geographically distinct isolated HearNPV, HearNPV-G4 and HearNPV-SP1. Two laboratory colonies were established using H. armigera collected from South African fields in the Belmont Valley near Grahamstown labelled as Albany colony and a colony provided from Haygrove Eden farm near George labelled as Haygrove colony. Biological studies were carried out using the Albany H. armigera colony comparing the rate of development, survival and fertility on bell green peppers, cabbage leaves and on artificial diet. From the biological studies, it was recorded that development and survivorship was best on artificial diet. Regular quality control was required for the maintenance of the colony and continuous generations of healthy larvae were eventually established. Diseased cadavers with signs of baculovirus infection were collected after bioprospecting from the Kwa-Zulu Natal Province in South Africa and were labelled KZN isolate; Belmont Valley near Grahamstown and were labelled Albany isolate; and Haygrove Eden farm near George and were labelled Haygrove isolate for the study. A fourth isolate made up of a crude extract of occlusion bodies (OBs) first described by Whitlock was also analysed and labelled Whitlock isolate. Occlusion bodies were extracted, purified and morphologically identified from the KZN, Albany, Haygrove and Whitlock isolates using TEM. Genomic DNA, which was extracted from the purified OBs. Using PCR, the identity of the OBs as HearNPV was confirmed. Genomic analyses were performed on HearNPV-Albany and HearNPV-KZN through genetic characterisation and comparison with other geographically distinct HearNPV genomes to confirm novelty and establish potential genetic relationships between the isolates through evolutionary distances. Full genomic sequencing of the isolated HearNPV and comparison with other geographically distinct HearNPV isolates identified genomic differences that showed that the HearNPV isolates were novel. HearNPV-Albany and HearNPV-KZN were successfully sequenced and identified as novel isolates with unique fragment patterns and unique gene sequences through deletions or insertions when compared to other geographically distinct HearNPV. This raised the potential for differences in biological activity against H. armigera larvae when tested through biological assays. HearNPV-Whit genome assembly had low quality data which resulted in many gaps and failed assembly. The biological activity of HearNPV isolates from Spain, China, South Africa and two commercial formulations were studied against the laboratory established H. armigera South African colony. The LC50 values of the different South African HearNPV isolates were established to be between 7.7 × 101 OBs.ml-1 for the most effective and 3.2 × 102 OBs.ml-1 for the least effective. The Spanish and Chinese HearNPV isolates resulted in LC50 values of 2.0 × 102 OBs.ml-1 and 1.2 × 101 OBs.ml-1 respectively. The commercial formulations resulted in the least virulence observed with an LC50 of 5.84× 102 OBs.ml-1 and 9.0 × 102 OBs.ml-1 for Helicovex® and Helicovir™ respectively. In this study, novel South African HearNPV isolates were isolated and identified. Through characterisation and bioassays against South African H. armigera populations the HearNPV isolates were shown to have different virulence in comparison to geographically distinct isolates. From this research, there is potential for development of new H. armigera biopesticides based on the novel isolates after field trial testing.
- Full Text:
- Date Issued: 2019
A lignocellulolytic enzyme system for fruit waste degradation : commercial enzyme mixture synergy and bioreactor design
- Authors: Gama, Repson
- Date: 2014
- Subjects: Enzymes -- Biotechnology , Enzymes -- Industrial applications , Lignocellulose -- Biodegradation , Biomass energy , Biomass conversion , Biochemical engineering , Agricultural wastes as fuel
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4114 , http://hdl.handle.net/10962/d1013073
- Description: Studies into sources of alternative liquid transport fuel energy have identified agro-industrial wastes, which are lignocellulosic in nature, as a potential feedstock for biofuel production against the background of depleting nonrenewable fossil fuels. In South Africa, large quantities of apple and other fruit wastes, called pomace, are generated from fruit and juice industries. Apple pomace is a rich source of cellulose, pectin and hemicellulose, making it a potential target for utilisation as a lignocellulosic feedstock for biofuel and biorefinery chemical production. Lignocellulosic biomass is recalcitrant in nature and therefore its degradation requires the synergistic action of a number of enzymes such as cellulases, hemicellulases, pectinases and ligninases. Commercial enzyme cocktails, containing some of these enzymes, are available and can be used for apple pomace degradation. In this study, the degradation of apple pomace using commercial enzyme cocktails was investigated. The main focus was the optimisation of the release of sugar monomers that could potentially be used for biofuel and biorefinery chemical production. There is no or little information reported in literature on the enzymatic degradation of fruit waste using commercial enzyme mixtures. This study first focused on the characterisation of the substrate (apple pomace) and the commercial enzyme cocktails. Apple pomace was found to contain mainly glucose, galacturonic acid, arabinose, galactose, lignin and low amounts of xylose and fructose. Three commercial enzyme cocktails were initially selected: Biocip Membrane, Viscozyme L (from Aspergillus aculeatus) and Celluclast 1.5L (a Trichoderma reesei ATCC 26921 cellulase preparation). The selection of the enzymes was based on activities declared by the manufacturers, cost and local availability. The enzymes were screened based on their synergistic cooperation in the degradation of apple pomace and the main enzymes present in each cocktail. Viscozyme L and Celluclast 1.5L, in a 50:50 ratio, resulted in the best degree of synergy (1.6) compared to any other combination. The enzyme ratios were determined on Viscozyme L and Celluclast 1.5L based on the protein ratio. Enzyme activity was determined as glucose equivalents using the dinitrosalicylic acid (DNS) method. Sugar monomers were determined using Megazyme assay kits. There is limited information available on the enzymes present in the commercial enzyme cocktails. Therefore, the main enzymes present in Viscozyme L and Celluclast 1.5L were identified using different substrates, each targeted for a specific enzyme and activity. Characterisation of the enzyme mixtures revealed a large number of enzymes required for apple pomace degradation and these included cellulases, pectinases, xylanases, arabinases and mannanases in different proportions. Viscozyme L contained mainly pectinases and hemicellulases, while Celluclast 1.5L displayed largely cellulase and xylanase activity, hence the high degree of synergy reported. The temperature optimum was 50ºC for both enzyme mixtures and pH optima were observed at pH 5.0 and pH 3.0 for Viscozyme L and Celluclast 1.5L, respectively. At 37ºC and pH 5.0, the enzymes retained more that 90% activity after 15 days of incubation, allowing the enzymes to be used together with less energy input. The enzymes were further characterised by determining the effect of various compounds, such as alcohols, sugars, phenolic compounds and metal ions at various concentrations on the activity of the enzymes during apple pomace hydrolysis. Apart from lignin, which had almost no effect on enzyme activity, all the compounds caused inhibition of the enzymes to varying degrees. The most inhibitory compounds were some organic acids and metal ions, as well as cellobiose and xylobiose. Using the best ratio for Viscozyme L and Celluclast 1.5L (50:50) for the hydrolysis of apple pomace, it was observed that synergy was highest at the initial stages of hydrolysis and decreased over time, though the sugar concentration increased. The type of synergy for optimal apple pomace hydrolysis was found to be simultaneous. There was no synergy observed between Viscozyme L and Celluclast 1.5L with ligninases - laccase, lignin peroxidase and manganese peroxidase. Hydrolysing apple pomace with ligninases prior to addition of Viscozyme L and Celluclast 1.5L did not improve degradation of the substrate. Immobilisation of the enzyme mixtures on different supports was performed with the aim of increasing stability and enabling reuse of the enzymes. Immobilisation methods were selected based on the chemical properties of the supports, availability, cost and applicability on heterogeneous and insoluble substrate like apple pomace. These methods included crosslinked enzyme aggregates (CLEAs), immobilisation on various supports such as nylon mesh, nylon beads, sodium alginate beads, chitin and silica gel beads. The immobilisation strategies were unsuccessful, mainly due to the low percentage of immobilisation of the enzyme on the matrix and loss of activity of the immobilised enzyme. Free enzymes were therefore used for the remainder of the study. Hydrolysis conditions for apple pomace degradation were optimised using different temperatures and buffer systems in 1 L volumes mixed with compressed air. Hydrolysis at room temperature, using an unbuffered system, gave a better performance as compared to a buffered system. Reactors operated in batch mode performed better (4.2 g/L (75% yield) glucose and 16.8 g/L (75%) reducing sugar) than fed-batch reactors (3.2 g/L (66%) glucose and 14.6 g/L (72.7% yield) reducing sugar) over 100 h using Viscozyme L and Celluclast 1.5L. Supplementation of β- glucosidase activity in Viscozyme L and Celluclast 1.5L with Novozyme 188 resulted in a doubling of the amount of glucose released. The main products released from apple pomace hydrolysis were galacturonic acid, glucose and arabinose and low amounts of galactose and xylose. These products are potential raw materials for biofuel and biorefinery chemical production. An artificial neural network (ANN) model was successfully developed and used for predicting the optimum conditions for apple pomace hydrolysis using Celluclast 1.5L, Viscozyme L and Novozyme 188. Four main conditions that affect apple pomace hydrolysis were selected, namely temperature, initial pH, enzyme loading and substrate loading, which were taken as inputs. The glucose and reducing sugars released as a result of each treatment and their combinations were taken as outputs for 1–100 h. An ANN with 20, 20 and 6 neurons in the first, second and third hidden layers, respectively, was constructed. The performance and predictive ability of the ANN was good, with a R² of 0.99 and a small mean square error (MSE). New data was successfully predicted and simulated. Optimal hydrolysis conditions predicted by ANN for apple pomace hydrolysis were at 30% substrate (wet w/v) and an enzyme loading of 0.5 mg/g and 0.2 mg/mL of substrate for glucose and reducing sugar, respectively, giving sugar concentrations of 6.5 mg/mL and 28.9 mg/mL for glucose and reducing sugar, respectively. ANN showed that enzyme and substrate loadings were the most important factors for the hydrolysis of apple pomace.
- Full Text:
- Date Issued: 2014
- Authors: Gama, Repson
- Date: 2014
- Subjects: Enzymes -- Biotechnology , Enzymes -- Industrial applications , Lignocellulose -- Biodegradation , Biomass energy , Biomass conversion , Biochemical engineering , Agricultural wastes as fuel
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4114 , http://hdl.handle.net/10962/d1013073
- Description: Studies into sources of alternative liquid transport fuel energy have identified agro-industrial wastes, which are lignocellulosic in nature, as a potential feedstock for biofuel production against the background of depleting nonrenewable fossil fuels. In South Africa, large quantities of apple and other fruit wastes, called pomace, are generated from fruit and juice industries. Apple pomace is a rich source of cellulose, pectin and hemicellulose, making it a potential target for utilisation as a lignocellulosic feedstock for biofuel and biorefinery chemical production. Lignocellulosic biomass is recalcitrant in nature and therefore its degradation requires the synergistic action of a number of enzymes such as cellulases, hemicellulases, pectinases and ligninases. Commercial enzyme cocktails, containing some of these enzymes, are available and can be used for apple pomace degradation. In this study, the degradation of apple pomace using commercial enzyme cocktails was investigated. The main focus was the optimisation of the release of sugar monomers that could potentially be used for biofuel and biorefinery chemical production. There is no or little information reported in literature on the enzymatic degradation of fruit waste using commercial enzyme mixtures. This study first focused on the characterisation of the substrate (apple pomace) and the commercial enzyme cocktails. Apple pomace was found to contain mainly glucose, galacturonic acid, arabinose, galactose, lignin and low amounts of xylose and fructose. Three commercial enzyme cocktails were initially selected: Biocip Membrane, Viscozyme L (from Aspergillus aculeatus) and Celluclast 1.5L (a Trichoderma reesei ATCC 26921 cellulase preparation). The selection of the enzymes was based on activities declared by the manufacturers, cost and local availability. The enzymes were screened based on their synergistic cooperation in the degradation of apple pomace and the main enzymes present in each cocktail. Viscozyme L and Celluclast 1.5L, in a 50:50 ratio, resulted in the best degree of synergy (1.6) compared to any other combination. The enzyme ratios were determined on Viscozyme L and Celluclast 1.5L based on the protein ratio. Enzyme activity was determined as glucose equivalents using the dinitrosalicylic acid (DNS) method. Sugar monomers were determined using Megazyme assay kits. There is limited information available on the enzymes present in the commercial enzyme cocktails. Therefore, the main enzymes present in Viscozyme L and Celluclast 1.5L were identified using different substrates, each targeted for a specific enzyme and activity. Characterisation of the enzyme mixtures revealed a large number of enzymes required for apple pomace degradation and these included cellulases, pectinases, xylanases, arabinases and mannanases in different proportions. Viscozyme L contained mainly pectinases and hemicellulases, while Celluclast 1.5L displayed largely cellulase and xylanase activity, hence the high degree of synergy reported. The temperature optimum was 50ºC for both enzyme mixtures and pH optima were observed at pH 5.0 and pH 3.0 for Viscozyme L and Celluclast 1.5L, respectively. At 37ºC and pH 5.0, the enzymes retained more that 90% activity after 15 days of incubation, allowing the enzymes to be used together with less energy input. The enzymes were further characterised by determining the effect of various compounds, such as alcohols, sugars, phenolic compounds and metal ions at various concentrations on the activity of the enzymes during apple pomace hydrolysis. Apart from lignin, which had almost no effect on enzyme activity, all the compounds caused inhibition of the enzymes to varying degrees. The most inhibitory compounds were some organic acids and metal ions, as well as cellobiose and xylobiose. Using the best ratio for Viscozyme L and Celluclast 1.5L (50:50) for the hydrolysis of apple pomace, it was observed that synergy was highest at the initial stages of hydrolysis and decreased over time, though the sugar concentration increased. The type of synergy for optimal apple pomace hydrolysis was found to be simultaneous. There was no synergy observed between Viscozyme L and Celluclast 1.5L with ligninases - laccase, lignin peroxidase and manganese peroxidase. Hydrolysing apple pomace with ligninases prior to addition of Viscozyme L and Celluclast 1.5L did not improve degradation of the substrate. Immobilisation of the enzyme mixtures on different supports was performed with the aim of increasing stability and enabling reuse of the enzymes. Immobilisation methods were selected based on the chemical properties of the supports, availability, cost and applicability on heterogeneous and insoluble substrate like apple pomace. These methods included crosslinked enzyme aggregates (CLEAs), immobilisation on various supports such as nylon mesh, nylon beads, sodium alginate beads, chitin and silica gel beads. The immobilisation strategies were unsuccessful, mainly due to the low percentage of immobilisation of the enzyme on the matrix and loss of activity of the immobilised enzyme. Free enzymes were therefore used for the remainder of the study. Hydrolysis conditions for apple pomace degradation were optimised using different temperatures and buffer systems in 1 L volumes mixed with compressed air. Hydrolysis at room temperature, using an unbuffered system, gave a better performance as compared to a buffered system. Reactors operated in batch mode performed better (4.2 g/L (75% yield) glucose and 16.8 g/L (75%) reducing sugar) than fed-batch reactors (3.2 g/L (66%) glucose and 14.6 g/L (72.7% yield) reducing sugar) over 100 h using Viscozyme L and Celluclast 1.5L. Supplementation of β- glucosidase activity in Viscozyme L and Celluclast 1.5L with Novozyme 188 resulted in a doubling of the amount of glucose released. The main products released from apple pomace hydrolysis were galacturonic acid, glucose and arabinose and low amounts of galactose and xylose. These products are potential raw materials for biofuel and biorefinery chemical production. An artificial neural network (ANN) model was successfully developed and used for predicting the optimum conditions for apple pomace hydrolysis using Celluclast 1.5L, Viscozyme L and Novozyme 188. Four main conditions that affect apple pomace hydrolysis were selected, namely temperature, initial pH, enzyme loading and substrate loading, which were taken as inputs. The glucose and reducing sugars released as a result of each treatment and their combinations were taken as outputs for 1–100 h. An ANN with 20, 20 and 6 neurons in the first, second and third hidden layers, respectively, was constructed. The performance and predictive ability of the ANN was good, with a R² of 0.99 and a small mean square error (MSE). New data was successfully predicted and simulated. Optimal hydrolysis conditions predicted by ANN for apple pomace hydrolysis were at 30% substrate (wet w/v) and an enzyme loading of 0.5 mg/g and 0.2 mg/mL of substrate for glucose and reducing sugar, respectively, giving sugar concentrations of 6.5 mg/mL and 28.9 mg/mL for glucose and reducing sugar, respectively. ANN showed that enzyme and substrate loadings were the most important factors for the hydrolysis of apple pomace.
