Development of a hydantoin-hydrolysing biocatalyst for the production of optically pure amino acids using Agrobacterium tumefaciens strain RU-ORPN1
- Authors: Foster, Ingrid Margaret
- Date: 2004
- Subjects: Agrobacterium tumefaciens Amino acids Hydantoin Hydrolysis Enzymes
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3943 , http://hdl.handle.net/10962/d1004002
- Description: A calcium alginate bead-immobilised biocatalyst was developed utilising the D-hydantoinase and D-N-carbamoylase from a novel, mutant Agrobacterium tumefaciens strain RU-ORPN1. The growth conditions for the inducer-independent strain were optimised for production of hydantoinase and N-carbamoylase activities. Methods for the preparation of crude enzyme extracts were evaluated in terms of hydantoinase and N-carbamoylase activities produced. After comparison of the enzyme activities and stabilities in various extracts from fresh and frozen cells, sonication of frozen cells for 5 minutes was found to be the best method for the production of the enzyme extract. The optimal pH and temperature for the hydantoinase activity were pH 10 and 30°C, respectively, while pH 9 and 40°C were optimal for Ncarbamoylase activity. The hydantoinase activity was enhanced by the addition of Mg^(2+) ions to the enzyme extract and the N-carbamoylase was enhanced by the addition of Mg^(2+), Mn^(2+) or Zn^(2+) ions to the enzyme extract. The enzyme activities increased in the presence of ATP suggesting that the enzymes may be ATP-dependent. The addition of DTT and PMSF to the enzyme extract enhanced the hydantoinase activity but had no effect on the N-carbamoylase activity. The N-carbamoylase was unstable at 40°C and was almost completely inactivated after 24 hours incubation at this temperature. The hydantoinase and N-carbamoylase appeared to be insoluble. Various techniques were investigated for the solubilisation of the enzymes including various cell lysis methods, cell lysis at extremes of pH and ionic strength, addition of a reducing agent and protease inhibitors, and treatment with hydrolysing enzymes and detergents. Treatment with Triton X-100 was most effective for the solubilisation of the enzymes indicating that the enzymes were membrane-bound. Hydropathy and transmembrane prediction plots of the predicted amino acid sequences for two identified N-carbamoylase genes from A. tumefaciens RU-ORPN1 revealed possible transmembrane regions in the amino acid sequences, and thus supported the hypothesis that the enzymes were membrane-bound. Various methods were evaluated for the immobilisation of the enzymes in whole cells and enzyme extracts. Immobilisation of the enzyme extract in calcium alginate beads was found to be the best method in terms of enzyme activity retention and stability. The hydantoinase retained 55% activity while the N-carbamoylase exhibited a remarkable sevenfold increase in activity after immobilisation by this method. Furthermore, the hydantoinase activity increased after storage at 4°C for 21 days, while the N-carbamoylase retained 30% activity after this storage period. The calcium alginate bead-immobilised enzymes were further biochemically characterised and then applied in a bioreactor system for the production of D-hydroxyphenylglycine (D-HPG) from D,L-5-hydroxyphenylhydantoin (D,L-5-HPH). The pH and temperature optima for the immobilised hydantoinase were pH 7 and 50°C, respectively, while pH 8 and 40°C were optimal for the immobilised N-carbamoylase enzyme. The immobilised enzymes showed improved thermostability at 40°C in comparison to the free enzymes and retained high levels of activity after five repeated batch reactions. Low levels of conversion were obtained in a packed-bed bioreactor containing the A. tumefaciens RU-ORPN1 biocatalyst due to the low hydantoinase activity present in the strain, relative to N-carbamoylase. A novel, packed-bed bioreactor system was therefore developed for the production of D-HPG from D,L-5-HPH using the A. tumefaciens biocatalyst in combination with a Pseudomonas sp. biocatalyst having high hydantoinase activity. A conversion yield of 22 to 30% was achieved for the production of D-HPG from D,L-5-HPH over 5 days operation demonstrating that the hydantoin-hydrolysing enzymes from A. tumefaciens RU-ORPN1 could be stabilised by immobilisation and, in combination with a biocatalyst with high hydantoinase activity, could be applied to the fully enzymatic conversion of D,L-5-HPH to D-HPG.
- Full Text:
- Date Issued: 2004
- Authors: Foster, Ingrid Margaret
- Date: 2004
- Subjects: Agrobacterium tumefaciens Amino acids Hydantoin Hydrolysis Enzymes
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3943 , http://hdl.handle.net/10962/d1004002
- Description: A calcium alginate bead-immobilised biocatalyst was developed utilising the D-hydantoinase and D-N-carbamoylase from a novel, mutant Agrobacterium tumefaciens strain RU-ORPN1. The growth conditions for the inducer-independent strain were optimised for production of hydantoinase and N-carbamoylase activities. Methods for the preparation of crude enzyme extracts were evaluated in terms of hydantoinase and N-carbamoylase activities produced. After comparison of the enzyme activities and stabilities in various extracts from fresh and frozen cells, sonication of frozen cells for 5 minutes was found to be the best method for the production of the enzyme extract. The optimal pH and temperature for the hydantoinase activity were pH 10 and 30°C, respectively, while pH 9 and 40°C were optimal for Ncarbamoylase activity. The hydantoinase activity was enhanced by the addition of Mg^(2+) ions to the enzyme extract and the N-carbamoylase was enhanced by the addition of Mg^(2+), Mn^(2+) or Zn^(2+) ions to the enzyme extract. The enzyme activities increased in the presence of ATP suggesting that the enzymes may be ATP-dependent. The addition of DTT and PMSF to the enzyme extract enhanced the hydantoinase activity but had no effect on the N-carbamoylase activity. The N-carbamoylase was unstable at 40°C and was almost completely inactivated after 24 hours incubation at this temperature. The hydantoinase and N-carbamoylase appeared to be insoluble. Various techniques were investigated for the solubilisation of the enzymes including various cell lysis methods, cell lysis at extremes of pH and ionic strength, addition of a reducing agent and protease inhibitors, and treatment with hydrolysing enzymes and detergents. Treatment with Triton X-100 was most effective for the solubilisation of the enzymes indicating that the enzymes were membrane-bound. Hydropathy and transmembrane prediction plots of the predicted amino acid sequences for two identified N-carbamoylase genes from A. tumefaciens RU-ORPN1 revealed possible transmembrane regions in the amino acid sequences, and thus supported the hypothesis that the enzymes were membrane-bound. Various methods were evaluated for the immobilisation of the enzymes in whole cells and enzyme extracts. Immobilisation of the enzyme extract in calcium alginate beads was found to be the best method in terms of enzyme activity retention and stability. The hydantoinase retained 55% activity while the N-carbamoylase exhibited a remarkable sevenfold increase in activity after immobilisation by this method. Furthermore, the hydantoinase activity increased after storage at 4°C for 21 days, while the N-carbamoylase retained 30% activity after this storage period. The calcium alginate bead-immobilised enzymes were further biochemically characterised and then applied in a bioreactor system for the production of D-hydroxyphenylglycine (D-HPG) from D,L-5-hydroxyphenylhydantoin (D,L-5-HPH). The pH and temperature optima for the immobilised hydantoinase were pH 7 and 50°C, respectively, while pH 8 and 40°C were optimal for the immobilised N-carbamoylase enzyme. The immobilised enzymes showed improved thermostability at 40°C in comparison to the free enzymes and retained high levels of activity after five repeated batch reactions. Low levels of conversion were obtained in a packed-bed bioreactor containing the A. tumefaciens RU-ORPN1 biocatalyst due to the low hydantoinase activity present in the strain, relative to N-carbamoylase. A novel, packed-bed bioreactor system was therefore developed for the production of D-HPG from D,L-5-HPH using the A. tumefaciens biocatalyst in combination with a Pseudomonas sp. biocatalyst having high hydantoinase activity. A conversion yield of 22 to 30% was achieved for the production of D-HPG from D,L-5-HPH over 5 days operation demonstrating that the hydantoin-hydrolysing enzymes from A. tumefaciens RU-ORPN1 could be stabilised by immobilisation and, in combination with a biocatalyst with high hydantoinase activity, could be applied to the fully enzymatic conversion of D,L-5-HPH to D-HPG.
- Full Text:
- Date Issued: 2004
Integrated anaerobic/aerobic bioprocess environments and the biodegradation of complex hydrocarbon wastes
- Authors: Ehlers, George A C
- Date: 2004
- Subjects: Hydrocarbons -- Biodegradation Sewage -- Purification -- Anaerobic treatment Water -- Purification -- Biological treatment Anaerobic bacteria Aerobic bacteria
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4011 , http://hdl.handle.net/10962/d1004071
- Description: An investigation of the biodegradation of complex hydrocarbon wastes, with emphasis on chlorinated aromatic compounds, in an anaerobic/aerobic bioprocess environment was made. A reactor configuration was developed consisting of linked anaerobic and aerobic reactors which served as the model for a proposed bioremediation strategy targeting subterranean soil/sediment/aquifer chlorinated phenol-contaminated environments. Here oxygen is frequently limited and sulphate is readily available, as occurs especially in marine sediment and intertidal habitats. In the anaerobic system the successful transformation and mobilization of the model contaminant, 2,4,6-trichlorophenol, was shown to rely on reductive dechlorination by a sulphate-reducing dependent dechlororespiring co-culture. This was followed in the aerobic system by degradation of the pollutant and its metabolites, 2,4-dichlorophenol, 4-chlorophenol and phenol, by immobilized white-rot fungi.The strategy was initially investigated separately in laboratory bench- and intermediate scale reactors whereafter reactors were linked to simulate the integrated biodegradation strategy. The application of the fungal reactor to treat an actual waste stream by degrading complex mixtures of hydrocarbons in a waste oil recycling effluent was also investigated. The mineralization of phenol and 2,4,6-TCP by immobilized fungal cultures was studied in pinewood chip and foam glass bead-packed trickling reactors. The reactors were operated in sequencing batch format. Removal efficiency increased over time and elevated influent phenol and TCP (800 and 85 mg.L⁻¹) concentrations were degraded by > 98 % in 24 – 30 h batch cycles. Comparable performance between the packing materials was shown. Uptake by the packing was negligible and stripping of compounds induced by aeration had a minimal effect on biodegradation efficiency. Reactor performances are discussed in relation to sequencing batch operation and nutrient requirements necessary to sustain fungal activity in inert vs. organic material packed systems. It was shown that a co-culture consisting of sulphate-reducing and dechlororespiring bacteria established in fed-batch and soil flasks, as well as pine chip-packed fluidized bed reactors. Results showed reductive dechlorination of 2,4,6-TCP to be in strict dependence on the activity of the sulphate-reducing population, sulphate and lactate concentrations. Transformation to 2,4-DCP, 4-CP and phenol was enhanced in sulphate deficient conditions. Dechlororespiring activity was found to be dependent on the fermentative activity of sulphate-reducing bacteria, and the culture was also shown to mobilize and dechlorinate TCP in soils contaminated with the pollutant. Linking the systems achieved degradation of the compound by > 99 % through fungal mineralization of metabolites produced in the dechlororespiring stage of the system. pH correction to the anaerobic reactor was found to be necessary since acidic effluent from the fungal reactor inhibited sulphate reduction and dechlorination. The fungal reactor system was evaluated at intermediate-scale using a complex waste oil recycling effluent. Substantial COD reduction (> 96 % in 48 h batch cycles) and removal of specific effluent hydrocarbon components was shown in diluted, undiluted (COD > 37 g.L⁻¹) and 2,4,6-TCP-spiked effluents. Industrial application of the fungal reactor was evaluated in a 14 m³ pilot plant operated on-site at a waste oil processing plant.