- Full Text:
- Date Issued: 2014
An investigation into the bacterial diversity associated with South African latrunculid sponges that produce bioactive secondary metabolites
- Authors: Walmsley, Tara Aisling
- Date: 2014
- Subjects: Sponges -- South Africa -- Algoa Bay , Sponges -- Classification , Metabolites -- South Africa -- Algoa Bay , Marine metabolites -- South Africa -- Algoa Bay , PQQ (Biochemistry) , Bacterial diversity
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4109 , http://hdl.handle.net/10962/d1012943
- Description: Algoa Bay Latrunculid sponges are well known for their production of cytotoxic pyrroloiminoquinones with speculation that these secondary metabolites may have a microbial origin. This study describes a thorough investigation into the bacterial community associated with Tsitsikamma favus, Tsitsikamma scurra a newly described Latrunculia sp. and a yellow encrusting sponge associated with T. scurra. Molecular and chemical characterisation were used in conjunction with traditional taxonomy in identification of the sponge specimens. The 28S rRNA and COX1 analysis confirmed the traditional taxonomy with T. favus and T. scurra being very closely related. Chemical analysis revealed that T. favus and T. scurra shared the discorhabdins 2,4-debromo-3-dihydrodiscorhabdin C, 7,8-dehydro-3-dihydrodiscorhabdin C and 14-bromo-1-hydroxy-discorhabdin V in common with each other and Tsitsikamma pedunculata indicating that these pyrroloiminoquinones are common to Tsitsikamma sponges in general. The bacterial community associated with T. favus was explored using 16S rRNA molecular techniques including DGGE, clonal libraries of full length 16S rRNA genes, as well as 454 pyrosequencing. DGGE analysis revealed that the bacterial community associated with T. favus appeared to be highly conserved, which was confirmed by both the clone library and 454 pyrosequencing, with the Betaproteobacteria as the most dominant class. Further exploration into T. favus, as well as T. scurra, Latrunculia sp. and the yellow encrusting sponge indicated that the bacterial populations associated with each of these sponge species were conserved and species specific. OTU analysis to the species level revealed that T. favus and T. scurra shared an abundant Spirochaete species in common while the most abundant species in the Latrunculia sp. and the yellow encrusting sponge belonged to the class Betaproteobacteria. The exclusivity of the tsitsikammamines to T. favus precipitated attempts to culture the T. favus associated bacteria, with a focus on the dominant betaproteobacterium as indicated by the 16S rRNA clone library. Actinobacteria associated with the Algoa Bay sponge specimens were also cultured and the actinobacterial isolates were sent for screening against Mycobacterium aurum with two Kocuria kristinae isolates and a Streptomyces albdioflavus isolate showing good antimycobacterial activity.
- Full Text:
- Date Issued: 2014
- Authors: Walmsley, Tara Aisling
- Date: 2014
- Subjects: Sponges -- South Africa -- Algoa Bay , Sponges -- Classification , Metabolites -- South Africa -- Algoa Bay , Marine metabolites -- South Africa -- Algoa Bay , PQQ (Biochemistry) , Bacterial diversity
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4109 , http://hdl.handle.net/10962/d1012943
- Description: Algoa Bay Latrunculid sponges are well known for their production of cytotoxic pyrroloiminoquinones with speculation that these secondary metabolites may have a microbial origin. This study describes a thorough investigation into the bacterial community associated with Tsitsikamma favus, Tsitsikamma scurra a newly described Latrunculia sp. and a yellow encrusting sponge associated with T. scurra. Molecular and chemical characterisation were used in conjunction with traditional taxonomy in identification of the sponge specimens. The 28S rRNA and COX1 analysis confirmed the traditional taxonomy with T. favus and T. scurra being very closely related. Chemical analysis revealed that T. favus and T. scurra shared the discorhabdins 2,4-debromo-3-dihydrodiscorhabdin C, 7,8-dehydro-3-dihydrodiscorhabdin C and 14-bromo-1-hydroxy-discorhabdin V in common with each other and Tsitsikamma pedunculata indicating that these pyrroloiminoquinones are common to Tsitsikamma sponges in general. The bacterial community associated with T. favus was explored using 16S rRNA molecular techniques including DGGE, clonal libraries of full length 16S rRNA genes, as well as 454 pyrosequencing. DGGE analysis revealed that the bacterial community associated with T. favus appeared to be highly conserved, which was confirmed by both the clone library and 454 pyrosequencing, with the Betaproteobacteria as the most dominant class. Further exploration into T. favus, as well as T. scurra, Latrunculia sp. and the yellow encrusting sponge indicated that the bacterial populations associated with each of these sponge species were conserved and species specific. OTU analysis to the species level revealed that T. favus and T. scurra shared an abundant Spirochaete species in common while the most abundant species in the Latrunculia sp. and the yellow encrusting sponge belonged to the class Betaproteobacteria. The exclusivity of the tsitsikammamines to T. favus precipitated attempts to culture the T. favus associated bacteria, with a focus on the dominant betaproteobacterium as indicated by the 16S rRNA clone library. Actinobacteria associated with the Algoa Bay sponge specimens were also cultured and the actinobacterial isolates were sent for screening against Mycobacterium aurum with two Kocuria kristinae isolates and a Streptomyces albdioflavus isolate showing good antimycobacterial activity.
- Full Text:
- Date Issued: 2014
Biochemical mechanisms towards understanding Alzheimer's disease
- Authors: Padayachee, Eden Rebecca
- Date: 2014
- Subjects: Alzheimer's disease Nitric-oxide synthase Biochemical markers Amyloid beta-protein Peptide hormones
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4103 , http://hdl.handle.net/10962/d1011092
- Description: The start of the amyloidogenic pathway in Alzheimer’s disease (AD) begins with the deposition of the Aβ₁₋₄₂ peptide surrounded by astrocytes. High levels of arginine and low amounts of neuronal nitric oxide synthase (nNOS) are associated with AD. These astrocytes store reserve arginine that is eventually metabolized by nNOS, within the vicinity of the Aβ₁₋₄₂ peptide. We propose the existence of an association vs. dissociation equilibrium between Aβ and nNOS such that nNOS is an amyloidogenic catalyst for fibrils. When Aβ binds to nNOS, it inhibits the activity of the enzyme (association phase). However when the amyloid peptide dissociates into a form that can no longer bind, later deduced as a fibril, the activity is restored. Thus, the interaction of Aβ with nNOS could serve to regulate the interaction between nNOS and arginine by restoring activity of the enzyme but at the same time promoting fibrillogenesis. Given this event occurring with the neuron, both nNOS and amyloid can serve as a biomarker for the early onset of AD. The enzyme nNOS catalyzed the formation of fibrils in the presence of Aβ peptides, while Ag nps were shown to reverse the fibril formation from Aβ peptides more so than Au and curcumin either through electrostatic or π-π stacking (aromatic) influences. Our studies have shown that the fragments of Aβ₁₋₄₂ i.e. the pentapeptide (Aβ₁₇₋₂₁) and the three glycine zipper peptides (Aβ₂₅₋₂₉, Aβ₂₉₋₃₃, Aβ₃₃₋₃₇) and the full length glycine zipper stretch (Aβ₂₅₋₃₇) all inhibited nNOS activity to varying degrees. The peptides Aβ₁₇₋₂₁ and Aβ₂₉₋₃₃ with their respective Ki values of 5.1 μM and 7.5 μM inhibited the enzyme the most. The Ki values for reversed sequenced peptides (Aβ₁₇₋₂₁r and Aβ₂₉₋₃₃r) were two fold greater than that of the original peptides while the Ki values for the polar forms (Aβ₁₇₋₂₁p and Aβ₂₉₋₃₃p) were between 3-4 fold greater than that of the original peptides. It was also found that Ag nps (Ki = 0.12 μM) inhibited the activity of nNOS the most compared to Au nps; (Ki = 0.15 μM) and curcumin (Ki = 0.25 μM). At 298K, all the ligands bound at a single site on the enzyme (n=1) and a single Trp residue (θ =1), (later identified as Trp678) was made available on the enzyme surface for quenching by the ligands. Increasing the temperature from 298K-313K, increased the value of Ksv and pointed to a dynamic quenching mechanism for Aβ peptides, nps and curcumin interaction with nNOS. The positive signs for entropy and enthalpy for all Aβ peptides nps and curcumin pointed to hydrophobic–hydrophobic interaction with the enzyme. The fact that Kd increased with temperature emphasized the endothermic nature of the binding reaction and the requirement of thermal energy to aid in diffusion of the ligand to the active site. It was concluded that the binding reaction between the ligands and nNOS was non-spontaneous and endothermic at low temperatures (+ΔG) but spontaneous at high temperatures (-ΔG). The two amino acids Tyr706 and Trp678 moved from their original positions, subject to ligand binding. Trp678 moved a minimum distance of 5 Å toward the heme while Tyr706 moved a maximum distance of 14 Å away from the heme. AutoDock 4.2 was a valuable tool in monitoring the distance of Trp678 within the enzyme interior and fluorescence resonance energy transfer (FRET) was efficient in monitoring the distance moved by Trp residues on the enzyme surface.
- Full Text:
- Date Issued: 2014
- Authors: Padayachee, Eden Rebecca
- Date: 2014
- Subjects: Alzheimer's disease Nitric-oxide synthase Biochemical markers Amyloid beta-protein Peptide hormones
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4103 , http://hdl.handle.net/10962/d1011092
- Description: The start of the amyloidogenic pathway in Alzheimer’s disease (AD) begins with the deposition of the Aβ₁₋₄₂ peptide surrounded by astrocytes. High levels of arginine and low amounts of neuronal nitric oxide synthase (nNOS) are associated with AD. These astrocytes store reserve arginine that is eventually metabolized by nNOS, within the vicinity of the Aβ₁₋₄₂ peptide. We propose the existence of an association vs. dissociation equilibrium between Aβ and nNOS such that nNOS is an amyloidogenic catalyst for fibrils. When Aβ binds to nNOS, it inhibits the activity of the enzyme (association phase). However when the amyloid peptide dissociates into a form that can no longer bind, later deduced as a fibril, the activity is restored. Thus, the interaction of Aβ with nNOS could serve to regulate the interaction between nNOS and arginine by restoring activity of the enzyme but at the same time promoting fibrillogenesis. Given this event occurring with the neuron, both nNOS and amyloid can serve as a biomarker for the early onset of AD. The enzyme nNOS catalyzed the formation of fibrils in the presence of Aβ peptides, while Ag nps were shown to reverse the fibril formation from Aβ peptides more so than Au and curcumin either through electrostatic or π-π stacking (aromatic) influences. Our studies have shown that the fragments of Aβ₁₋₄₂ i.e. the pentapeptide (Aβ₁₇₋₂₁) and the three glycine zipper peptides (Aβ₂₅₋₂₉, Aβ₂₉₋₃₃, Aβ₃₃₋₃₇) and the full length glycine zipper stretch (Aβ₂₅₋₃₇) all inhibited nNOS activity to varying degrees. The peptides Aβ₁₇₋₂₁ and Aβ₂₉₋₃₃ with their respective Ki values of 5.1 μM and 7.5 μM inhibited the enzyme the most. The Ki values for reversed sequenced peptides (Aβ₁₇₋₂₁r and Aβ₂₉₋₃₃r) were two fold greater than that of the original peptides while the Ki values for the polar forms (Aβ₁₇₋₂₁p and Aβ₂₉₋₃₃p) were between 3-4 fold greater than that of the original peptides. It was also found that Ag nps (Ki = 0.12 μM) inhibited the activity of nNOS the most compared to Au nps; (Ki = 0.15 μM) and curcumin (Ki = 0.25 μM). At 298K, all the ligands bound at a single site on the enzyme (n=1) and a single Trp residue (θ =1), (later identified as Trp678) was made available on the enzyme surface for quenching by the ligands. Increasing the temperature from 298K-313K, increased the value of Ksv and pointed to a dynamic quenching mechanism for Aβ peptides, nps and curcumin interaction with nNOS. The positive signs for entropy and enthalpy for all Aβ peptides nps and curcumin pointed to hydrophobic–hydrophobic interaction with the enzyme. The fact that Kd increased with temperature emphasized the endothermic nature of the binding reaction and the requirement of thermal energy to aid in diffusion of the ligand to the active site. It was concluded that the binding reaction between the ligands and nNOS was non-spontaneous and endothermic at low temperatures (+ΔG) but spontaneous at high temperatures (-ΔG). The two amino acids Tyr706 and Trp678 moved from their original positions, subject to ligand binding. Trp678 moved a minimum distance of 5 Å toward the heme while Tyr706 moved a maximum distance of 14 Å away from the heme. AutoDock 4.2 was a valuable tool in monitoring the distance of Trp678 within the enzyme interior and fluorescence resonance energy transfer (FRET) was efficient in monitoring the distance moved by Trp residues on the enzyme surface.
- Full Text:
- Date Issued: 2014
Characterization of the Hsp40 partner proteins of Plasmodium falciparum Hsp70
- Authors: Njunge, James Mwangi
- Date: 2014
- Subjects: Plasmodium falciparum , Heat shock proteins , Malaria -- Chemotherapy , Protein-protein interactions , Erythrocytes -- Biotechnology , Molecular chaperones , Host-parasite relationships , Mitochondria
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4117 , http://hdl.handle.net/10962/d1013186
- Description: Human malaria is an economically important disease caused by single-celled parasites of the Plasmodium genus whose biology displays great evolutionary adaptation to both its mammalian host and transmitting vectors. This thesis details the 70 kDa heat shock protein (Hsp70) and J protein chaperone complements in malaria parasites affecting humans, primates and rodents. Heat shock proteins comprise a family of evolutionary conserved and structurally related proteins that play a crucial role in maintaining the structural integrity of proteins during normal and stress conditions. They are considered future therapeutic targets in various cellular systems including Plasmodium falciparum. J proteins (Hsp40) canonically partner with Hsp70s during protein synthesis and folding, trafficking or targeting of proteins for degradation. However, in P. falciparum, these classes of proteins have also been implicated in aiding the active transport of parasite proteins to the erythrocyte cytosol following erythrocyte entry by the parasite. This host-parasite “cross-talk” results in tremendous modifications of the infected erythrocyte, imparting properties that allow it to adhere to the endothelium, preventing splenic clearance. The genome of P. falciparum encodes six Hsp70 homologues and a large number of J proteins that localize to the various intracellular compartments or are exported to the infected erythrocyte cytosol. Understanding the Hsp70-J protein interactions and/or partnerships is an essential step for drug target validation and illumination of parasite biology. A review of these chaperone complements across the Plasmodium species shows that P. falciparum possesses an expanded Hsp70-J protein complement compared to the rodent and primate infecting species. It further highlights how unique the P. falciparum chaperone complement is compared to the other Plasmodium species included in the analysis. In silico analysis showed that the genome of P. falciparum encodes approximately 49 J proteins, 19 of which contain a PEXEL motif that has been implicated in routing proteins to the infected erythrocyte. Most of these PEXEL containing J proteins are unique with no homologues in the human system and are considered as attractive drug targets. Very few of the predicted J proteins in P. falciparum have been experimentally characterized. To this end, cell biological and biochemical approaches were employed to characterize PFB0595w and PFD0462w (Pfj1) J proteins. The uniqueness of Pfj1 and the controversy in literature regarding its localization formed the basis for the experimental work. This is the first study showing that Pfj1 localizes to the mitochondrion in the intraerythrocytic stage of development of P. falciparum and has further proposed PfHsp70-3 as a potential Hsp70 partner. Indeed, attempts to heterologously express and purify Pfj1 for its characterization are described. It is also the first study that details the successful expression and purification of PfHsp70-3. Further, research findings have described for the first time the expression and localization of PFB0595w in the intraerythrocytic stages of P. falciparum development. Based on the cytosolic localization of both PFB0595w and PfHsp70-1, a chaperone – cochaperone partnership was proposed that formed the basis for the in vitro experiments. PFB0595w was shown for the first time to stimulate the ATPase activity of PfHsp70-1 pointing to a functional interaction. Preliminary surface plasmon spectroscopy analysis has revealed a potential interaction between PFB0595w and PfHsp70-1 but highlights the need for further related experiments to support the findings. Gel filtration analysis showed that PFB0595w exists as a dimer thereby confirming in silico predictions. Based on these observations, we conclude that PFB0595w may regulate the chaperone activity of PfHsp70-1 in the cytosol while Pfj1 may play a co-chaperoning role for PfHsp70-3 in the mitochondrion. Overall, this data is expected to increase the knowledge of the Hsp70-J protein partnerships in the erythrocytic stage of P. falciparum development, thereby enhancing the understanding of parasite biology.