- Full Text:
- Date Issued: 2004
- Authors: Ehlers, George A C
- Date: 2004
- Subjects: Hydrocarbons -- Biodegradation Sewage -- Purification -- Anaerobic treatment Water -- Purification -- Biological treatment Anaerobic bacteria Aerobic bacteria
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4011 , http://hdl.handle.net/10962/d1004071
- Description: An investigation of the biodegradation of complex hydrocarbon wastes, with emphasis on chlorinated aromatic compounds, in an anaerobic/aerobic bioprocess environment was made. A reactor configuration was developed consisting of linked anaerobic and aerobic reactors which served as the model for a proposed bioremediation strategy targeting subterranean soil/sediment/aquifer chlorinated phenol-contaminated environments. Here oxygen is frequently limited and sulphate is readily available, as occurs especially in marine sediment and intertidal habitats. In the anaerobic system the successful transformation and mobilization of the model contaminant, 2,4,6-trichlorophenol, was shown to rely on reductive dechlorination by a sulphate-reducing dependent dechlororespiring co-culture. This was followed in the aerobic system by degradation of the pollutant and its metabolites, 2,4-dichlorophenol, 4-chlorophenol and phenol, by immobilized white-rot fungi.The strategy was initially investigated separately in laboratory bench- and intermediate scale reactors whereafter reactors were linked to simulate the integrated biodegradation strategy. The application of the fungal reactor to treat an actual waste stream by degrading complex mixtures of hydrocarbons in a waste oil recycling effluent was also investigated. The mineralization of phenol and 2,4,6-TCP by immobilized fungal cultures was studied in pinewood chip and foam glass bead-packed trickling reactors. The reactors were operated in sequencing batch format. Removal efficiency increased over time and elevated influent phenol and TCP (800 and 85 mg.L⁻¹) concentrations were degraded by > 98 % in 24 – 30 h batch cycles. Comparable performance between the packing materials was shown. Uptake by the packing was negligible and stripping of compounds induced by aeration had a minimal effect on biodegradation efficiency. Reactor performances are discussed in relation to sequencing batch operation and nutrient requirements necessary to sustain fungal activity in inert vs. organic material packed systems. It was shown that a co-culture consisting of sulphate-reducing and dechlororespiring bacteria established in fed-batch and soil flasks, as well as pine chip-packed fluidized bed reactors. Results showed reductive dechlorination of 2,4,6-TCP to be in strict dependence on the activity of the sulphate-reducing population, sulphate and lactate concentrations. Transformation to 2,4-DCP, 4-CP and phenol was enhanced in sulphate deficient conditions. Dechlororespiring activity was found to be dependent on the fermentative activity of sulphate-reducing bacteria, and the culture was also shown to mobilize and dechlorinate TCP in soils contaminated with the pollutant. Linking the systems achieved degradation of the compound by > 99 % through fungal mineralization of metabolites produced in the dechlororespiring stage of the system. pH correction to the anaerobic reactor was found to be necessary since acidic effluent from the fungal reactor inhibited sulphate reduction and dechlorination. The fungal reactor system was evaluated at intermediate-scale using a complex waste oil recycling effluent. Substantial COD reduction (> 96 % in 48 h batch cycles) and removal of specific effluent hydrocarbon components was shown in diluted, undiluted (COD > 37 g.L⁻¹) and 2,4,6-TCP-spiked effluents. Industrial application of the fungal reactor was evaluated in a 14 m³ pilot plant operated on-site at a waste oil processing plant.
- Full Text:
- Date Issued: 2004
Over-expression, purification and biochemical characterization of DOXP reductoisomerase and the rational design of novel anti-malarial drugs
- Authors: Tanner, Delia Caroline
- Date: 2004
- Subjects: Malaria -- Prevention , Malaria -- Drug therapy , Antimalarials -- Therapeutic use , Proteins -- Purification , Amino acids -- Analysis
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3931 , http://hdl.handle.net/10962/d1003990 , Malaria -- Prevention , Malaria -- Drug therapy , Antimalarials -- Therapeutic use , Proteins -- Purification , Amino acids -- Analysis
- Description: Malaria poses the greatest threat of all parasites to human life. Current vaccines and efficacious drugs are available however their use is limited due to toxicity, emergence of drug resistance, and cost. The discovery of an alternative pathway of isoprenoid biosynthesis, the non-mevalonate pathway, within the malarial parasite has resulted in development of novel anti-malarial drugs. 1-Deoxy-D-xylulose-5-phosphate (DOXP) reductoisomerase, the second enzyme in this pathway, is responsible for the synthesis of 2-C-methyl-D-erythritol 4-phosphate (MEP) in an intramolecular rearrangement step followed by a reduction process involving NADPH as a hydrogen donor and divalent cations as co-factors. Fosmidomycin and FR900098 have been identified as inhibitors of DOXP reductoisomerase. However, they lack clinical efficacy. In this investigation recombinant DOXP reductoisomerase from Escherichia coli (EcDXR) and Plasmodium falciparum (pfDXR) were biochemically characterized as potential targets for inhibition. (His)6-EcDXR was successfully purified using nickel-chelate affinity chromatography with a specific activity of 1.77 μmoles/min/mg and Km value 282 μM. Utilizing multiple sequence alignment, previous structural data predictions and homology modeling approaches, critical active site amino acid residues were identified and their role in the catalytic activity investigated utilizing site-directed mutagenesis techniques. We have shown evidence that suggests that Trp212 and Met214 interact to maintain the active site architecture and hydrophobic interactions necessary for substrate binding, cofactor binding and enzyme activity. Replacement of Trp212 with Tyr, Phe, and Leu reduced specific activity relative to EcDXR. EcDXR(W212F) and EcDXR(W212Y) had an increased Km relative to EcDXR indicative of loss in affinity toward DOXP, whereas EcDXR(W212L) had a lower Km of ~8 μM indicative of increased affinity for DOXP. The W212L substitution possibly removed contacts necessary for full catalytic activity, but could be considered a non-disruptive substitution in that it maintained active site architecture sufficient for DOXP reductoisomerase activity. EcDXR(M214I) had 36-fold reduced enzyme activity relative to EcDXR, while its Km (~8 μM) was found to be lower than that of EcDXR. This suggested that the M214I substitution had maintained (perhaps improved) substrate and active site architecture, but may have perturbed interactions with NADPH. Rational drug design strategies and docking methods have been utilized in the development of furan derivatives as DOXP reductoisomerase inhibitors, and the synthesis of phosphorylated derivatives (5) and (6) has been achieved. Future inhibitor studies using these novel potential DOXP reductoisomerase inhibitors may lead to the development of effective anti-malarial drug candidates.
- Full Text:
- Date Issued: 2004
- Authors: Tanner, Delia Caroline
- Date: 2004
- Subjects: Malaria -- Prevention , Malaria -- Drug therapy , Antimalarials -- Therapeutic use , Proteins -- Purification , Amino acids -- Analysis
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3931 , http://hdl.handle.net/10962/d1003990 , Malaria -- Prevention , Malaria -- Drug therapy , Antimalarials -- Therapeutic use , Proteins -- Purification , Amino acids -- Analysis
- Description: Malaria poses the greatest threat of all parasites to human life. Current vaccines and efficacious drugs are available however their use is limited due to toxicity, emergence of drug resistance, and cost. The discovery of an alternative pathway of isoprenoid biosynthesis, the non-mevalonate pathway, within the malarial parasite has resulted in development of novel anti-malarial drugs. 1-Deoxy-D-xylulose-5-phosphate (DOXP) reductoisomerase, the second enzyme in this pathway, is responsible for the synthesis of 2-C-methyl-D-erythritol 4-phosphate (MEP) in an intramolecular rearrangement step followed by a reduction process involving NADPH as a hydrogen donor and divalent cations as co-factors. Fosmidomycin and FR900098 have been identified as inhibitors of DOXP reductoisomerase. However, they lack clinical efficacy. In this investigation recombinant DOXP reductoisomerase from Escherichia coli (EcDXR) and Plasmodium falciparum (pfDXR) were biochemically characterized as potential targets for inhibition. (His)6-EcDXR was successfully purified using nickel-chelate affinity chromatography with a specific activity of 1.77 μmoles/min/mg and Km value 282 μM. Utilizing multiple sequence alignment, previous structural data predictions and homology modeling approaches, critical active site amino acid residues were identified and their role in the catalytic activity investigated utilizing site-directed mutagenesis techniques. We have shown evidence that suggests that Trp212 and Met214 interact to maintain the active site architecture and hydrophobic interactions necessary for substrate binding, cofactor binding and enzyme activity. Replacement of Trp212 with Tyr, Phe, and Leu reduced specific activity relative to EcDXR. EcDXR(W212F) and EcDXR(W212Y) had an increased Km relative to EcDXR indicative of loss in affinity toward DOXP, whereas EcDXR(W212L) had a lower Km of ~8 μM indicative of increased affinity for DOXP. The W212L substitution possibly removed contacts necessary for full catalytic activity, but could be considered a non-disruptive substitution in that it maintained active site architecture sufficient for DOXP reductoisomerase activity. EcDXR(M214I) had 36-fold reduced enzyme activity relative to EcDXR, while its Km (~8 μM) was found to be lower than that of EcDXR. This suggested that the M214I substitution had maintained (perhaps improved) substrate and active site architecture, but may have perturbed interactions with NADPH. Rational drug design strategies and docking methods have been utilized in the development of furan derivatives as DOXP reductoisomerase inhibitors, and the synthesis of phosphorylated derivatives (5) and (6) has been achieved. Future inhibitor studies using these novel potential DOXP reductoisomerase inhibitors may lead to the development of effective anti-malarial drug candidates.
- Full Text:
- Date Issued: 2004
The enzymology of enhanced hydrolysis within the biosulphidogenic recycling sludge bed reactor (RSBR)
- Authors: Enongene, Godlove Nkwelle
- Date: 2004
- Subjects: Hydrolysis , Sewage sludge , Sewage -- Purification -- Anaerobic treatment , Water -- Purification -- Biological treatment
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4132 , http://hdl.handle.net/10962/d1015744
- Description: The hydrolysis of complex organic heteropolymers contained in municipal wastewater to simpler monomers by extracellular hydrolytic enzymes is generally considered the rate-limiting step of the biodegradation process. Previous studies of the Recycling Sludge Bed Reactor (RSBR) revealed that the hydrolysis of complex particulate organics, such as those contained in primary sludge (PS), was enhanced under anaerobic biosulphidogenic conditions. Although the mechanism was not fully understood, it appeared to involve the interaction of sulfide and sludge flocs. The current study was conducted using a 3500 ml laboratory-scale RSBR fed sieved PS at a loading rate of 0.5 kg COD/m³.day and an initial chemical oxygen demand (COD) to sulfate ratio (COD:SO₄) of 1:1. There was no significant accumulation of undigested sludge in the reactor over the 60-day experimental period and the quantity of SO₄ reduced indicated that the yield of soluble products from PS was at least as high as those reported previously for this system (> 50%). In the current study, the specific activities of a range of extracellular hydrolytic enzymes (L-alanine aminopeptidase, L-leucine aminopeptidase, arylsulphatase, α-glucosidase, β- glucosidase, protease and lipase) were monitored in a sulfide gradient within a biosulphidogenic RSBR. Data obtained indicated that the specific enzymatic activities increased with the depth of the RSBR and also correlated with a number of the physicochemical parameters including sulfide, alkalinity and sulfate. The activities of α- glucosidase and β-glucosidase were higher than that of the other enzymes studied. Lipase activity was relatively low and studies conducted on the enzyme-enzyme interaction using specific enzyme inhibitors indicated that lipases were probably being digested by the proteases. Further studies to determine the impact of sulfide on the enzymes, showed an increase in the enzyme activity with increasing sulfide concentration. Possible direct affects were investigated by looking for changes in the Michaelis constant (Km) and the maximal velocity (Vmax) of the crude enzymes with varying sulfide concentrations (250, 400 and 500 mg/l) using natural and synthetic substrates. The results showed no significant difference in both the Km and the Vmax for any of the hydrolytic enzymes except for the protease. The latter showed a statistically significant increase in the Km with increasing sulfide concentration. Although this indicated a direct interaction, this difference was not large enough to be of biochemical significance and was consequently not solely responsible for the enhanced hydrolysis observed in the RSBR. Investigation into the floc characteristics indicated that the biosulphidogenic RSBR flocs were generally small in size and became more dendritic with the depth of the RSBR. Based on the above data, the previously proposed descriptive models of enhanced hydrolysis of particulate organic matter in a biosulphidogenic RSBR has been revised. It is thought that the effect of sulfide on the hydrolysis step is primarily indirect and that the reduction in floc size and alteration of the floc shape to a more dendritic form is central to the success of the process.