- Full Text:
- Date Issued: 2014
- Authors: Njunge, James Mwangi
- Date: 2014
- Subjects: Plasmodium falciparum , Heat shock proteins , Malaria -- Chemotherapy , Protein-protein interactions , Erythrocytes -- Biotechnology , Molecular chaperones , Host-parasite relationships , Mitochondria
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4117 , http://hdl.handle.net/10962/d1013186
- Description: Human malaria is an economically important disease caused by single-celled parasites of the Plasmodium genus whose biology displays great evolutionary adaptation to both its mammalian host and transmitting vectors. This thesis details the 70 kDa heat shock protein (Hsp70) and J protein chaperone complements in malaria parasites affecting humans, primates and rodents. Heat shock proteins comprise a family of evolutionary conserved and structurally related proteins that play a crucial role in maintaining the structural integrity of proteins during normal and stress conditions. They are considered future therapeutic targets in various cellular systems including Plasmodium falciparum. J proteins (Hsp40) canonically partner with Hsp70s during protein synthesis and folding, trafficking or targeting of proteins for degradation. However, in P. falciparum, these classes of proteins have also been implicated in aiding the active transport of parasite proteins to the erythrocyte cytosol following erythrocyte entry by the parasite. This host-parasite “cross-talk” results in tremendous modifications of the infected erythrocyte, imparting properties that allow it to adhere to the endothelium, preventing splenic clearance. The genome of P. falciparum encodes six Hsp70 homologues and a large number of J proteins that localize to the various intracellular compartments or are exported to the infected erythrocyte cytosol. Understanding the Hsp70-J protein interactions and/or partnerships is an essential step for drug target validation and illumination of parasite biology. A review of these chaperone complements across the Plasmodium species shows that P. falciparum possesses an expanded Hsp70-J protein complement compared to the rodent and primate infecting species. It further highlights how unique the P. falciparum chaperone complement is compared to the other Plasmodium species included in the analysis. In silico analysis showed that the genome of P. falciparum encodes approximately 49 J proteins, 19 of which contain a PEXEL motif that has been implicated in routing proteins to the infected erythrocyte. Most of these PEXEL containing J proteins are unique with no homologues in the human system and are considered as attractive drug targets. Very few of the predicted J proteins in P. falciparum have been experimentally characterized. To this end, cell biological and biochemical approaches were employed to characterize PFB0595w and PFD0462w (Pfj1) J proteins. The uniqueness of Pfj1 and the controversy in literature regarding its localization formed the basis for the experimental work. This is the first study showing that Pfj1 localizes to the mitochondrion in the intraerythrocytic stage of development of P. falciparum and has further proposed PfHsp70-3 as a potential Hsp70 partner. Indeed, attempts to heterologously express and purify Pfj1 for its characterization are described. It is also the first study that details the successful expression and purification of PfHsp70-3. Further, research findings have described for the first time the expression and localization of PFB0595w in the intraerythrocytic stages of P. falciparum development. Based on the cytosolic localization of both PFB0595w and PfHsp70-1, a chaperone – cochaperone partnership was proposed that formed the basis for the in vitro experiments. PFB0595w was shown for the first time to stimulate the ATPase activity of PfHsp70-1 pointing to a functional interaction. Preliminary surface plasmon spectroscopy analysis has revealed a potential interaction between PFB0595w and PfHsp70-1 but highlights the need for further related experiments to support the findings. Gel filtration analysis showed that PFB0595w exists as a dimer thereby confirming in silico predictions. Based on these observations, we conclude that PFB0595w may regulate the chaperone activity of PfHsp70-1 in the cytosol while Pfj1 may play a co-chaperoning role for PfHsp70-3 in the mitochondrion. Overall, this data is expected to increase the knowledge of the Hsp70-J protein partnerships in the erythrocytic stage of P. falciparum development, thereby enhancing the understanding of parasite biology.
- Full Text:
- Date Issued: 2014
Purification and characterization of TbHsp70.c, a novel Hsp70 from Trypanosoma brucei
- Authors: Burger, Adélle
- Date: 2014
- Subjects: African trypanosomiasis -- Research Heat shock proteins -- Research Trypanosoma brucei -- Research Mycobacterial diseases -- Research -- Africa Parasitic diseases -- Africa -- Prevention Parasites -- Physiology Developing countries -- Economic conditions
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4105 , http://hdl.handle.net/10962/d1011618
- Description: One of Africa’s neglected tropical diseases, African Trypanosomiasis, is not only fatal but also has a crippling impact on economic development. Heat shock proteins play a wide range of roles in the cell and they are required to assist the parasite as it moves from a cold blooded insect vector to a warm blooded mammalian host. The expression of heat shock proteins increases during these heat shock conditions, and this is considered to play a role in differentiation of these vector-borne parasites. Heat shock protein 70 (Hsp70) is an important molecular chaperone that is involved in protein homeostasis, Hsp40 acts as a co-chaperone and stimulates its intrinsically weak ATPase activity. In silico analysis of the T. brucei genome has revealed the existence of 12 Hsp70 proteins and 65 Hsp40 proteins to date. A novel Hsp70, TbHsp70.c, was recently identified in T. brucei. Different from the prototypical Hsp70, TbHsp70.c contains an acidic substrate binding domain and lacks the C-terminal EEVD motif. By implication the substrate range and mechanism by which the substrates are recognized may be novel. The ability of a Type I Hsp40, Tbj2, to function as a co-chaperone of TbHsp70.c was investigated. The main objective of this study was to biochemically characterize TbHsp70.c and its partnership with Tbj2 to further enhance our knowledge of parasite biology. TbHsp70.c and Tbj2 were heterologously expressed and purified and both proteins displayed chaperone activities in their ability to suppress aggregation of thermolabile MDH. TbHsp70.c also suppressed aggregation of rhodanese. ATPase assays revealed that the ATPase activity of TbHsp70.c was stimulated by Tbj2. The targeted inhibition of the function of heat shock proteins is emerging as a tool to combat disease. The small molecule modulators quercetin and methylene blue are known to inhibit the ATPase activity of Hsp70. However, methylene blue did not significantly inhibit the ATPase activity of TbHsp70.c; while quercetin, did inhibit the ATPase activity. In vivo heat stress experiments indicated an up-regulation of the expression levels of TbHsp70.c. RNA interference studies showed partial knockdown of TbHsp70.c with no detrimental effect on the parasite. Fluorescence microscopy studies of TbHsp70.c showed a probable cytoplasmic subcellular localization. In this study both TbHsp70.c and Tbj2 demonstrated chaperone activity and Tbj2 possibly functions as a co-chaperone of TbHsp70.c.
- Full Text:
- Date Issued: 2014
- Authors: Burger, Adélle
- Date: 2014
- Subjects: African trypanosomiasis -- Research Heat shock proteins -- Research Trypanosoma brucei -- Research Mycobacterial diseases -- Research -- Africa Parasitic diseases -- Africa -- Prevention Parasites -- Physiology Developing countries -- Economic conditions
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4105 , http://hdl.handle.net/10962/d1011618
- Description: One of Africa’s neglected tropical diseases, African Trypanosomiasis, is not only fatal but also has a crippling impact on economic development. Heat shock proteins play a wide range of roles in the cell and they are required to assist the parasite as it moves from a cold blooded insect vector to a warm blooded mammalian host. The expression of heat shock proteins increases during these heat shock conditions, and this is considered to play a role in differentiation of these vector-borne parasites. Heat shock protein 70 (Hsp70) is an important molecular chaperone that is involved in protein homeostasis, Hsp40 acts as a co-chaperone and stimulates its intrinsically weak ATPase activity. In silico analysis of the T. brucei genome has revealed the existence of 12 Hsp70 proteins and 65 Hsp40 proteins to date. A novel Hsp70, TbHsp70.c, was recently identified in T. brucei. Different from the prototypical Hsp70, TbHsp70.c contains an acidic substrate binding domain and lacks the C-terminal EEVD motif. By implication the substrate range and mechanism by which the substrates are recognized may be novel. The ability of a Type I Hsp40, Tbj2, to function as a co-chaperone of TbHsp70.c was investigated. The main objective of this study was to biochemically characterize TbHsp70.c and its partnership with Tbj2 to further enhance our knowledge of parasite biology. TbHsp70.c and Tbj2 were heterologously expressed and purified and both proteins displayed chaperone activities in their ability to suppress aggregation of thermolabile MDH. TbHsp70.c also suppressed aggregation of rhodanese. ATPase assays revealed that the ATPase activity of TbHsp70.c was stimulated by Tbj2. The targeted inhibition of the function of heat shock proteins is emerging as a tool to combat disease. The small molecule modulators quercetin and methylene blue are known to inhibit the ATPase activity of Hsp70. However, methylene blue did not significantly inhibit the ATPase activity of TbHsp70.c; while quercetin, did inhibit the ATPase activity. In vivo heat stress experiments indicated an up-regulation of the expression levels of TbHsp70.c. RNA interference studies showed partial knockdown of TbHsp70.c with no detrimental effect on the parasite. Fluorescence microscopy studies of TbHsp70.c showed a probable cytoplasmic subcellular localization. In this study both TbHsp70.c and Tbj2 demonstrated chaperone activity and Tbj2 possibly functions as a co-chaperone of TbHsp70.c.
- Full Text:
- Date Issued: 2014
The plasmodium falciparum exported Hsp40 co-chaperone, PFA0660w
- Authors: Daniyan, Michael Oluwatoyin
- Date: 2014
- Subjects: Molecular chaperones Heat shock proteins Proteins -- Analysis Proteins -- Structure Plasmodium Plasmodium falciparum Malaria -- Prevention -- Research
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4108 , http://hdl.handle.net/10962/d1011780
- Description: Plasmodium falciparum is the pathogen that is responsible for the most virulent, severe and dangerous form of human malaria infection, accounting for nearly a million deaths every year. To survive and develop in the unusual environment of the red blood cells, the parasite causes structural remodelling of the host cell and biochemical changes through the export of virulence factors. Among the exportome are the molecular chaperones of the heat shock protein family, of which Hsp40s and Hsp70s are prominent. PF A0660w, a type II P. falciparum Hsp40, has been shown to be exported in complex with PfHsp70-x into the infected erythrocyte, suggesting possible functional interactions. However, the chaperone properties of PF A0660w and its interactions with proteins of parasite and human origin are yet to be investigated. Using a codon optimised coding region, PF A0660w was successfully expressed in E. coli M 15 [pREP4] cells. However, the expressed protein was largely deposited as insoluble pellet, and analysis of the pellets revealed a high percentage of PF A0660w, characteristic of inclusion body formation. PF A0660w was purified from inclusion bodies using additive enhanced solubilisation and refolding buffers followed by nickel affinity chromatography. SDS-PAGE and western analysis revealed that the purified protein was of high purity. Size exclusion chromatography showed that the protein existed as a monomer in solution and the secondary structure analysis using Fourier transformed infrared spectroscopy (FTIR) confirmed the success of the refolding approach. Its monomeric state suggests that PF A0660w may be functionally different from other Hsp40 that form dimers and that for PF A0660w, dimer formation may not be needed to maintain the stability of the protein in solution, but may occur in response to functional necessities during its interaction with partner Hsp70. PFA0660w was able to significantly stimulate the ATPase activity ofPfl-Isp70-x but not Pfl-Isp70-1 or human Hsp70 (HsHsp70), suggesting a specific functional interaction. Also, PF A0660w produced a dose dependent suppression of rhodanese aggregation and cooperated with Pfl-Isp70-1, PfHsp70-x and HsHsp70 to cause enhanced aggregation suppression. Its ability to independently suppress aggregation may help to maintain substrates in an unfolded conformation for eventual transfer to partner Hsp70s during refolding processes. Also, the in vivo characterisation using a PF A0660w peptide specific antibody confirmed that PF A0660w was exported into the cytosol of infected erythrocytes. Its lack of induction upon heat shock suggests that PF A0660w may not be involved in the response of the parasite to heat stress. Overall, this study has provided the first heterologous over-expression, purification and biochemical evidence for the possible functional role of PF A0660w, and has thereby provided the needed background for further exploration of this protein as a potential target for drug discovery.
- Full Text:
- Date Issued: 2014
- Authors: Daniyan, Michael Oluwatoyin
- Date: 2014
- Subjects: Molecular chaperones Heat shock proteins Proteins -- Analysis Proteins -- Structure Plasmodium Plasmodium falciparum Malaria -- Prevention -- Research
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4108 , http://hdl.handle.net/10962/d1011780
- Description: Plasmodium falciparum is the pathogen that is responsible for the most virulent, severe and dangerous form of human malaria infection, accounting for nearly a million deaths every year. To survive and develop in the unusual environment of the red blood cells, the parasite causes structural remodelling of the host cell and biochemical changes through the export of virulence factors. Among the exportome are the molecular chaperones of the heat shock protein family, of which Hsp40s and Hsp70s are prominent. PF A0660w, a type II P. falciparum Hsp40, has been shown to be exported in complex with PfHsp70-x into the infected erythrocyte, suggesting possible functional interactions. However, the chaperone properties of PF A0660w and its interactions with proteins of parasite and human origin are yet to be investigated. Using a codon optimised coding region, PF A0660w was successfully expressed in E. coli M 15 [pREP4] cells. However, the expressed protein was largely deposited as insoluble pellet, and analysis of the pellets revealed a high percentage of PF A0660w, characteristic of inclusion body formation. PF A0660w was purified from inclusion bodies using additive enhanced solubilisation and refolding buffers followed by nickel affinity chromatography. SDS-PAGE and western analysis revealed that the purified protein was of high purity. Size exclusion chromatography showed that the protein existed as a monomer in solution and the secondary structure analysis using Fourier transformed infrared spectroscopy (FTIR) confirmed the success of the refolding approach. Its monomeric state suggests that PF A0660w may be functionally different from other Hsp40 that form dimers and that for PF A0660w, dimer formation may not be needed to maintain the stability of the protein in solution, but may occur in response to functional necessities during its interaction with partner Hsp70. PFA0660w was able to significantly stimulate the ATPase activity ofPfl-Isp70-x but not Pfl-Isp70-1 or human Hsp70 (HsHsp70), suggesting a specific functional interaction. Also, PF A0660w produced a dose dependent suppression of rhodanese aggregation and cooperated with Pfl-Isp70-1, PfHsp70-x and HsHsp70 to cause enhanced aggregation suppression. Its ability to independently suppress aggregation may help to maintain substrates in an unfolded conformation for eventual transfer to partner Hsp70s during refolding processes. Also, the in vivo characterisation using a PF A0660w peptide specific antibody confirmed that PF A0660w was exported into the cytosol of infected erythrocytes. Its lack of induction upon heat shock suggests that PF A0660w may not be involved in the response of the parasite to heat stress. Overall, this study has provided the first heterologous over-expression, purification and biochemical evidence for the possible functional role of PF A0660w, and has thereby provided the needed background for further exploration of this protein as a potential target for drug discovery.