- Full Text:
- Date Issued: 2004
- Authors: Enongene, Godlove Nkwelle
- Date: 2004
- Subjects: Hydrolysis , Sewage sludge , Sewage -- Purification -- Anaerobic treatment , Water -- Purification -- Biological treatment
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4132 , http://hdl.handle.net/10962/d1015744
- Description: The hydrolysis of complex organic heteropolymers contained in municipal wastewater to simpler monomers by extracellular hydrolytic enzymes is generally considered the rate-limiting step of the biodegradation process. Previous studies of the Recycling Sludge Bed Reactor (RSBR) revealed that the hydrolysis of complex particulate organics, such as those contained in primary sludge (PS), was enhanced under anaerobic biosulphidogenic conditions. Although the mechanism was not fully understood, it appeared to involve the interaction of sulfide and sludge flocs. The current study was conducted using a 3500 ml laboratory-scale RSBR fed sieved PS at a loading rate of 0.5 kg COD/m³.day and an initial chemical oxygen demand (COD) to sulfate ratio (COD:SO₄) of 1:1. There was no significant accumulation of undigested sludge in the reactor over the 60-day experimental period and the quantity of SO₄ reduced indicated that the yield of soluble products from PS was at least as high as those reported previously for this system (> 50%). In the current study, the specific activities of a range of extracellular hydrolytic enzymes (L-alanine aminopeptidase, L-leucine aminopeptidase, arylsulphatase, α-glucosidase, β- glucosidase, protease and lipase) were monitored in a sulfide gradient within a biosulphidogenic RSBR. Data obtained indicated that the specific enzymatic activities increased with the depth of the RSBR and also correlated with a number of the physicochemical parameters including sulfide, alkalinity and sulfate. The activities of α- glucosidase and β-glucosidase were higher than that of the other enzymes studied. Lipase activity was relatively low and studies conducted on the enzyme-enzyme interaction using specific enzyme inhibitors indicated that lipases were probably being digested by the proteases. Further studies to determine the impact of sulfide on the enzymes, showed an increase in the enzyme activity with increasing sulfide concentration. Possible direct affects were investigated by looking for changes in the Michaelis constant (Km) and the maximal velocity (Vmax) of the crude enzymes with varying sulfide concentrations (250, 400 and 500 mg/l) using natural and synthetic substrates. The results showed no significant difference in both the Km and the Vmax for any of the hydrolytic enzymes except for the protease. The latter showed a statistically significant increase in the Km with increasing sulfide concentration. Although this indicated a direct interaction, this difference was not large enough to be of biochemical significance and was consequently not solely responsible for the enhanced hydrolysis observed in the RSBR. Investigation into the floc characteristics indicated that the biosulphidogenic RSBR flocs were generally small in size and became more dendritic with the depth of the RSBR. Based on the above data, the previously proposed descriptive models of enhanced hydrolysis of particulate organic matter in a biosulphidogenic RSBR has been revised. It is thought that the effect of sulfide on the hydrolysis step is primarily indirect and that the reduction in floc size and alteration of the floc shape to a more dendritic form is central to the success of the process.
- Full Text:
- Date Issued: 2004
The enzymology of sludge solubilisation under biosulphidogenic conditions : isolation, characterisation and partial purification of endoglucanases
- Authors: Oyekola, Oluwaseun Oyekanmi
- Date: 2004
- Subjects: Sewage -- Purification -- Anaerobic treatment , Anaerobic bacteria , Sewage sludge , Hydrolysis
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3921 , http://hdl.handle.net/10962/d1003980 , Sewage -- Purification -- Anaerobic treatment , Anaerobic bacteria , Sewage sludge , Hydrolysis
- Description: Endoglucanases play an important function in cellulose hydrolysis and catalyse the initial attack on the polymer by randomly hydrolysing the β-1,4 glucosidic bonds within the amorphous regions of cellulose chains. Cellulolytic bacteria have been isolated and characterised from the sewage sludge and the activation of several hydrolytic enzymes under biosulphidogenic conditions of sewage hydrolysis has been reported. The aims of this study were to: identify, induce production, locate and isolate, characterise (physicochemical and kinetic) and purify endoglucanases from anaerobic biosulphidogenic sludge. The endoglucanase activities were shown to be associated with the pellet particulate matter and exhibited a pH optimum of 6 and temperature optimum of 50 °C. The enzymes were thermally more stable when immobilised to the floc matrix of the sludge than when they were released into the aqueous solution via sonication. For both immobilised and released enzymes, sulphate was slightly inhibitory; activity was reduced to 84 % and 77.5 % of the initial activity at sulphate concentrations between 200 and 1000 mg/l, respectively. Sulphite was stimulatory to the immobilised enzymes between 200 and 1000 mg/l. Sulphide stimulated the activities of the immobilised endoglucanases, but inhibited activities of the soluble enzymes above 200 mg/l. The enzyme fraction did not hydrolyse avicel (a crystalline substrate), indicating the absence of any exocellulase activity. For CMC (carboxymethylcellulose) and HEC (hydroxylethylcellulose) the enzyme had K_m,app_ values of 4 and 5.1 mg/ml respectively and V_max,app_ values of 0.297 and 0.185 μmol/min/ml respectively. Divalent ions (Cu²⁺, Ni²⁺ and Zn²⁺) proved to be inhibitory while Fe²⁺, Mg²⁺ and Ca²⁺ stimulated the enzyme at concentrations between 200 and 1000 mg/l. All the volatile fatty acids studied (acetic acid, butyric acid, propionic acid and valeric acid) inhibited the enzymes, with acetic acid eliciting the highest degree of inhibition. Sonication released ~74.9 % of the total enzyme activities into solution and this was partially purified by PEG 20 000 concentration followed by DEAE-Cellulose ion exchange chromatography, which resulted in an appreciable purity as measured by the purification factor, 25.4 fold.