- Full Text:
- Date Issued: 2014
Investigation into the technical feasibility of biological treatment of precious metal refining wastewater
- Authors: Moore, Bronwyn Ann
- Date: 2013
- Subjects: Sewage -- Purification -- Biological treatment -- South Africa Sewage -- Purification -- Activated sludge process -- South Africa Water reuse -- South Africa Flotation -- South Africa Platinum mines and mining -- Waste disposal -- South Africa Platinum mines and mining -- Economic aspects -- South Africa Mine water -- Environmental aspects -- South Africa Platinum mines and mining -- Waste minimization -- South Africa
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3888 , http://hdl.handle.net/10962/d1002013
- Description: The hydrometallurgical refining of platinum group metals results in large volumes of liquid waste that requires suitable treatment before any disposal can be contemplated. The wastewater streams are characterized by extremes of pH, high inorganic ion content (such as chloride), significant residual metal loads and small amounts of entrained organic compounds. Historically these effluents were housed in evaporation reservoirs, however lack of space and growing water demands have led Anglo Platinum to consider treatment of these effluents. The aim of this study was to investigate whether biological wastewater treatment could produce water suitable for onsite reuse. Bench-scale activated sludge and anaerobic digestion for co-treatment of an acidic refinery waste stream with domestic wastewater were used to give preliminary data. Activated sludge showed better water treatment at lab scale in terms of removal efficiencies of ammonia (approximately 25%, cf. 20% in anaerobic digestion) and COD (70% cf. 43% in digestion) and greater robustness when biomass health was compared. Activated sludge was consequently selected for a pilot plant trial. The pilot plant was operated on-site and performed comparably with the bench-scale system, however challenges in the clarifier design led to losses of biomass and poor effluent quality (suspended solids washout). The pilot plant was unable to alter the pH of the feed, but a two week maturation period resulted in the pH increasing from 5.3 to 7.0. Tests on algal treatment as an alternative or follow-on unit operation to activated sludge showed it not to be a viable process. The activated sludge effluent was assessed for onsite reuse in flotation and it was found that there was no significant difference between its flotation performance and that of the process water currently used, indicating the effluent generated by the biological treatment system can be used successfully for flotation. Flotation is the method whereby minerals refining operations recover minerals of interest from ore through the addition of chemicals and aeration of the ore slurry. Target minerals adhere to the bubbles and can be removed from the process.
- Full Text:
- Date Issued: 2013
- Authors: Moore, Bronwyn Ann
- Date: 2013
- Subjects: Sewage -- Purification -- Biological treatment -- South Africa Sewage -- Purification -- Activated sludge process -- South Africa Water reuse -- South Africa Flotation -- South Africa Platinum mines and mining -- Waste disposal -- South Africa Platinum mines and mining -- Economic aspects -- South Africa Mine water -- Environmental aspects -- South Africa Platinum mines and mining -- Waste minimization -- South Africa
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3888 , http://hdl.handle.net/10962/d1002013
- Description: The hydrometallurgical refining of platinum group metals results in large volumes of liquid waste that requires suitable treatment before any disposal can be contemplated. The wastewater streams are characterized by extremes of pH, high inorganic ion content (such as chloride), significant residual metal loads and small amounts of entrained organic compounds. Historically these effluents were housed in evaporation reservoirs, however lack of space and growing water demands have led Anglo Platinum to consider treatment of these effluents. The aim of this study was to investigate whether biological wastewater treatment could produce water suitable for onsite reuse. Bench-scale activated sludge and anaerobic digestion for co-treatment of an acidic refinery waste stream with domestic wastewater were used to give preliminary data. Activated sludge showed better water treatment at lab scale in terms of removal efficiencies of ammonia (approximately 25%, cf. 20% in anaerobic digestion) and COD (70% cf. 43% in digestion) and greater robustness when biomass health was compared. Activated sludge was consequently selected for a pilot plant trial. The pilot plant was operated on-site and performed comparably with the bench-scale system, however challenges in the clarifier design led to losses of biomass and poor effluent quality (suspended solids washout). The pilot plant was unable to alter the pH of the feed, but a two week maturation period resulted in the pH increasing from 5.3 to 7.0. Tests on algal treatment as an alternative or follow-on unit operation to activated sludge showed it not to be a viable process. The activated sludge effluent was assessed for onsite reuse in flotation and it was found that there was no significant difference between its flotation performance and that of the process water currently used, indicating the effluent generated by the biological treatment system can be used successfully for flotation. Flotation is the method whereby minerals refining operations recover minerals of interest from ore through the addition of chemicals and aeration of the ore slurry. Target minerals adhere to the bubbles and can be removed from the process.
- Full Text:
- Date Issued: 2013
Modulation of Plasmodium falciparum chaperones PfHsp70-1 and PfHsp70-x by small molecules
- Authors: Cockburn, Ingrid Louise
- Date: 2013
- Subjects: Plasmodium falciparum Heat shock proteins Molecular chaperones Homeostasis Protein folding Malaria Antimalarials Escherichia coli
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3887 , http://hdl.handle.net/10962/d1001747
- Description: The heat shock proteins of ~ 70 kDa (Hsp70s) are a conserved group of molecular chaperones important in maintaining the protein homeostasis in cells, carrying out functions including refolding of misfolded or unfolded proteins. Hsp70s function in conjunction with a number of other proteins including Hsp40 cochaperones. Central to the regulation Hsp70 activity is the Hsp70 ATPase cycle, involving ATP hydrolysis by Hsp70, and stimulation of this ATP hydrolysis by Hsp40. PfHsp70-1, the major cytosolic Hsp70 in the malaria parasite, Plasmodium falciparum, and PfHsp70-x, a novel malarial Hsp70 recently found to be exported to the host cell cytosol during the erythrocytic stages of the P. falciparum lifecycle, are both thought to play important roles in the malaria parasite’s survival and virulence, and thus represent novel antimalarial targets. Modulation of the function of these proteins by small molecules could thus lead to the development of antimalarials with novel targets and mechanisms. In the present study, malarial Hsp70s (PfHsp70-1 and PfHsp70-x), human Hsp70 (HSPA1A), malarial Hsp40 (PfHsp40) and human Hsp40 (Hsj1a) were recombinantly produced in Escherichia coli. In a characterisation of the chaperone activity of recombinant PfHsp70-x, the protein was found to have a basal ATPase activity (15.7 nmol ATP/min/mg protein) comparable to that previously described for PfHsp70-1, and an aggregation suppression activity significantly higher than that of PfHsp70-1. In vitro assays were used to screen five compounds of interest (lapachol, bromo-β-lapachona and malonganenones A, B and C) belonging to two compound classes (1,4 naphthoquinones and prenylated alkaloids) for modulatory effects on PfHsp70-1, PfHsp70-x and HsHsp70. A wide range of effects by compounds on the chaperone activities of Hsp70s was observed, including differential effects by compounds on different Hsp70s despite high conservation (≥ 70 % sequence identity) between the Hsp70s. The five compounds were shown to interact with all three Hsp70s in in vitro binding studies. Differential modulation by compounds was observed between the Hsj1a-stimulated ATPase activities of different Hsp70s, suggestive of not only a high degree of specificity of compounds to chaperone systems, but also distinct interactions between different Hsp70s and Hjs1a. The effects of compounds on the survival of P. falciparum parasites as well as mammalian cells was assessed. Bromo-β-lapachona was found to have broad effects across all systems, modulating the chaperone activities of all three Hsp70s, and showing significant toxicity toward both P. falciparum parasites and mammalian cells in culture. Malonganenone A was found to modulate only the malarial Hsp70s, not human Hsp70, showing significant toxicity toward malarial parasites (IC₅₀ ~ 0.8 μM), and comparatively low toxicity toward mammalian cells, representing therefore a novel starting point for a new class of antimalarials potentially targeting a new antimalarial drug target, Hsp70.
- Full Text:
- Date Issued: 2013
- Authors: Cockburn, Ingrid Louise
- Date: 2013
- Subjects: Plasmodium falciparum Heat shock proteins Molecular chaperones Homeostasis Protein folding Malaria Antimalarials Escherichia coli
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3887 , http://hdl.handle.net/10962/d1001747
- Description: The heat shock proteins of ~ 70 kDa (Hsp70s) are a conserved group of molecular chaperones important in maintaining the protein homeostasis in cells, carrying out functions including refolding of misfolded or unfolded proteins. Hsp70s function in conjunction with a number of other proteins including Hsp40 cochaperones. Central to the regulation Hsp70 activity is the Hsp70 ATPase cycle, involving ATP hydrolysis by Hsp70, and stimulation of this ATP hydrolysis by Hsp40. PfHsp70-1, the major cytosolic Hsp70 in the malaria parasite, Plasmodium falciparum, and PfHsp70-x, a novel malarial Hsp70 recently found to be exported to the host cell cytosol during the erythrocytic stages of the P. falciparum lifecycle, are both thought to play important roles in the malaria parasite’s survival and virulence, and thus represent novel antimalarial targets. Modulation of the function of these proteins by small molecules could thus lead to the development of antimalarials with novel targets and mechanisms. In the present study, malarial Hsp70s (PfHsp70-1 and PfHsp70-x), human Hsp70 (HSPA1A), malarial Hsp40 (PfHsp40) and human Hsp40 (Hsj1a) were recombinantly produced in Escherichia coli. In a characterisation of the chaperone activity of recombinant PfHsp70-x, the protein was found to have a basal ATPase activity (15.7 nmol ATP/min/mg protein) comparable to that previously described for PfHsp70-1, and an aggregation suppression activity significantly higher than that of PfHsp70-1. In vitro assays were used to screen five compounds of interest (lapachol, bromo-β-lapachona and malonganenones A, B and C) belonging to two compound classes (1,4 naphthoquinones and prenylated alkaloids) for modulatory effects on PfHsp70-1, PfHsp70-x and HsHsp70. A wide range of effects by compounds on the chaperone activities of Hsp70s was observed, including differential effects by compounds on different Hsp70s despite high conservation (≥ 70 % sequence identity) between the Hsp70s. The five compounds were shown to interact with all three Hsp70s in in vitro binding studies. Differential modulation by compounds was observed between the Hsj1a-stimulated ATPase activities of different Hsp70s, suggestive of not only a high degree of specificity of compounds to chaperone systems, but also distinct interactions between different Hsp70s and Hjs1a. The effects of compounds on the survival of P. falciparum parasites as well as mammalian cells was assessed. Bromo-β-lapachona was found to have broad effects across all systems, modulating the chaperone activities of all three Hsp70s, and showing significant toxicity toward both P. falciparum parasites and mammalian cells in culture. Malonganenone A was found to modulate only the malarial Hsp70s, not human Hsp70, showing significant toxicity toward malarial parasites (IC₅₀ ~ 0.8 μM), and comparatively low toxicity toward mammalian cells, representing therefore a novel starting point for a new class of antimalarials potentially targeting a new antimalarial drug target, Hsp70.
- Full Text:
- Date Issued: 2013
The screening and characterisation of compounds for modulators of heat shock protein (Hsp90) in a breast cancer cell model
- Authors: Moyo, Buhle
- Date: 2013 , 2013-07-18
- Subjects: Heat shock proteins Breast -- Cancer Breast -- Cancer -- Chemotherapy Breast -- Cancer -- Treatment Cancer cells Naphthoquinone PQQ (Biochemistry)
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4060 , http://hdl.handle.net/10962/d1004129
- Description: Breast cancer is a leading cause of cancer death in Africa. Hsp90 has been identified as a target for anti-cancer treatments as its inhibition results in the disruption and ubiquitin–proteasome degradation of activated oncoproteins. Currently, there are no US Food and Drug Administration approved Hsp90 inhibitor drugs and existing Hsp90 inhibitors such as geldanamycin and novobiocin are hepatotoxic and display a low affinity for Hsp90, respectively. Therefore, there is a need for the development of Hsp90 inhibitors with improved inhibitory properties. In this study twelve natural compounds bearing a quinone nucleus were screened and characterised for the modulation of Hsp90. The compounds analysed formed three series; the sargaquinoic acid (SQA), naphthoquinone, and pyrroloiminoquinone alkaloid series. Certain compounds exhibited half maximal inhibitory concentrations of between 3.32 μM and 12.4 μM, while others showed no antiproliferative activity at concentrations of up to 500 μM in the MDA-MB-231 breast adenocarcinoma cell line. Immunofluorescence and Western analyses indicated that the modulation of Hsp90 and partner proteins by SQA was more similar to that of novobiocin. Isothermal titration calorimetry analyses suggested that SQA interacted with Hsp90β with a low affinity, and saturation-transfer difference nuclear magnetic resonance confirmed that this interaction with Hsp90β occurred through the methyl moiety bound to 1, 4 benzoquinone of SQA. Pulldown assays indicated SQA disrupted the association between Hsp90 and Hop dose-dependently, more similarly to novobiocin. Immunofluorescence and Western analyses performed on naphthoquinone and pyrroloiminoquinone alkaloid compounds indicated modulation of Hsp90 and Hsp90 partner proteins by the compounds. Naphthoquinone compounds were prioritised for analysis for binding to Hsp90β over the pyrroloiminoquinone alkaloid compounds. Lapachol interacted with Hsp90β with a low affinity however; this interaction was thought to be too weak to disrupt the association of Hsp90 and Hop. The remaining naphthoquinone compounds showed no interaction with Hsp90β, thus allowing the determination of a preliminary structure-activity relationship for these compounds. To the best of our knowledge, this is the first study to describe a systematic subcellular analysis of the effects of geldanamycin and novobiocin in comparison to sargaquinoic acid and compounds of the naphthoquinone and pyrroloquinoline scaffold on Hsp90 and its partner proteins. , Microsoft� Word 2010 , Adobe Acrobat 9.54 Paper Capture Plug-in
- Full Text:
- Date Issued: 2013
- Authors: Moyo, Buhle
- Date: 2013 , 2013-07-18
- Subjects: Heat shock proteins Breast -- Cancer Breast -- Cancer -- Chemotherapy Breast -- Cancer -- Treatment Cancer cells Naphthoquinone PQQ (Biochemistry)
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4060 , http://hdl.handle.net/10962/d1004129
- Description: Breast cancer is a leading cause of cancer death in Africa. Hsp90 has been identified as a target for anti-cancer treatments as its inhibition results in the disruption and ubiquitin–proteasome degradation of activated oncoproteins. Currently, there are no US Food and Drug Administration approved Hsp90 inhibitor drugs and existing Hsp90 inhibitors such as geldanamycin and novobiocin are hepatotoxic and display a low affinity for Hsp90, respectively. Therefore, there is a need for the development of Hsp90 inhibitors with improved inhibitory properties. In this study twelve natural compounds bearing a quinone nucleus were screened and characterised for the modulation of Hsp90. The compounds analysed formed three series; the sargaquinoic acid (SQA), naphthoquinone, and pyrroloiminoquinone alkaloid series. Certain compounds exhibited half maximal inhibitory concentrations of between 3.32 μM and 12.4 μM, while others showed no antiproliferative activity at concentrations of up to 500 μM in the MDA-MB-231 breast adenocarcinoma cell line. Immunofluorescence and Western analyses indicated that the modulation of Hsp90 and partner proteins by SQA was more similar to that of novobiocin. Isothermal titration calorimetry analyses suggested that SQA interacted with Hsp90β with a low affinity, and saturation-transfer difference nuclear magnetic resonance confirmed that this interaction with Hsp90β occurred through the methyl moiety bound to 1, 4 benzoquinone of SQA. Pulldown assays indicated SQA disrupted the association between Hsp90 and Hop dose-dependently, more similarly to novobiocin. Immunofluorescence and Western analyses performed on naphthoquinone and pyrroloiminoquinone alkaloid compounds indicated modulation of Hsp90 and Hsp90 partner proteins by the compounds. Naphthoquinone compounds were prioritised for analysis for binding to Hsp90β over the pyrroloiminoquinone alkaloid compounds. Lapachol interacted with Hsp90β with a low affinity however; this interaction was thought to be too weak to disrupt the association of Hsp90 and Hop. The remaining naphthoquinone compounds showed no interaction with Hsp90β, thus allowing the determination of a preliminary structure-activity relationship for these compounds. To the best of our knowledge, this is the first study to describe a systematic subcellular analysis of the effects of geldanamycin and novobiocin in comparison to sargaquinoic acid and compounds of the naphthoquinone and pyrroloquinoline scaffold on Hsp90 and its partner proteins. , Microsoft� Word 2010 , Adobe Acrobat 9.54 Paper Capture Plug-in
- Full Text:
- Date Issued: 2013
Biological synthesis of metallic nanoparticles and their interactions with various biomedical targets
- Authors: Sennuga, Afolake Temitope
- Date: 2012
- Subjects: Nanoparticles Biosynthesis Nanotechnology Biomineralization Morphology Ceruloplasmin Ribonucleases Adenosine triphosphatase Acetylcholinesterase Platinum Gold Silver
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4009 , http://hdl.handle.net/10962/d1004069
- Description: The synthesis of nanostructured materials, especially metallic nanoparticles, has accrued utmost interest over the past decade owing to their unique properties that make them applicable in different fields of science and technology. The limitation to the use of these nanoparticles is the paucity of an effective method of synthesis that will produce homogeneous size and shape nanoparticles as well as particles with limited or no toxicity to the human health and the environment. The biological method of nanoparticle synthesis is a relatively simple, cheap and environmentally friendly method than the conventional chemical method of synthesis and thus gains an upper hand. The biomineralization of nanoparticles in protein cages is one of such biological approaches used in the generation of nanoparticles. This method of synthesis apart from being a safer method in the production of nanoparticles is also able to control particle morphology. In this study, a comparative biological synthesis, characterization and biomedical effects of metallic nanoparticles of platinum, gold and silver were investigated. Metallic nanoparticles were biologically synthesized using cage-like (apoferritin), barrel-like (GroEL) and non-caged (ribonuclease) proteins. Nanoparticles generated were characterized using common techniques such as UV-visible spectroscopy, scanning and transmission electron microscopy, inductively coupled optical emission spectroscopy, Fourier transform infra-red spectroscopy and energy dispersion analysis of X-rays (EDAX). Nanoparticles synthesised biologically using apoferritin, GroEL and RNase with exhibited similar chemical and physical properties as thoses nanoparticles generated chemically. In addition, the metallic nanoparticles fabricated within the cage-like and barrel-like cavities of apoferritin and GroEL respectively, resulted in nanoparticles with relatively uniform morphology as opposed to those obtained with the non-caged ribonuclease. The enzymatic (ferroxidase) activity of apoferritin was found to be greatly enhanced with platinum (9-fold), gold (7-fold) and silver (54-fold) nanoparticles. The ATPase activity of GroEL was inhibited by silver nanoparticles (64%), was moderately activated by gold nanoparticles (47%) and considerably enhanced by platinum nanoparticles (85%). The hydrolytic activity of RNase was however, lowered by these metallic nanoparticles (90% in Ag nanoparticles) and to a higher degree with platinum (95%) and gold nanoparticles (~100%). The effect of synthesized nanoparticles on the respective enzyme activities of these proteins was also investigated and the potential neurotoxic property of these particles was also determined by an in vitro interaction with acetylcholinesterase. Protein encapsulated nanoparticles with apoferrtin and GroEL showed a decreased inhibition of acetylcholinesterase (<50%) compared with nanoparticles attached to ribonuclease (>50%). Thus, it can be concluded that the cavities of apoferitin and GroEL acted as nanobiofactories for the synthesis and confinement of the size and shape of nanoparticles. Furthermore, the interior of these proteins provided a shielding effect for these nanoparticles and thus reduced/prevented their possible neurotoxic effect and confirmed safety in their method of production and application. The findings from this study would prove beneficial in the application of these nanoparticles as a potential drug/drug delivery vehicle for the prevention, treatment/management of diseases associated with these enzymes/proteins.
- Full Text:
- Date Issued: 2012
- Authors: Sennuga, Afolake Temitope
- Date: 2012
- Subjects: Nanoparticles Biosynthesis Nanotechnology Biomineralization Morphology Ceruloplasmin Ribonucleases Adenosine triphosphatase Acetylcholinesterase Platinum Gold Silver
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4009 , http://hdl.handle.net/10962/d1004069
- Description: The synthesis of nanostructured materials, especially metallic nanoparticles, has accrued utmost interest over the past decade owing to their unique properties that make them applicable in different fields of science and technology. The limitation to the use of these nanoparticles is the paucity of an effective method of synthesis that will produce homogeneous size and shape nanoparticles as well as particles with limited or no toxicity to the human health and the environment. The biological method of nanoparticle synthesis is a relatively simple, cheap and environmentally friendly method than the conventional chemical method of synthesis and thus gains an upper hand. The biomineralization of nanoparticles in protein cages is one of such biological approaches used in the generation of nanoparticles. This method of synthesis apart from being a safer method in the production of nanoparticles is also able to control particle morphology. In this study, a comparative biological synthesis, characterization and biomedical effects of metallic nanoparticles of platinum, gold and silver were investigated. Metallic nanoparticles were biologically synthesized using cage-like (apoferritin), barrel-like (GroEL) and non-caged (ribonuclease) proteins. Nanoparticles generated were characterized using common techniques such as UV-visible spectroscopy, scanning and transmission electron microscopy, inductively coupled optical emission spectroscopy, Fourier transform infra-red spectroscopy and energy dispersion analysis of X-rays (EDAX). Nanoparticles synthesised biologically using apoferritin, GroEL and RNase with exhibited similar chemical and physical properties as thoses nanoparticles generated chemically. In addition, the metallic nanoparticles fabricated within the cage-like and barrel-like cavities of apoferritin and GroEL respectively, resulted in nanoparticles with relatively uniform morphology as opposed to those obtained with the non-caged ribonuclease. The enzymatic (ferroxidase) activity of apoferritin was found to be greatly enhanced with platinum (9-fold), gold (7-fold) and silver (54-fold) nanoparticles. The ATPase activity of GroEL was inhibited by silver nanoparticles (64%), was moderately activated by gold nanoparticles (47%) and considerably enhanced by platinum nanoparticles (85%). The hydrolytic activity of RNase was however, lowered by these metallic nanoparticles (90% in Ag nanoparticles) and to a higher degree with platinum (95%) and gold nanoparticles (~100%). The effect of synthesized nanoparticles on the respective enzyme activities of these proteins was also investigated and the potential neurotoxic property of these particles was also determined by an in vitro interaction with acetylcholinesterase. Protein encapsulated nanoparticles with apoferrtin and GroEL showed a decreased inhibition of acetylcholinesterase (<50%) compared with nanoparticles attached to ribonuclease (>50%). Thus, it can be concluded that the cavities of apoferitin and GroEL acted as nanobiofactories for the synthesis and confinement of the size and shape of nanoparticles. Furthermore, the interior of these proteins provided a shielding effect for these nanoparticles and thus reduced/prevented their possible neurotoxic effect and confirmed safety in their method of production and application. The findings from this study would prove beneficial in the application of these nanoparticles as a potential drug/drug delivery vehicle for the prevention, treatment/management of diseases associated with these enzymes/proteins.
- Full Text:
- Date Issued: 2012
Identification of novel marine algal compounds with differential anti-cancer activity: towards a cancer stem-cell specific chemotherapy
- Authors: De la Mare, Jo-Anne
- Date: 2012
- Subjects: Breast -- Cancer , Stem cells -- Research , Chemotherapy , Algae -- Biotechnology
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4143 , http://hdl.handle.net/10962/d1016250
- Description: Breast cancer remains the leading cause of cancer-related death in women worldwide. Furthermore, it has been demonstrated that the treatment-resistant ER-PR-HER2/neu- sub-type is more common among women of African descent, necessitating the search for novel chemotherapies for this form of the disease. The secondary metabolites produced by marine algae represent a rich source of structurally unique compounds with chemotherapeutic potential, particularly in South Africa, whose oceans allegedly host 15 % of the total number of species in the world. Indeed, a recent study reported the isolation of a range of novel compounds from South African red and brown algae of the Plocamium, Portiera and Sargassum genera which displayed cytotoxicity against oesophageal cancer cells in vitro. The molecular mechanisms mediating this toxicity were unknown, as was the effect of these and similar compounds on metastatic ER-PR-HER2/neu- breast cancer cell lines or breast cancer stem cells. The current study aimed to address these questions by screening a library of twenty-two novel marine algal compounds for the ability to inhibit MDA-MB-231 and Hs578T breast cancer cells, while having no adverse effects on non-cancerous MCF12A breast epithelial cells. While twelve of these were toxic in the micromolar range against breast cancer cells, only the polyhalogenated monoterpenes RU004 and RU007, and the tetraprenylated quinone sargaquinoic acid (SQA) were identified as hit compounds based on the criteria that their cytotoxicity was specific to breast cancer and not healthy breast cells in vitro. On the other hand, the halogenated monoterpene RU015 was found to be highly toxic to both breast cancer and non-cancerous breast cell lines, while the halogenated monoterpene stereoisomers RU017 and RU018 were non-toxic to either of these cell lines. The mode of action of RU004, RU007, RU015 and SQA, together with the previously characterized carotenoid fucoxanthin (FXN), was assessed in terms of the type of cell death induced and the effect on cell cycle distribution of these compounds. Flow cytometric analysis of the extent of Hoescht 33342 and propidium iodide staining along with PARP cleavage studies suggested that SQA induced apoptosis in MDA-MB-231 cells. On the other hand, the highly toxic compound RU015 appeared to induce necrosis as evidenced by 50 kDa PARP cleavage product in MDA-MB-231 cells. The flow cytometry profiles of MDA-MB-231 and Hst578T cells treated with the hit compounds RU004 and RU007 were suggestive of the induction of apoptosis by these compounds. Cell cycle analysis by flow cytometry with propidium iodide staining revealed that both SQA and FXN induced G0-G1 arrest together with an increase in the apoptotic sub-G0 population, which agreed with previous reports in the literature. The molecular mechanism of action of SQA and FXN were further investigated by the identification of specific signal transducer molecules involved in mediating their anti-cancer activities. SQA was found to require the activity of numerous caspases, including caspase-3, -6, -8, -9, -10 and -13, for its cytotoxicity and was demonstrated to decrease the level of the antiapoptotic protein Bcl-2. On the other hand, FXN was shown to require caspase-1, -2, -3, -9 and - 10 for its toxicity. This, together with the ability to decrease the levels of Bcl-2, pointed to the involvement of the intrinsic pathway in particular in mediating the activity of FXN. The screening of algal compounds against non-cancerous breast epithelial cells carried out in this study, together with the investigation into their mechanisms of action, represent one of the few reports in which characterization of algal metabolites goes beyond the initial cytotoxicity assays. Finally, in order to assess the potential anti-cancer stem cell activity of the marine algal compounds, a subset of these was screened using a mammosphere assay technique developed in this study. The cancer stem cell (CSC) theory proposes that cancers arise from and are maintained by a specific subpopulation of cells able to undergo asymmetric cell division and termed CSCs. These CSCs are capable of anchorage-independent growth in serum-free culture conditions, such as those in the mammosphere assay. Using this assay, the novel halogenated monoterpene stereoisomers RU017 and RU018 were demonstrated to possess putative anti- CSC activity as evidenced by their ability to completely eliminate mammosphere formation in vitro. Furthermore, since RU017 and RU018 were non-toxic to both breast cancer and healthy breast cells, it appeared that the activity of the compounds was potentially specific to the CSCs. The results require further validation, but represent the first report of selective anti-CSC activity.
- Full Text:
- Date Issued: 2012
- Authors: De la Mare, Jo-Anne
- Date: 2012
- Subjects: Breast -- Cancer , Stem cells -- Research , Chemotherapy , Algae -- Biotechnology
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4143 , http://hdl.handle.net/10962/d1016250
- Description: Breast cancer remains the leading cause of cancer-related death in women worldwide. Furthermore, it has been demonstrated that the treatment-resistant ER-PR-HER2/neu- sub-type is more common among women of African descent, necessitating the search for novel chemotherapies for this form of the disease. The secondary metabolites produced by marine algae represent a rich source of structurally unique compounds with chemotherapeutic potential, particularly in South Africa, whose oceans allegedly host 15 % of the total number of species in the world. Indeed, a recent study reported the isolation of a range of novel compounds from South African red and brown algae of the Plocamium, Portiera and Sargassum genera which displayed cytotoxicity against oesophageal cancer cells in vitro. The molecular mechanisms mediating this toxicity were unknown, as was the effect of these and similar compounds on metastatic ER-PR-HER2/neu- breast cancer cell lines or breast cancer stem cells. The current study aimed to address these questions by screening a library of twenty-two novel marine algal compounds for the ability to inhibit MDA-MB-231 and Hs578T breast cancer cells, while having no adverse effects on non-cancerous MCF12A breast epithelial cells. While twelve of these were toxic in the micromolar range against breast cancer cells, only the polyhalogenated monoterpenes RU004 and RU007, and the tetraprenylated quinone sargaquinoic acid (SQA) were identified as hit compounds based on the criteria that their cytotoxicity was specific to breast cancer and not healthy breast cells in vitro. On the other hand, the halogenated monoterpene RU015 was found to be highly toxic to both breast cancer and non-cancerous breast cell lines, while the halogenated monoterpene stereoisomers RU017 and RU018 were non-toxic to either of these cell lines. The mode of action of RU004, RU007, RU015 and SQA, together with the previously characterized carotenoid fucoxanthin (FXN), was assessed in terms of the type of cell death induced and the effect on cell cycle distribution of these compounds. Flow cytometric analysis of the extent of Hoescht 33342 and propidium iodide staining along with PARP cleavage studies suggested that SQA induced apoptosis in MDA-MB-231 cells. On the other hand, the highly toxic compound RU015 appeared to induce necrosis as evidenced by 50 kDa PARP cleavage product in MDA-MB-231 cells. The flow cytometry profiles of MDA-MB-231 and Hst578T cells treated with the hit compounds RU004 and RU007 were suggestive of the induction of apoptosis by these compounds. Cell cycle analysis by flow cytometry with propidium iodide staining revealed that both SQA and FXN induced G0-G1 arrest together with an increase in the apoptotic sub-G0 population, which agreed with previous reports in the literature. The molecular mechanism of action of SQA and FXN were further investigated by the identification of specific signal transducer molecules involved in mediating their anti-cancer activities. SQA was found to require the activity of numerous caspases, including caspase-3, -6, -8, -9, -10 and -13, for its cytotoxicity and was demonstrated to decrease the level of the antiapoptotic protein Bcl-2. On the other hand, FXN was shown to require caspase-1, -2, -3, -9 and - 10 for its toxicity. This, together with the ability to decrease the levels of Bcl-2, pointed to the involvement of the intrinsic pathway in particular in mediating the activity of FXN. The screening of algal compounds against non-cancerous breast epithelial cells carried out in this study, together with the investigation into their mechanisms of action, represent one of the few reports in which characterization of algal metabolites goes beyond the initial cytotoxicity assays. Finally, in order to assess the potential anti-cancer stem cell activity of the marine algal compounds, a subset of these was screened using a mammosphere assay technique developed in this study. The cancer stem cell (CSC) theory proposes that cancers arise from and are maintained by a specific subpopulation of cells able to undergo asymmetric cell division and termed CSCs. These CSCs are capable of anchorage-independent growth in serum-free culture conditions, such as those in the mammosphere assay. Using this assay, the novel halogenated monoterpene stereoisomers RU017 and RU018 were demonstrated to possess putative anti- CSC activity as evidenced by their ability to completely eliminate mammosphere formation in vitro. Furthermore, since RU017 and RU018 were non-toxic to both breast cancer and healthy breast cells, it appeared that the activity of the compounds was potentially specific to the CSCs. The results require further validation, but represent the first report of selective anti-CSC activity.
- Full Text:
- Date Issued: 2012
An investigation into the replication biology of Helicoverpa armigera stunt virus
- Authors: Short, James Roswell
- Date: 2011
- Subjects: Helicoverpa armigera RNA viruses Viruses -- Reproduction Lepidoptera -- Viruses
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3967 , http://hdl.handle.net/10962/d1004026
- Description: Tetraviruses are a family of small non-enveloped positive sense RNA viruses that exclusively infect members of the order Lepidoptera. Their replication biology is poorly studied because, with the exception of Providence virus (PrV), tetraviruses are unable to replicate in tissue culture cells. The overall aim of the research described in this thesis was to develop a fundamental understanding of the replication of tetraviruses, focussing on the site of replication within host cells and in particular, the subcellular localisation of the viral replicase. Helicoverpa armigera stunt virus (HaSV, Genus: Omegatetravirus) was chosen for this study because it is the only tetravirus for which the cDNAs have been shown to be infectious. In the absence of tissue culture cell lines susceptible to HaSV infection, the approach was to use confocal fluorescence microscopy to examine the subcellular localisation of the HaSV replicase fused to enhanced green fluorescent protein (EGFP) in mammalian and insect tissue culture cells. The replicase (with EGFP fused at its C-terminus) localised to punctate structures throughout the cytoplasm of transfected HeLa and Sf9 cells. These structures were then shown – using live cell imaging and time lapse photography – to behave similarly to cellular endocytic organelles and fluorescence partially overlapped with membranes containing the late endosomal marker protein CD63. Biochemical fractionation of Sf9 cells expressing the replicase via a recombinant baculovirus (as well as transfected HeLa and Sf9 cells expressing EGFP-replicase fusion proteins) demonstrated that the replicase was strongly associated with detergentresistant membranes (DRMs) in these cells. Deletion analysis of the replicase coding sequence revealed two regions involved in the generation of the punctuate structures. Firstly, the C-terminal half of the replicase RNAdependant RNA polymerase domain was found to be essential for targeting and the tight association with DRMs while the second region, within the Nterminal 44 amino acids, enhanced localisation through a combination of secondary structural elements and sequence-specific functions. A comparative immunofluorescence study on PrV, which replicates as a persistent infection in an insect midgut cell line, showed that the PrV replicase also localised to punctate structures in the cytoplasm. Biochemical fractionation showed that the replicase was also strongly associated with DRMs. This thesis describes the development of new experimental systems for the study of tetravirus replication biology and the data lead to the conclusion that the HaSV replicase associates with DRMs derived from alternate endocytic pathway organelles.