- Full Text:
- Date Issued: 2004
- Authors: Oyekola, Oluwaseun Oyekanmi
- Date: 2004
- Subjects: Sewage -- Purification -- Anaerobic treatment , Anaerobic bacteria , Sewage sludge , Hydrolysis
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3921 , http://hdl.handle.net/10962/d1003980 , Sewage -- Purification -- Anaerobic treatment , Anaerobic bacteria , Sewage sludge , Hydrolysis
- Description: Endoglucanases play an important function in cellulose hydrolysis and catalyse the initial attack on the polymer by randomly hydrolysing the β-1,4 glucosidic bonds within the amorphous regions of cellulose chains. Cellulolytic bacteria have been isolated and characterised from the sewage sludge and the activation of several hydrolytic enzymes under biosulphidogenic conditions of sewage hydrolysis has been reported. The aims of this study were to: identify, induce production, locate and isolate, characterise (physicochemical and kinetic) and purify endoglucanases from anaerobic biosulphidogenic sludge. The endoglucanase activities were shown to be associated with the pellet particulate matter and exhibited a pH optimum of 6 and temperature optimum of 50 °C. The enzymes were thermally more stable when immobilised to the floc matrix of the sludge than when they were released into the aqueous solution via sonication. For both immobilised and released enzymes, sulphate was slightly inhibitory; activity was reduced to 84 % and 77.5 % of the initial activity at sulphate concentrations between 200 and 1000 mg/l, respectively. Sulphite was stimulatory to the immobilised enzymes between 200 and 1000 mg/l. Sulphide stimulated the activities of the immobilised endoglucanases, but inhibited activities of the soluble enzymes above 200 mg/l. The enzyme fraction did not hydrolyse avicel (a crystalline substrate), indicating the absence of any exocellulase activity. For CMC (carboxymethylcellulose) and HEC (hydroxylethylcellulose) the enzyme had K_m,app_ values of 4 and 5.1 mg/ml respectively and V_max,app_ values of 0.297 and 0.185 μmol/min/ml respectively. Divalent ions (Cu²⁺, Ni²⁺ and Zn²⁺) proved to be inhibitory while Fe²⁺, Mg²⁺ and Ca²⁺ stimulated the enzyme at concentrations between 200 and 1000 mg/l. All the volatile fatty acids studied (acetic acid, butyric acid, propionic acid and valeric acid) inhibited the enzymes, with acetic acid eliciting the highest degree of inhibition. Sonication released ~74.9 % of the total enzyme activities into solution and this was partially purified by PEG 20 000 concentration followed by DEAE-Cellulose ion exchange chromatography, which resulted in an appreciable purity as measured by the purification factor, 25.4 fold.
- Full Text:
- Date Issued: 2004
The toxic effect of heavy metals on algal biomass (Spirulina sp.) and carbonic anhydrase activity, an enzyme which is central to algal application in metal precipitation
- Authors: Nightingale, Leigh
- Date: 2004
- Subjects: Heavy metals -- Toxicology , Spirulina , Carbonic anhydrase , Algae -- Metabolism , Photosynthesis
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4093 , http://hdl.handle.net/10962/d1007858 , Heavy metals -- Toxicology , Spirulina , Carbonic anhydrase , Algae -- Metabolism , Photosynthesis
- Description: Acid rmne drainage (AMD) is a major pollution problem througbout the world, adversely affecting both surface and groundwaters. AMD is principally associated with the mining of sulphide ores. The most commonly associated minerals being sulphur, copper, zinc, silver, gold, lead and uranium. As conventional methods for removing heavy metals from wastewater are often prohibitively expensive, the implementation of biological processes for the removal of heavy metals has become a realistic practice. The objectives of this project was firstly to establish the effect of copper, lead and nickel, heavy metals commonly found in AMD waters, on the enzyme carbonic anhydrase, which is an integral part of the carbon concentrating mechanism (CCM) and secondly, to determine the feasibility of using the alkalinity generated by Spindina for the precipitation of heavy metals from solution. Initially, batch flask experiments were performed and it was found that the algae were able to utilise the bicarbonate supplied in the medium, under CO, limiting conditions, through the induction of their CCM, resulting in the generation of carbonate. The effect of the inhibitors, acetazolamide (AZ) and ethoxyzolamide (EZ), were also investigated in order to determine the importance of carbonic anhydrase (CA) in inorganic carbon accumulation and photosynthesis. Results obtained were consistent with those observed in literature and it was found that at IOOf.LM AZ and EZ, complete inhibition of photosynthesis and carbonic anhydrase occurred, with no oxygen being evolved. The results obtained from the inhibitor experiments substantiate the findings that carbonic anhydrase is an important part of the CCM, and that the dehydration of bicarbonate to carbon dioxide and hydroxide ions, is in fact an enzymatic process regulated by the enzyme carbonic anhydrase and is essential for efficient photosynthesis. The effect of heavy metals on Spirulina was also investigated. Lead, copper and nickel were all found to cause a reduction in the synthesis of chlorophyll a, which resulted in a decrease in photosynthetic efficiency and eventually death of the culture. The morphology of the algae was also severely affected by heavy metals, with degradation and aJmost complete disintegration of the algal filaments occurring. Using the Wilbur-Anderson assay method, carbonic anhydrase activity was found to be lower in the experimental flasks containing heavy metals, than the control flasks, reducing the algae's ability to utilise the bicarbonate in solution for effective photosynthesis. The Wilbur-Anderson assay method did not prove to be a reliable method for measuring changes in enzyme activity as results were found to be erratic. Therefore attempts were made to use an oxygen electrode as an alternative method for determining the effects of various parameters on enzyme activity and photosynthesis, this proved to be more successful. Because of the toxic effects of heavy metals on Spirulina it was decided that the use of the biogenic alkalinity generated by the algae for the precipitation of heavy metals may be successfully employed as an alternative method for bioremediation and metal recovery. Carbonate reacts readily with metals, therefore the carbonate produced by this algal system was used for the precipitation of metals. It was possible to categorise the precipitation reactions observed into three groups, namely those metals which, a) precipitate as hydroxides, b) precipitate as carbonates generated from the dissociation of bicarbonate and c) metals which can only precipitate if there is free carbonate present in solution.