- Full Text:
- Date Issued: 2011
- Authors: Short, James Roswell
- Date: 2011
- Subjects: Helicoverpa armigera RNA viruses Viruses -- Reproduction Lepidoptera -- Viruses
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3967 , http://hdl.handle.net/10962/d1004026
- Description: Tetraviruses are a family of small non-enveloped positive sense RNA viruses that exclusively infect members of the order Lepidoptera. Their replication biology is poorly studied because, with the exception of Providence virus (PrV), tetraviruses are unable to replicate in tissue culture cells. The overall aim of the research described in this thesis was to develop a fundamental understanding of the replication of tetraviruses, focussing on the site of replication within host cells and in particular, the subcellular localisation of the viral replicase. Helicoverpa armigera stunt virus (HaSV, Genus: Omegatetravirus) was chosen for this study because it is the only tetravirus for which the cDNAs have been shown to be infectious. In the absence of tissue culture cell lines susceptible to HaSV infection, the approach was to use confocal fluorescence microscopy to examine the subcellular localisation of the HaSV replicase fused to enhanced green fluorescent protein (EGFP) in mammalian and insect tissue culture cells. The replicase (with EGFP fused at its C-terminus) localised to punctate structures throughout the cytoplasm of transfected HeLa and Sf9 cells. These structures were then shown – using live cell imaging and time lapse photography – to behave similarly to cellular endocytic organelles and fluorescence partially overlapped with membranes containing the late endosomal marker protein CD63. Biochemical fractionation of Sf9 cells expressing the replicase via a recombinant baculovirus (as well as transfected HeLa and Sf9 cells expressing EGFP-replicase fusion proteins) demonstrated that the replicase was strongly associated with detergentresistant membranes (DRMs) in these cells. Deletion analysis of the replicase coding sequence revealed two regions involved in the generation of the punctuate structures. Firstly, the C-terminal half of the replicase RNAdependant RNA polymerase domain was found to be essential for targeting and the tight association with DRMs while the second region, within the Nterminal 44 amino acids, enhanced localisation through a combination of secondary structural elements and sequence-specific functions. A comparative immunofluorescence study on PrV, which replicates as a persistent infection in an insect midgut cell line, showed that the PrV replicase also localised to punctate structures in the cytoplasm. Biochemical fractionation showed that the replicase was also strongly associated with DRMs. This thesis describes the development of new experimental systems for the study of tetravirus replication biology and the data lead to the conclusion that the HaSV replicase associates with DRMs derived from alternate endocytic pathway organelles.
- Full Text:
- Date Issued: 2011
Effect of alkaline pre-treatments on the synergistic enzymatic hydrolysis of sugarcane (Saccharum officinarum) bagasse by Clostridium cellulovorans XynA, ManA and ArfA
- Authors: Beukes, Natasha
- Date: 2011
- Subjects: Sugarcane -- Biotechnology Lignocellulose -- Biotechnology Renewable energy sources Hydrolysis Enzymes
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3952 , http://hdl.handle.net/10962/d1004011
- Description: The continual increase in industrialization and global population has increased the dependency and demand on traditional fossil fuels for energy; however, there are limited amounts of fossil fuels available. The slow depletion of fossil fuels has sparked a fresh interest in renewable sources such as lignocellulose to produce a variety of biofuels, such as biogases (e.g. methane), bioethanol, biodiesel and a variety of other solvents and economically valuable by-products. Agricultural crop wastes produced in surplus are typically lignocellulosic in composition and thus partially recalcitrant to enzymatic degradation. The recalcitrant nature of plant biomass and the inability to obtain complete enzymatic hydrolysis has led to the establishment of various pre-treatment strategies. Alkaline pre-treatments increase the accessibility of the exposed surface to enzymatic hydrolysis through the removal of acetyl and uronic acid substituents on hemicellulose. Unlike the use of steam and acid pre-treatments, alkaline pre-treatments solubilize lignin and a small percentage of the hemicellulose, increasing enzyme accessibility and thus the hydrolysis of lignocellulose. The majority of Clostridium cellulovorans associated enzyme synergy studies have been devoted to an understanding of the cellulolytic and hemi-cellulolytic degradation of plant cell walls. However, little is known about the effect of various physical and chemical pre-treatments on the synergistic enzymatic degradation of plant biomass and possible depolymerization of plant cell walls. This study investigates the use of slake lime, sodium hydroxide and ammonium hydroxide to pre-treat sugarcane bagasse under mild conditions and elucidates potentially important synergistic associations between the C. cellulovorans enzymes for the enhanced degradation of lignocellulose. The primary aims of the study were addressed using of a variety of techniques. This included suitable vector constructs for the expression and purification of recombinant C. cellulovorans enzymes, identification of the effects of various pre-treatments on enzyme synergy, and identification of the resultant reducing sugars and phenolic compounds (released during the pre-treatment of the bagasse). This study also made use of physical and chemical pre-treatment methods, protein purification using affinity, high performance liquid and thin layer chromatography, mass spectrometry, sodium dodecyl sulphate and fluorophore-assisted polyacrylamide gel electrophoresis (FACE) , enzymatic degradation and synergy studies with various substrates indirectly using the 3, 4-dinitrosalicylic acid (DNS) reducing sugar assay. From this investigation, the following conclusions were made: alkaline pre-treatment successfully solublised, redistributed and removed lignin from the bagasse, increasing the digestibility of the substrates. In summary, the most effective pre-treatment employed 0.114 M ammonium hydroxide / gram bagasse at 70°C for 36 hours, followed by hydrolysis with an enzyme cocktail containing 25% ManA and 75% XynA. This increased the production of sugars approximately 13-fold. Analysis of the sugars produced by the synergistic hydrolysis of sugarcane bagasse (SCB) indicated the presence of xylose, indicating that the enzymes are potentially bifunctional under certain conditions. This study indicated that the use of mild pre-treatment conditions sufficiently removed a large portion of lignin without affecting the hemicellulose moiety of the SCB. This facilitated the potential use of the hemicellulose component for the production of valuable products (e.g. xylitol) in addition to the production of bioethanol. Thus, the potential use of additional components of holocellulose may generate an additional biotechnological benefit and allow a certain degree of flexibility in the biofuel industry, depending on consumer and industrial needs.
- Full Text:
- Date Issued: 2011
- Authors: Beukes, Natasha
- Date: 2011
- Subjects: Sugarcane -- Biotechnology Lignocellulose -- Biotechnology Renewable energy sources Hydrolysis Enzymes
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3952 , http://hdl.handle.net/10962/d1004011
- Description: The continual increase in industrialization and global population has increased the dependency and demand on traditional fossil fuels for energy; however, there are limited amounts of fossil fuels available. The slow depletion of fossil fuels has sparked a fresh interest in renewable sources such as lignocellulose to produce a variety of biofuels, such as biogases (e.g. methane), bioethanol, biodiesel and a variety of other solvents and economically valuable by-products. Agricultural crop wastes produced in surplus are typically lignocellulosic in composition and thus partially recalcitrant to enzymatic degradation. The recalcitrant nature of plant biomass and the inability to obtain complete enzymatic hydrolysis has led to the establishment of various pre-treatment strategies. Alkaline pre-treatments increase the accessibility of the exposed surface to enzymatic hydrolysis through the removal of acetyl and uronic acid substituents on hemicellulose. Unlike the use of steam and acid pre-treatments, alkaline pre-treatments solubilize lignin and a small percentage of the hemicellulose, increasing enzyme accessibility and thus the hydrolysis of lignocellulose. The majority of Clostridium cellulovorans associated enzyme synergy studies have been devoted to an understanding of the cellulolytic and hemi-cellulolytic degradation of plant cell walls. However, little is known about the effect of various physical and chemical pre-treatments on the synergistic enzymatic degradation of plant biomass and possible depolymerization of plant cell walls. This study investigates the use of slake lime, sodium hydroxide and ammonium hydroxide to pre-treat sugarcane bagasse under mild conditions and elucidates potentially important synergistic associations between the C. cellulovorans enzymes for the enhanced degradation of lignocellulose. The primary aims of the study were addressed using of a variety of techniques. This included suitable vector constructs for the expression and purification of recombinant C. cellulovorans enzymes, identification of the effects of various pre-treatments on enzyme synergy, and identification of the resultant reducing sugars and phenolic compounds (released during the pre-treatment of the bagasse). This study also made use of physical and chemical pre-treatment methods, protein purification using affinity, high performance liquid and thin layer chromatography, mass spectrometry, sodium dodecyl sulphate and fluorophore-assisted polyacrylamide gel electrophoresis (FACE) , enzymatic degradation and synergy studies with various substrates indirectly using the 3, 4-dinitrosalicylic acid (DNS) reducing sugar assay. From this investigation, the following conclusions were made: alkaline pre-treatment successfully solublised, redistributed and removed lignin from the bagasse, increasing the digestibility of the substrates. In summary, the most effective pre-treatment employed 0.114 M ammonium hydroxide / gram bagasse at 70°C for 36 hours, followed by hydrolysis with an enzyme cocktail containing 25% ManA and 75% XynA. This increased the production of sugars approximately 13-fold. Analysis of the sugars produced by the synergistic hydrolysis of sugarcane bagasse (SCB) indicated the presence of xylose, indicating that the enzymes are potentially bifunctional under certain conditions. This study indicated that the use of mild pre-treatment conditions sufficiently removed a large portion of lignin without affecting the hemicellulose moiety of the SCB. This facilitated the potential use of the hemicellulose component for the production of valuable products (e.g. xylitol) in addition to the production of bioethanol. Thus, the potential use of additional components of holocellulose may generate an additional biotechnological benefit and allow a certain degree of flexibility in the biofuel industry, depending on consumer and industrial needs.
- Full Text:
- Date Issued: 2011
Fundamental investigations into the factors affecting the response of laccase-based electrochemical biosensors
- Authors: Fogel, Ronen
- Date: 2011
- Subjects: Laccase Phenols Biosensors
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4073 , http://hdl.handle.net/10962/d1007166
- Description: Given their widespread effects and distribution in both natural and industrial environments, the monitoring of phenolic compounds is of considerable analytical interest. Electrochemical biosensor technologies, in particular those comprising laccase enzymes, afford many potential benefits to address this analytical need. However, several key factors affecting sensor response currently limit their applicability. This Thesis reports on the fabrication and optimisation of an electrochemical laccase-based biosensor towards the application of the monitoring of phenolic compounds. Selected factors considered to affect sensor response were investigated using the optimised biosensor. These included: electrochemical, biochemical and substrate-dependent factors, which were found to intersect in modulating biosensor response signals. Through the application of transducer-dependent and substrate-dependent parameters, the selective and simultaneous detection of a mixture of different phenolic analytes is successfully demonstrated. This Thesis also investigates the use of Quartz-Crystal Microbalance with Dissipation (QCM-D) technology, an analytical technique that measures physical parameters of thin-film structures, towards the successful monitoring of enzyme immobilisation strategies. These strategies are fundamental to the successful fabrication of biosensors, and the real-time monitoring of immobilised film formations is of considerable research interest. In the studies reported on in this Thesis, QCM-D technology was demonstrated to be an effective complementary technology in the prediction of film immobilisation techniques on the resultant biochemical kinetics of immobilised enzymes.
- Full Text:
- Date Issued: 2011
- Authors: Fogel, Ronen
- Date: 2011
- Subjects: Laccase Phenols Biosensors
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4073 , http://hdl.handle.net/10962/d1007166
- Description: Given their widespread effects and distribution in both natural and industrial environments, the monitoring of phenolic compounds is of considerable analytical interest. Electrochemical biosensor technologies, in particular those comprising laccase enzymes, afford many potential benefits to address this analytical need. However, several key factors affecting sensor response currently limit their applicability. This Thesis reports on the fabrication and optimisation of an electrochemical laccase-based biosensor towards the application of the monitoring of phenolic compounds. Selected factors considered to affect sensor response were investigated using the optimised biosensor. These included: electrochemical, biochemical and substrate-dependent factors, which were found to intersect in modulating biosensor response signals. Through the application of transducer-dependent and substrate-dependent parameters, the selective and simultaneous detection of a mixture of different phenolic analytes is successfully demonstrated. This Thesis also investigates the use of Quartz-Crystal Microbalance with Dissipation (QCM-D) technology, an analytical technique that measures physical parameters of thin-film structures, towards the successful monitoring of enzyme immobilisation strategies. These strategies are fundamental to the successful fabrication of biosensors, and the real-time monitoring of immobilised film formations is of considerable research interest. In the studies reported on in this Thesis, QCM-D technology was demonstrated to be an effective complementary technology in the prediction of film immobilisation techniques on the resultant biochemical kinetics of immobilised enzymes.
- Full Text:
- Date Issued: 2011
Nanomaterial modified electrodes : optimization of voltammetric sensors for pharmaceutical and industrial application
- Authors: Brimecombe, Rory Dennis
- Date: 2011
- Subjects: Voltammetry , Electrochemistry , Nanotubes , Nanostructured materials
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4101 , http://hdl.handle.net/10962/d1009721
- Description: Nanomaterials, in particular carbon nanotubes have been shown to exhibit favourable properties for the enhancement of electrochemical detection of target analytes in complex matrices. There is however scope for improvement in terms of the optimization thereof in electrochemical sensors surface modification. The aim of this thesis was to examine methods that would result in increased current response, lowered passivation and application of such modified surfaces with application to pharmaceutically and industrially relevant analytes. Current methods for enhancing the performance of carbon nanotubes include acid functionalization which not only increases the hydrophilicity of the nanotubes, and consequently their ability to provide stable (aqueous) suspensions, but also introduces electrochemically active sites. This particular approach is however not normalized in the literature. Over-exposure to acid treatment results in loss of structural integrity of the carbon nanotubes, and as such a fine balance exists between achieving these dual outcomes. Guided by high resolution scanning electron microscopy, atomic force microscopy, voltammetric and impedance studies, this thesis examined the role of the length of time of the acid functionalization process as well as the impact of activation of carbon nanotubes and fullerenes on electrochemical sensor performance. Based on desired charge transfer resistances, rate transfer coefficients and sensitivity towards redox probes the optimal length of acid functionalization for multiwalled carbon nanotubes was 9 hours and 4 hours for single-walled carbon nanotubes. Further improvements in the desired outcomes were achieved through electrochemical activation of the modified electrode surface by cycling in the presence of catechol, in a novel approach. By employing electrochemical impedance spectroscopy it was observed that catechol activation resulted in lowered charge transfer resistance, before and after activation, with functionalized multi-walled carbon nanotubes (9 hours) exhibiting the greatest decrease of 90 % and functionalized single-walled carbon nanotubes (4 hours), a 50 % decrease. Corresponding increases in the heterologous rate transfer coefficient showed a 770 % increase for functionalized multi-walled carbon nanotubes (9 hours), following catechol activation. Comparative observations for fullerenes following partial reduction in potassium hydroxide yielded a 30 % decrease in charge transfer resistance, with an increased heterologous rate transfer coefficient at a fullerene modified surface The performance of the nanomaterial modified electrodes was applied to the detection of wortmannin with applications in bioprocess control and in the pharmaceutical sector as well as to the detection and monitoring of the industrial dye Reactive red. Of particular relevance to these analytes was the assessment of the nanomaterial modified electrodes for enhanced stability, reproducibility, sensitivity and decreased passivation effects. In this study the first known account of wortmannin detection through electrochemical methods is reported. Voltammetric characterization of wortmannin revealed an irreversible cathodic process with a total number of 4 electrons and a diffusion coefficient of 1.19 x 10-7 cm².s⁻¹. At a functionalized multiwalled carbon nanotubes modified glassy carbon electrode a limit of detection of 0.128 nmol.cm⁻³ was obtained, and with limited surface passivation the detection scheme afforded pertinent analyses in biological media representing a substantial improvement over chromatographic detection methods. This study also provided the first account of the voltammetric detection of reactive red, competing favourably with traditional spectroscopic methods for monitoring biodegradation of this compound in real time.
- Full Text:
- Date Issued: 2011
- Authors: Brimecombe, Rory Dennis
- Date: 2011
- Subjects: Voltammetry , Electrochemistry , Nanotubes , Nanostructured materials
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4101 , http://hdl.handle.net/10962/d1009721
- Description: Nanomaterials, in particular carbon nanotubes have been shown to exhibit favourable properties for the enhancement of electrochemical detection of target analytes in complex matrices. There is however scope for improvement in terms of the optimization thereof in electrochemical sensors surface modification. The aim of this thesis was to examine methods that would result in increased current response, lowered passivation and application of such modified surfaces with application to pharmaceutically and industrially relevant analytes. Current methods for enhancing the performance of carbon nanotubes include acid functionalization which not only increases the hydrophilicity of the nanotubes, and consequently their ability to provide stable (aqueous) suspensions, but also introduces electrochemically active sites. This particular approach is however not normalized in the literature. Over-exposure to acid treatment results in loss of structural integrity of the carbon nanotubes, and as such a fine balance exists between achieving these dual outcomes. Guided by high resolution scanning electron microscopy, atomic force microscopy, voltammetric and impedance studies, this thesis examined the role of the length of time of the acid functionalization process as well as the impact of activation of carbon nanotubes and fullerenes on electrochemical sensor performance. Based on desired charge transfer resistances, rate transfer coefficients and sensitivity towards redox probes the optimal length of acid functionalization for multiwalled carbon nanotubes was 9 hours and 4 hours for single-walled carbon nanotubes. Further improvements in the desired outcomes were achieved through electrochemical activation of the modified electrode surface by cycling in the presence of catechol, in a novel approach. By employing electrochemical impedance spectroscopy it was observed that catechol activation resulted in lowered charge transfer resistance, before and after activation, with functionalized multi-walled carbon nanotubes (9 hours) exhibiting the greatest decrease of 90 % and functionalized single-walled carbon nanotubes (4 hours), a 50 % decrease. Corresponding increases in the heterologous rate transfer coefficient showed a 770 % increase for functionalized multi-walled carbon nanotubes (9 hours), following catechol activation. Comparative observations for fullerenes following partial reduction in potassium hydroxide yielded a 30 % decrease in charge transfer resistance, with an increased heterologous rate transfer coefficient at a fullerene modified surface The performance of the nanomaterial modified electrodes was applied to the detection of wortmannin with applications in bioprocess control and in the pharmaceutical sector as well as to the detection and monitoring of the industrial dye Reactive red. Of particular relevance to these analytes was the assessment of the nanomaterial modified electrodes for enhanced stability, reproducibility, sensitivity and decreased passivation effects. In this study the first known account of wortmannin detection through electrochemical methods is reported. Voltammetric characterization of wortmannin revealed an irreversible cathodic process with a total number of 4 electrons and a diffusion coefficient of 1.19 x 10-7 cm².s⁻¹. At a functionalized multiwalled carbon nanotubes modified glassy carbon electrode a limit of detection of 0.128 nmol.cm⁻³ was obtained, and with limited surface passivation the detection scheme afforded pertinent analyses in biological media representing a substantial improvement over chromatographic detection methods. This study also provided the first account of the voltammetric detection of reactive red, competing favourably with traditional spectroscopic methods for monitoring biodegradation of this compound in real time.
- Full Text:
- Date Issued: 2011
Structural and functional characterisation of the protein inhibitor of activated STAT3 (PIAS3)
- Authors: Mautsa, Nicodemus
- Date: 2011
- Subjects: Cytokines Immune response Proteins Cancer cells -- Growth -- Regulation
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3991 , http://hdl.handle.net/10962/d1004050
- Description: The signal transducer and activator of transcription (STAT) and protein inhibitor of STAT(PIAS) system represent an elegant regulatory mechanism of transcriptional control IN mammalian cytokine signalling. Abnormal activation of the system is associated with immune disorders and a large group of diverse tumours. PIAS3 is a multiple domain protein with distinct functions involved in regulation of cytokine-mediated gene activation pathways.Its over-expression significantly inhibits cell growth and renders cancer cells more sensitive to drugs. The objective of this study was to structurally and biochemically characterise the function of the PIAS3 protein using in silico, in vivo and in vitro analysis approaches.The conservation pattern of the PIAS protein family and critical conserved residues in the PINIT (Proline, Isoleucine, Asparagine, Isoleucine, Tyrosine) domain were identified. The PINIT domain model was generated based on the PINIT domain structure of yeast PIAS3 homologue Siz1 and structural determinants in the PIAS3-STAT3 interaction were evaluated.Guided by the in silico findings, in vivo analysis of the localisation of the PIAS3, mutantderivatives of PIAS3 (PIAS3-L97A, PIAS3-R99N, PIAS3-R99Q), PINIT and acidic domain was conducted. PIAS3 was completely localised in the nucleus while PIAS3 mutants appeared to exhibit diffuse cytoplasmic distribution. The PINIT domain was predominantly localised in the nucleus with some apparent perinuclear staining while the acidic domain exhibited a predominantly perinuclear staining pattern. Further analysis of the PINIT domain and the effect of the mutants on PIAS3-STAT3 interaction were assessed by in vitro analysis. Guided by in silico analysis, the PINIT domain and mutant derivatives of PINIT domain (PINIT-L97A, PINIT-R99N, and PINIT-R99Q) were heterologously expressed in Escherichia coli and subsequently purified using a combination of immobilized metal affinity and size exclusion based chromatography. The size and structural elements of the PINIT domain and its mutants were characterised. The 23 kDa PINIT domain was found to exist as a monomer in solution and its secondary structure was shown to consist of 66 % β-sheets by fourier transformed infrared spectroscopy consistent with the generated homology model.Using surface plasmonresonance spectroscopy (SPR) the PINIT domain was shown to bind to STAT3 in a specific concentration dependent manner. Recombinant PINIT-L97A,PINITR99N and PINIT-R99Q mutants, which exhibited similar structural integrity to the wildtype, were found to abrogate binding to STAT3. These findings suggest that these residues form part of a potential binding surface for stat3. In conclusion, this study has provided evidence that the PINIT domain is an important determinant of PIAS3 interaction with STAT3 and that the interaction is mediated by defined conserved residues directly involved in the PINITSTAT3 interaction.
- Full Text:
- Date Issued: 2011
- Authors: Mautsa, Nicodemus
- Date: 2011
- Subjects: Cytokines Immune response Proteins Cancer cells -- Growth -- Regulation
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3991 , http://hdl.handle.net/10962/d1004050
- Description: The signal transducer and activator of transcription (STAT) and protein inhibitor of STAT(PIAS) system represent an elegant regulatory mechanism of transcriptional control IN mammalian cytokine signalling. Abnormal activation of the system is associated with immune disorders and a large group of diverse tumours. PIAS3 is a multiple domain protein with distinct functions involved in regulation of cytokine-mediated gene activation pathways.Its over-expression significantly inhibits cell growth and renders cancer cells more sensitive to drugs. The objective of this study was to structurally and biochemically characterise the function of the PIAS3 protein using in silico, in vivo and in vitro analysis approaches.The conservation pattern of the PIAS protein family and critical conserved residues in the PINIT (Proline, Isoleucine, Asparagine, Isoleucine, Tyrosine) domain were identified. The PINIT domain model was generated based on the PINIT domain structure of yeast PIAS3 homologue Siz1 and structural determinants in the PIAS3-STAT3 interaction were evaluated.Guided by the in silico findings, in vivo analysis of the localisation of the PIAS3, mutantderivatives of PIAS3 (PIAS3-L97A, PIAS3-R99N, PIAS3-R99Q), PINIT and acidic domain was conducted. PIAS3 was completely localised in the nucleus while PIAS3 mutants appeared to exhibit diffuse cytoplasmic distribution. The PINIT domain was predominantly localised in the nucleus with some apparent perinuclear staining while the acidic domain exhibited a predominantly perinuclear staining pattern. Further analysis of the PINIT domain and the effect of the mutants on PIAS3-STAT3 interaction were assessed by in vitro analysis. Guided by in silico analysis, the PINIT domain and mutant derivatives of PINIT domain (PINIT-L97A, PINIT-R99N, and PINIT-R99Q) were heterologously expressed in Escherichia coli and subsequently purified using a combination of immobilized metal affinity and size exclusion based chromatography. The size and structural elements of the PINIT domain and its mutants were characterised. The 23 kDa PINIT domain was found to exist as a monomer in solution and its secondary structure was shown to consist of 66 % β-sheets by fourier transformed infrared spectroscopy consistent with the generated homology model.Using surface plasmonresonance spectroscopy (SPR) the PINIT domain was shown to bind to STAT3 in a specific concentration dependent manner. Recombinant PINIT-L97A,PINITR99N and PINIT-R99Q mutants, which exhibited similar structural integrity to the wildtype, were found to abrogate binding to STAT3. These findings suggest that these residues form part of a potential binding surface for stat3. In conclusion, this study has provided evidence that the PINIT domain is an important determinant of PIAS3 interaction with STAT3 and that the interaction is mediated by defined conserved residues directly involved in the PINITSTAT3 interaction.
- Full Text:
- Date Issued: 2011
The druggable antimalarial target 1-deoxy-D-xylulose-5-phosphate reductoisomerase: purfication, kinetic characterization and inhibition studies
- Authors: Goble, Jessica Leigh
- Date: 2011
- Subjects: Antimalarials -- Development Plasmodium falciparum Drug development Plasmodium falciparum -- Purification Plasmodium falciparum -- Inhibitors Enzyme kinetics Malaria -- Chemotherapy
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3949 , http://hdl.handle.net/10962/d1004008
- Description: Plasmodium falciparum 1–deoxy–D–xylulose–5 phosphatereductoisomerase (PfDXR) plays a role in isoprenoid biosynthesis in the malaria parasite and is absent in the human host, making this parasite enzyme an attractive target for antimalarial drug design. To characterize PfDXR, it is necessary to produce large quantities of the enzyme in a soluble and functional form. However, the over–production of malarial proteins in prokaryotic host systems often results in the formation of truncated proteins or insoluble protein aggregates. A heterologous expression system was developed for the production of active PfDXR using codon harmonization and tight control of expression in the presence of lac repressor. Yields of up to 2 mg/l of enzyme were reported using the optimised expression system, which is 8 to 10– fold greater than previously reported yields. The kinetic parameters Km, Vmax and kcat were determined for PfDXR; values reported in this study were consistent with those reported in the literature for other DXR enzymes. A three–dimensional model of the malarial drug target protein PfDXR was generated, and validated using structure–checking programs and protein docking studies. Structural and functional features unique to PfDXR were identified using the model and comparative sequence analyses with apicomplexan and non–apicomplexan DXR proteins. Residues Val44 and Asn45, essential for NADPH binding; and catalytic hatch residues Lys224 and Lys226, which are unique to the species of Plasmodium, were mutated to resemble those of E. coli DXR. Interestingly,these mutations resulted in significant reductions in substrate affinity, when compared to the unmutated PfDXR. Mutant enzymes PfDXR(VN43,44AG) and PfDXR(KK224,226NS) also demonstrated a decreased ability to turnover substrate by 4–fold and 2–fold respectively. This study indicates a difference in the role of the catalytic hatch of PfDXR with regards to the way in which it captures substrates. The study also highlights subtle differences in cofactor binding to PfDXR, compared with the well characterized EcDXR enzyme. The validated PfDXR model was also used to develop a novel efficient in silico screening method for potential tool compounds for use in the rational design of novel DXR inhibitors. Following in silico screening of 46 potential DXR inhibitors, a two–tiered in vitro screening approach was undertaken. DXR inhibition was assessed for the 46 novel compounds using an NADPH– ependant DXP enzyme inhibition assay and antimalarial potential was assessed using P.falciparum–infected erythrocyte growth assays. Select compounds were tested in human cells in order to determine whether they were toxic to the host. From the parallel in silico and in vitro drug screening, it was evident that only a single compound demonstrated reasonable potential binding to DXR (determined using in silico docking), inhibited DXR in vitro and inhibited P. falciparum growth, without being toxic to human cells. Its potential as a lead compound in antimalarial drug development is therefore feasible. Two outcomes were evident from this work. Firstly, analogues of known antimalarial natural products can be screened against malaria, which may then lead towards the rational design of novel compounds that are effective against a specific antimalarial drug target enzyme, such as PfDXR. Secondly, the rational design of novel compounds against a specific antimalarial drug target enzyme can be untaken by adopting a coupled in silico and in vitro approach to drug discovery.
- Full Text:
- Date Issued: 2011
- Authors: Goble, Jessica Leigh
- Date: 2011
- Subjects: Antimalarials -- Development Plasmodium falciparum Drug development Plasmodium falciparum -- Purification Plasmodium falciparum -- Inhibitors Enzyme kinetics Malaria -- Chemotherapy
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3949 , http://hdl.handle.net/10962/d1004008
- Description: Plasmodium falciparum 1–deoxy–D–xylulose–5 phosphatereductoisomerase (PfDXR) plays a role in isoprenoid biosynthesis in the malaria parasite and is absent in the human host, making this parasite enzyme an attractive target for antimalarial drug design. To characterize PfDXR, it is necessary to produce large quantities of the enzyme in a soluble and functional form. However, the over–production of malarial proteins in prokaryotic host systems often results in the formation of truncated proteins or insoluble protein aggregates. A heterologous expression system was developed for the production of active PfDXR using codon harmonization and tight control of expression in the presence of lac repressor. Yields of up to 2 mg/l of enzyme were reported using the optimised expression system, which is 8 to 10– fold greater than previously reported yields. The kinetic parameters Km, Vmax and kcat were determined for PfDXR; values reported in this study were consistent with those reported in the literature for other DXR enzymes. A three–dimensional model of the malarial drug target protein PfDXR was generated, and validated using structure–checking programs and protein docking studies. Structural and functional features unique to PfDXR were identified using the model and comparative sequence analyses with apicomplexan and non–apicomplexan DXR proteins. Residues Val44 and Asn45, essential for NADPH binding; and catalytic hatch residues Lys224 and Lys226, which are unique to the species of Plasmodium, were mutated to resemble those of E. coli DXR. Interestingly,these mutations resulted in significant reductions in substrate affinity, when compared to the unmutated PfDXR. Mutant enzymes PfDXR(VN43,44AG) and PfDXR(KK224,226NS) also demonstrated a decreased ability to turnover substrate by 4–fold and 2–fold respectively. This study indicates a difference in the role of the catalytic hatch of PfDXR with regards to the way in which it captures substrates. The study also highlights subtle differences in cofactor binding to PfDXR, compared with the well characterized EcDXR enzyme. The validated PfDXR model was also used to develop a novel efficient in silico screening method for potential tool compounds for use in the rational design of novel DXR inhibitors. Following in silico screening of 46 potential DXR inhibitors, a two–tiered in vitro screening approach was undertaken. DXR inhibition was assessed for the 46 novel compounds using an NADPH– ependant DXP enzyme inhibition assay and antimalarial potential was assessed using P.falciparum–infected erythrocyte growth assays. Select compounds were tested in human cells in order to determine whether they were toxic to the host. From the parallel in silico and in vitro drug screening, it was evident that only a single compound demonstrated reasonable potential binding to DXR (determined using in silico docking), inhibited DXR in vitro and inhibited P. falciparum growth, without being toxic to human cells. Its potential as a lead compound in antimalarial drug development is therefore feasible. Two outcomes were evident from this work. Firstly, analogues of known antimalarial natural products can be screened against malaria, which may then lead towards the rational design of novel compounds that are effective against a specific antimalarial drug target enzyme, such as PfDXR. Secondly, the rational design of novel compounds against a specific antimalarial drug target enzyme can be untaken by adopting a coupled in silico and in vitro approach to drug discovery.