- Full Text:
- Date Issued: 2004
- Authors: Nightingale, Leigh
- Date: 2004
- Subjects: Heavy metals -- Toxicology , Spirulina , Carbonic anhydrase , Algae -- Metabolism , Photosynthesis
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4093 , http://hdl.handle.net/10962/d1007858 , Heavy metals -- Toxicology , Spirulina , Carbonic anhydrase , Algae -- Metabolism , Photosynthesis
- Description: Acid rmne drainage (AMD) is a major pollution problem througbout the world, adversely affecting both surface and groundwaters. AMD is principally associated with the mining of sulphide ores. The most commonly associated minerals being sulphur, copper, zinc, silver, gold, lead and uranium. As conventional methods for removing heavy metals from wastewater are often prohibitively expensive, the implementation of biological processes for the removal of heavy metals has become a realistic practice. The objectives of this project was firstly to establish the effect of copper, lead and nickel, heavy metals commonly found in AMD waters, on the enzyme carbonic anhydrase, which is an integral part of the carbon concentrating mechanism (CCM) and secondly, to determine the feasibility of using the alkalinity generated by Spindina for the precipitation of heavy metals from solution. Initially, batch flask experiments were performed and it was found that the algae were able to utilise the bicarbonate supplied in the medium, under CO, limiting conditions, through the induction of their CCM, resulting in the generation of carbonate. The effect of the inhibitors, acetazolamide (AZ) and ethoxyzolamide (EZ), were also investigated in order to determine the importance of carbonic anhydrase (CA) in inorganic carbon accumulation and photosynthesis. Results obtained were consistent with those observed in literature and it was found that at IOOf.LM AZ and EZ, complete inhibition of photosynthesis and carbonic anhydrase occurred, with no oxygen being evolved. The results obtained from the inhibitor experiments substantiate the findings that carbonic anhydrase is an important part of the CCM, and that the dehydration of bicarbonate to carbon dioxide and hydroxide ions, is in fact an enzymatic process regulated by the enzyme carbonic anhydrase and is essential for efficient photosynthesis. The effect of heavy metals on Spirulina was also investigated. Lead, copper and nickel were all found to cause a reduction in the synthesis of chlorophyll a, which resulted in a decrease in photosynthetic efficiency and eventually death of the culture. The morphology of the algae was also severely affected by heavy metals, with degradation and aJmost complete disintegration of the algal filaments occurring. Using the Wilbur-Anderson assay method, carbonic anhydrase activity was found to be lower in the experimental flasks containing heavy metals, than the control flasks, reducing the algae's ability to utilise the bicarbonate in solution for effective photosynthesis. The Wilbur-Anderson assay method did not prove to be a reliable method for measuring changes in enzyme activity as results were found to be erratic. Therefore attempts were made to use an oxygen electrode as an alternative method for determining the effects of various parameters on enzyme activity and photosynthesis, this proved to be more successful. Because of the toxic effects of heavy metals on Spirulina it was decided that the use of the biogenic alkalinity generated by the algae for the precipitation of heavy metals may be successfully employed as an alternative method for bioremediation and metal recovery. Carbonate reacts readily with metals, therefore the carbonate produced by this algal system was used for the precipitation of metals. It was possible to categorise the precipitation reactions observed into three groups, namely those metals which, a) precipitate as hydroxides, b) precipitate as carbonates generated from the dissociation of bicarbonate and c) metals which can only precipitate if there is free carbonate present in solution.
- Full Text:
- Date Issued: 2004
An investigation into the biological treatment of platinum refinery effluent using the plant Azolla Filiculoides
- Authors: Marran, Vernon Edward
- Date: 2003
- Subjects: Uncatalogued
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/193453 , vital:45333
- Description: In order to understand the effects of metals contained in effluent and to define effluent quality suitable for safe discharge to natural water streams, it is essential to understand the effects of the interaction of metal ions with plants. The availability of metal ions and their ability to bind to plants are dependent on the chemical speciation of metals and on the biological factors governing the availability of metals within the plant cells. This thesis will address both aspects and thereby propose a combination of an appropriate chemical and biological approach to the investigation of bioaccumulation of the plant Azolla Filiculoides. Laboratory studies have shown that varying concentrations of free metal ions in solution determine efficiency of metal uptake and that metal toxicity can also be detrimental to plant life and efficiency of metal recovery from solution. Many questions however, remain unanswered with regard to the application of a biological treatment for effluent discharge. This thesis includes the determination of metal speciation combined with the study of bioaccumulation of metals in plants and their effects from test- work utilising effluent generated from a Precious Metals Refinery (PMR). Plant species are known to differ widely in their tolerance to metals, however despite an abundant knowledge on molecular, biochemical and physiological effects of metals to plants, only a few general principles have been proposed to guide the prediction of tolerance differences. The properties of protective cellular responses as well as of the molecular target sites are important components in determining the intrinsic tolerance of a particular species to a metal. The role of the whole assembly of cellular ligands in buffering metal ions within the cells will be evaluated. Standard preparation methods combined with use of Inductively Coupled Plasma Emission Spectrophotometer (1CP) used for analytical analysis will be included to reflect analytical data in providing evidence to support a conclusion. The outcome of the test work utilising the aquatic plant Azoila has proven that it can be used as a process step to re-mediate effluent generated from Precious Metal Refining operations. This process offers an alternative to the classical chemical methods widely used in the Precious Metals Refining industry proving economically viable and ensuring environmental sustainability in comparison to the current known methods of effluent treatment. , Thesis (MSc) -- Faculty of Science, Biochemistry, Microbiology and Biotechnology, 2003
- Full Text:
- Date Issued: 2003
- Authors: Marran, Vernon Edward
- Date: 2003
- Subjects: Uncatalogued
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/193453 , vital:45333