- Full Text:
- Date Issued: 2011
Isolation and evolution of novel nucleoside phosphorylases
- Authors: Visser, Daniel Finsch
- Date: 2010
- Subjects: AIDS (Disease) -- Treatment -- Africa HIV Infections -- Treatment -- Africa AIDS (Disease) -- Patients -- Africa HIV-Positive persons -- Africa Antiretroviral agents Pyrimidine nucleotides
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3972 , http://hdl.handle.net/10962/d1004031
- Description: Approximately 33.4 million people are living with HIV/AIDS. Of those, 97% live in low and middle income countries, with 22.4 million in sub-Saharan Africa. Only 42% of the people who require anti-retrovirals (ARVs) in low to middle income countries are receiving anti-retroviral therapy (ART). There is a need to develop novel and cost effective methods for producing antiretroviral drugs. Stavudine and azidothymidine (AZT) were identified as potential targets because they could both be produced through a common intermediate – 5 methyluridine (5-MU). It has been established that the biocatalytic production of 5-methyluridine is possible through a reaction known as transglycosylation, in a process which has not previously been demonstrated as commercially viable. A selection of biocatalysts were expressed either in recombinant E. coli strains or in the wild type organisms, purified and then screened for their ability to produce 5-MU. A combination of Bacillus halodurans purine nucleoside phosphorylase 1 (BHPNP1) and E. coli uridine phosphorylase (EcUP) gave the highest 5-MU yield (80%). This result represents the first combination of free enzymes from different organisms, giving high yields of 5-MU under high substrate conditions. Both enzymes were purified and successfully characterised. The established pH optimum was pH 7.0 for both enzymes. Temperature optima and stability data for BHPNP1 (70 C and t1/2 at 60 C of 20.8 h) indicated that the biocatalytic step was operating within the capabilities of this enzyme and would operate well at elevated temperatures (up to 60 C). Conversely, the temperature optimum and stability data for EcUP (optimum of 40 C and t1/2 at 60 C of 9.9 h) indicated that the enzyme remained active at 40 C for the duration of a 25 h biotransformation, but at 60 C would only be operating at 20% of its optimum activity and would lose activity rapidly. BHPNP1 and EcUP were used in a bench scale (650 ml) transglycosylation for the production of 5-MU. A 5-MU yield of 79.1% was obtained at this scale with a reactor productivity of 1.37 g.l-1.h-1. Iterative saturation mutagenesis was used to rapidly evolve EcUP for improved thermostability. A moderately high throughput colorimetric method was developed for screening the mutants based on the release of p-nitrophenol upon phosphorolysis of a pyrimidine nucleoside analogue. By screening under 20 000 clones the mutant UPL8 was isolated. The mutant enzyme showed an optimum temperature of 60 C and improved stability at 60 C (t1/2 = 17.3 h). The increase in stability of UPL8 is due to only 2 mutations (Lys235Arg, Gln236Ala). These mutations may have caused an increase in stability due to interactions with other structural units in the protein, stabilization of the entrance to the binding pocket, or by decreasing the flexibility of the α-helix at the N-terminus. Transglycosylation experiments showed that the mutant enzyme UPL8 is a superior catalyst for the production of 5-MU. A 300% increase in reactor productivity was noted when free enzyme preparations of UPL8 was combined with BHPNP1 at 1.5% m.m-1 substrate loading. The high yield of 5-MU (75-80% mol.mol-1) was maintained at 9% m.m-1 substrate loading. A commercially viable productivity of 31 g.l-1.h-1 was thus realised. Further optimisation of the process could produce still higher productivities. Future work in directed evolution of nucleoside phosphorylases is envisaged for improved stability and enhanced substrate range for application to other commercially relevant transglycosylation reactions.
- Full Text:
- Date Issued: 2010
- Authors: Visser, Daniel Finsch
- Date: 2010
- Subjects: AIDS (Disease) -- Treatment -- Africa HIV Infections -- Treatment -- Africa AIDS (Disease) -- Patients -- Africa HIV-Positive persons -- Africa Antiretroviral agents Pyrimidine nucleotides
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3972 , http://hdl.handle.net/10962/d1004031
- Description: Approximately 33.4 million people are living with HIV/AIDS. Of those, 97% live in low and middle income countries, with 22.4 million in sub-Saharan Africa. Only 42% of the people who require anti-retrovirals (ARVs) in low to middle income countries are receiving anti-retroviral therapy (ART). There is a need to develop novel and cost effective methods for producing antiretroviral drugs. Stavudine and azidothymidine (AZT) were identified as potential targets because they could both be produced through a common intermediate – 5 methyluridine (5-MU). It has been established that the biocatalytic production of 5-methyluridine is possible through a reaction known as transglycosylation, in a process which has not previously been demonstrated as commercially viable. A selection of biocatalysts were expressed either in recombinant E. coli strains or in the wild type organisms, purified and then screened for their ability to produce 5-MU. A combination of Bacillus halodurans purine nucleoside phosphorylase 1 (BHPNP1) and E. coli uridine phosphorylase (EcUP) gave the highest 5-MU yield (80%). This result represents the first combination of free enzymes from different organisms, giving high yields of 5-MU under high substrate conditions. Both enzymes were purified and successfully characterised. The established pH optimum was pH 7.0 for both enzymes. Temperature optima and stability data for BHPNP1 (70 C and t1/2 at 60 C of 20.8 h) indicated that the biocatalytic step was operating within the capabilities of this enzyme and would operate well at elevated temperatures (up to 60 C). Conversely, the temperature optimum and stability data for EcUP (optimum of 40 C and t1/2 at 60 C of 9.9 h) indicated that the enzyme remained active at 40 C for the duration of a 25 h biotransformation, but at 60 C would only be operating at 20% of its optimum activity and would lose activity rapidly. BHPNP1 and EcUP were used in a bench scale (650 ml) transglycosylation for the production of 5-MU. A 5-MU yield of 79.1% was obtained at this scale with a reactor productivity of 1.37 g.l-1.h-1. Iterative saturation mutagenesis was used to rapidly evolve EcUP for improved thermostability. A moderately high throughput colorimetric method was developed for screening the mutants based on the release of p-nitrophenol upon phosphorolysis of a pyrimidine nucleoside analogue. By screening under 20 000 clones the mutant UPL8 was isolated. The mutant enzyme showed an optimum temperature of 60 C and improved stability at 60 C (t1/2 = 17.3 h). The increase in stability of UPL8 is due to only 2 mutations (Lys235Arg, Gln236Ala). These mutations may have caused an increase in stability due to interactions with other structural units in the protein, stabilization of the entrance to the binding pocket, or by decreasing the flexibility of the α-helix at the N-terminus. Transglycosylation experiments showed that the mutant enzyme UPL8 is a superior catalyst for the production of 5-MU. A 300% increase in reactor productivity was noted when free enzyme preparations of UPL8 was combined with BHPNP1 at 1.5% m.m-1 substrate loading. The high yield of 5-MU (75-80% mol.mol-1) was maintained at 9% m.m-1 substrate loading. A commercially viable productivity of 31 g.l-1.h-1 was thus realised. Further optimisation of the process could produce still higher productivities. Future work in directed evolution of nucleoside phosphorylases is envisaged for improved stability and enhanced substrate range for application to other commercially relevant transglycosylation reactions.
- Full Text:
- Date Issued: 2010
The biotechnology of hard coal utilization as a bioprocess substrate
- Mutambanengwe, Cecil Clifford Zvandada
- Authors: Mutambanengwe, Cecil Clifford Zvandada
- Date: 2010
- Subjects: Coal -- Biotechnology Acid mine drainage Coal mines and mining -- Environmental aspects
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3934 , http://hdl.handle.net/10962/d1003993
- Description: The development of coal biotechnology, using hard coal as a substrate, has been impeded by its low reactivity in biological processes. As a result, the more successful application studies have focused on lignitic soft coals. However, new studies have reported using biologically or geologically oxidized hard coal as a functional substrate option for bioprocess applications on a large scale. This study undertook a preliminary investigation into the feasibility of environmental applications of coal biotechnology using oxidized hard coal substrates in both anaerobic and aerobic processes with carbon dioxide, sulfate and oxygen as terminal electron acceptors. A preliminary characterization of the oxidized hard coal substrates was undertaken to determine and predict their viability and behavior as electron donors and carbon sources for environmental bioprocess applications of direct interest to the coal mining industry. Both biologically and geologically oxidized coal substrates showed loss of up to 17% and 52% carbon respectively and incorporation of oxygen ranging from 0.9 – 24%. The latter substrate showed greater loss of carbon and increased oxygenation. The biologically and geologically oxidized hard coal substrates were shown to partition readily into 23% and 32% organic humic acid, a 0.1% fulvic acid fraction and 65% and 59% inorganic and humin fractions respectively. These organic components were shown to be potentially available for biological consumption. In the unmodified hard coal substrate, partitioning was not observed and it did not perform as a functional substrate for any of the bioprocesses investigated. Where carbon dioxide was used as a terminal electron acceptor, methane production ranging from 9 – 26 mg CH4.g substrate-1 was demonstrated from both oxidized coal substrates. Geologically oxidized coal produced 30% more methane than biologically oxidized coal. Methane yields from the geologically oxidized coal in the presence and absence of a co-substrate were 5 – 13-fold higher than previous studies that used hard coal for methanogenesis. Based on these results, and that the development and optimization of the biological oxidation process is currently ongoing, further applications investigated in this study were undertaken using geologically oxidized coal. It was shown using pyrolysis gas chromatography mass spectrometry that the methanogenic system was dependent on the presence of an effective co-substrate supporting the breakdown of the complex organic structures within the oxidized hard coal substrate. Also that the accumulation of aromatic intermediate breakdown compounds predominantly including toluene, furfural, styrene and 2-methoxy vinyl phenol appeared to become inhibitory to both methanogenic and sulfidogenic reactions. This was shown to be a more likely cause of reactor failure rather than substrate exhaustion over time. Evidence of a reductive degradation pathway of the complex organic structures within the oxidized hard coal substrates was shown through the production, accumulation and utilization of volatile fatty acids including acetic, formic, propionic, butyric and valeric acids. Comparative analysis of the volatile fatty acids produced in this system showed that geologically oxidized coal produced 20% more of the volatile fatty acids profiled and double the total concentration compared to the biologically oxidized coal. The use of geologically oxidized hard coal as a functional substrate for biological sulfate reduction was demonstrated in the neutralization of a simulated acid mine drainage wastewater in both batch and continuous process operations. Results showed an increase in pH from pH 4.0 to ~ pH 8.0 with sulfide production rates of ~ 86 mgL-1.day-1 in the batch reactions, while the pH increased to pH 9.0 and sulfide production rates of up to 450 mgL-1.day-1 were measured in the continuous process studies using sand and coal up-flow packed bed reactors. Again, the requirement for an effective co-substrate was demonstrated with lactate shown to function as a true co-substrate in this system. However, a low cost alternative to lactate would need to emerge if the process was to function in large-scale commercial environmental treatment applications. In this regard, the aerobic growth and production of Neosartorya fischeri biomass (0.64 g.biomass.g SOC-1) was demonstrated using oxidized hard coal and glutamate as a co-substrate. Both can be produced from wastes generated on coal mines, with the fungal biomass generated in potentially large volumes. Preliminary demonstration of the use of the fungal biomass as a carbon and electron donor source for biological sulfate reduction was shown and thus that this could serve as an effective substrate for anaerobic environmental treatment processes. Based on these findings, an Integrated Coal Bioprocess model was proposed using oxidized hard coal as a substrate for environmental remediation applications on coal mines. In this approach, potential applications included methane recovery from waste coal, use of waste coal in the treatment of acid mine drainage waste waters and the recovery and use of humic acids in the rehabilitation of open cast mining soils. This study provided a first report demonstrating the use of biologically and geologically oxidized hard coals as bioprocess substrates in environmental bioremediation applications. It also provided an indication that follow-up bioengineering studies to investigate scaled-up applications of these findings would be warranted.
- Full Text:
- Date Issued: 2010
- Authors: Mutambanengwe, Cecil Clifford Zvandada
- Date: 2010
- Subjects: Coal -- Biotechnology Acid mine drainage Coal mines and mining -- Environmental aspects
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3934 , http://hdl.handle.net/10962/d1003993
- Description: The development of coal biotechnology, using hard coal as a substrate, has been impeded by its low reactivity in biological processes. As a result, the more successful application studies have focused on lignitic soft coals. However, new studies have reported using biologically or geologically oxidized hard coal as a functional substrate option for bioprocess applications on a large scale. This study undertook a preliminary investigation into the feasibility of environmental applications of coal biotechnology using oxidized hard coal substrates in both anaerobic and aerobic processes with carbon dioxide, sulfate and oxygen as terminal electron acceptors. A preliminary characterization of the oxidized hard coal substrates was undertaken to determine and predict their viability and behavior as electron donors and carbon sources for environmental bioprocess applications of direct interest to the coal mining industry. Both biologically and geologically oxidized coal substrates showed loss of up to 17% and 52% carbon respectively and incorporation of oxygen ranging from 0.9 – 24%. The latter substrate showed greater loss of carbon and increased oxygenation. The biologically and geologically oxidized hard coal substrates were shown to partition readily into 23% and 32% organic humic acid, a 0.1% fulvic acid fraction and 65% and 59% inorganic and humin fractions respectively. These organic components were shown to be potentially available for biological consumption. In the unmodified hard coal substrate, partitioning was not observed and it did not perform as a functional substrate for any of the bioprocesses investigated. Where carbon dioxide was used as a terminal electron acceptor, methane production ranging from 9 – 26 mg CH4.g substrate-1 was demonstrated from both oxidized coal substrates. Geologically oxidized coal produced 30% more methane than biologically oxidized coal. Methane yields from the geologically oxidized coal in the presence and absence of a co-substrate were 5 – 13-fold higher than previous studies that used hard coal for methanogenesis. Based on these results, and that the development and optimization of the biological oxidation process is currently ongoing, further applications investigated in this study were undertaken using geologically oxidized coal. It was shown using pyrolysis gas chromatography mass spectrometry that the methanogenic system was dependent on the presence of an effective co-substrate supporting the breakdown of the complex organic structures within the oxidized hard coal substrate. Also that the accumulation of aromatic intermediate breakdown compounds predominantly including toluene, furfural, styrene and 2-methoxy vinyl phenol appeared to become inhibitory to both methanogenic and sulfidogenic reactions. This was shown to be a more likely cause of reactor failure rather than substrate exhaustion over time. Evidence of a reductive degradation pathway of the complex organic structures within the oxidized hard coal substrates was shown through the production, accumulation and utilization of volatile fatty acids including acetic, formic, propionic, butyric and valeric acids. Comparative analysis of the volatile fatty acids produced in this system showed that geologically oxidized coal produced 20% more of the volatile fatty acids profiled and double the total concentration compared to the biologically oxidized coal. The use of geologically oxidized hard coal as a functional substrate for biological sulfate reduction was demonstrated in the neutralization of a simulated acid mine drainage wastewater in both batch and continuous process operations. Results showed an increase in pH from pH 4.0 to ~ pH 8.0 with sulfide production rates of ~ 86 mgL-1.day-1 in the batch reactions, while the pH increased to pH 9.0 and sulfide production rates of up to 450 mgL-1.day-1 were measured in the continuous process studies using sand and coal up-flow packed bed reactors. Again, the requirement for an effective co-substrate was demonstrated with lactate shown to function as a true co-substrate in this system. However, a low cost alternative to lactate would need to emerge if the process was to function in large-scale commercial environmental treatment applications. In this regard, the aerobic growth and production of Neosartorya fischeri biomass (0.64 g.biomass.g SOC-1) was demonstrated using oxidized hard coal and glutamate as a co-substrate. Both can be produced from wastes generated on coal mines, with the fungal biomass generated in potentially large volumes. Preliminary demonstration of the use of the fungal biomass as a carbon and electron donor source for biological sulfate reduction was shown and thus that this could serve as an effective substrate for anaerobic environmental treatment processes. Based on these findings, an Integrated Coal Bioprocess model was proposed using oxidized hard coal as a substrate for environmental remediation applications on coal mines. In this approach, potential applications included methane recovery from waste coal, use of waste coal in the treatment of acid mine drainage waste waters and the recovery and use of humic acids in the rehabilitation of open cast mining soils. This study provided a first report demonstrating the use of biologically and geologically oxidized hard coals as bioprocess substrates in environmental bioremediation applications. It also provided an indication that follow-up bioengineering studies to investigate scaled-up applications of these findings would be warranted.
- Full Text:
- Date Issued: 2010