- Description: In order to understand the effects of metals contained in effluent and to define effluent quality suitable for safe discharge to natural water streams, it is essential to understand the effects of the interaction of metal ions with plants. The availability of metal ions and their ability to bind to plants are dependent on the chemical speciation of metals and on the biological factors governing the availability of metals within the plant cells. This thesis will address both aspects and thereby propose a combination of an appropriate chemical and biological approach to the investigation of bioaccumulation of the plant Azolla Filiculoides. Laboratory studies have shown that varying concentrations of free metal ions in solution determine efficiency of metal uptake and that metal toxicity can also be detrimental to plant life and efficiency of metal recovery from solution. Many questions however, remain unanswered with regard to the application of a biological treatment for effluent discharge. This thesis includes the determination of metal speciation combined with the study of bioaccumulation of metals in plants and their effects from test- work utilising effluent generated from a Precious Metals Refinery (PMR). Plant species are known to differ widely in their tolerance to metals, however despite an abundant knowledge on molecular, biochemical and physiological effects of metals to plants, only a few general principles have been proposed to guide the prediction of tolerance differences. The properties of protective cellular responses as well as of the molecular target sites are important components in determining the intrinsic tolerance of a particular species to a metal. The role of the whole assembly of cellular ligands in buffering metal ions within the cells will be evaluated. Standard preparation methods combined with use of Inductively Coupled Plasma Emission Spectrophotometer (1CP) used for analytical analysis will be included to reflect analytical data in providing evidence to support a conclusion. The outcome of the test work utilising the aquatic plant Azoila has proven that it can be used as a process step to re-mediate effluent generated from Precious Metal Refining operations. This process offers an alternative to the classical chemical methods widely used in the Precious Metals Refining industry proving economically viable and ensuring environmental sustainability in comparison to the current known methods of effluent treatment. , Thesis (MSc) -- Faculty of Science, Biochemistry, Microbiology and Biotechnology, 2003
- Full Text:
- Date Issued: 2003
An investigation of the isolation, characterisation and application of hydantoinases for the industrial production of amino acids
- Authors: Kirchmann, Shaun
- Date: 2003
- Subjects: Hydantoin Amino acids Hydrolysis
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3969 , http://hdl.handle.net/10962/d1004028
- Description: This thesis describes a series of investigations into the hydantoin-hydrolysing activity of bacterial strains RU-KM1 and RU-OR, which were previously isolated for their ability to hydrolyse hydantoins to amino acids. The main aim of the study was to develop biotransformations with potential application in the production of enantiomerically pure amino acids using a bioreactor based system utilising the hydantoin hydrolysing enzymes of the two isolated microorganisms. Different substituted hydantoins may be used as substrates by these enzymes for the production of a variety of amino acids. These are not only important for amino acid production, but they may be used for production of other industrially important compounds, such as semisynthetic penicillin/ampicillin, L-aspartame (sweetener), Fluvalinate (insecticide), Enalapril (ACE inhibitor). Thus, the ability of the above-mentioned strains to hydrolyse these substrates was investigated, with the view to utilizing the maximum potential of these biocatalysts. Hydantoin conversion involves a two-step hydrolysis reaction which yields, initially, an N-carbamylamino acid intermediate, and subsequently, an amino acid. The hydantoin-hydrolysing enzymes of a Pseudomonas sp. RU-KM1, and an Agrobacterium sp. RU-OR were characterised as whole cells and in a crude extract preparation, and reaction conditions for its biocatalytic application were optimised. The optimum conditions for conversion of hydantoin to glycine were found to be 1 hour at 40 °C, with conversion yields greater than 30 % achieved. The enzymes of RU-KM1 demonstrated considerable stability, retaining 80 % of their activity after storage for 2 weeks at 4 °C. The activities of the enzymes were increased by the addition of a detergent to the extraction medium, suggesting that the enzymes might be membrane-bound. The results of the determination of the metal-dependence of the hydantoinase and N-carbamoylase of RU-KM1 suggested that these enzymes required metal ions for activity, with metal ions such as Cu[superscript (2+)], Fe[superscript (2+)], and Co[superscript (2+)] resulting in no significant change in enzyme activity, however there was an activation of the enzymes when Mn[superscript (2+)] was added to the enzymes. The stereoselectivity of the enzymes was investigated, and the results suggested that the hydantoinase was D-selective, whereas the N-carbamoylase was shown to be L-selective by other researchers. The hydantoin substrate selectivity of RU-KM1 and RU-OR was investigated, and the organisms were shown to be able to hydrolyse all of the seven substrates tested. However, there was a difference in activity levels between crude extract preparations and whole cells, with crude extracts generally showing slightly lower activity than whole cells in RU-KM1, and the whole cells or RU-OR showing the lower activity than its crude extract. Some difference was also observed in the order of preference of substrates between whole cells and crude extracts. The preferred substrate for RU-KM1 whole cells was isopropylhydantoin, whereas the crude extract preparation preferentially hydrolysed p-hydroxyphenylhydantoin, achieving 57 % and 52 % conversions respectively. RU-OR whole cells preferred methylhydantoin where as the crude extract preferred isopropylhydantoin, and showed 49 % and 51 % conversions respectively. The enzymes were characterised in terms of their temperature and pH optima, inducer requirements, and product inhibition studies. The hydantoinase of RU-KM1 was shown to be inducible with low levels of hydantoin, and thermostable upto 75 °C with its optima between 60 and 70 °C. The N-carbamoylase was shown to have its optima at 50 °C. The addition of ATP (0.5 mM), DTT (1 mM) and a protease inhibitor (2 mg.mL[superscript (-1)]) all increased the hydantoinase activity of RU-KM1 crude extract, however they had very little effect on the N-carbamoylase activity. The hydantoinase enzyme from extracts of RU-KM1 was partially purified by development of cell disruption methods using mechanical and lysing enzymes, followed by precipitation and chromatographic resolution. The results obtained showed a hydantoinase enzyme of between 48 and 66 kDa. RU-KM1 was grown under fermentation conditions using different minimal media. The activity and yields under these conditions were low. Previous attempt to grow the organism in a rich medium had resulted in an increase in biomass but no hydantoinase activity. A rich medium was developed by carbon and nitrogen optimisation and yielded biomass up to 30 g.L[superscript (-1)] dry cell weight. The hydantoinase activity was restored by nitrogen starvation in stationary phase. This resulted in high biomass with increased activity. This data is currently in press. Crude extract and whole cells were immobilised on flat sheet membranes, hollow fibre membranes and in alginate beads. Low hydantoinase activity was measured in bioreactors using membranes in different configurations. A significant increase in hydantoinase activity was measured when the crude extract was immobilised in sodium alginate, as a result of stabilisation of the N-carbamoylase. Temperature and pH optima were unaffected by the immobilisation procedure, however the durability of the enzymes increased 2-fold. Different configurations of the bioreactor were investigated, as well as a hydroxyphenylhydantoin as an alternative substrate in this study. The bioreactors showed a near 95 % conversion of the hydantoin to glycine, and a 99 % conversion using HPG. In conclusion, the hydantoin-hydrolysing enzymes of RU-KM1 have been shown to be possibly membrane associated, which is a novel finding. This study has shown that the hydantoinase of RU-KM1 is D-stereoselective, with high temperature stability. A growth medium was developed for the rapid production of active biomass. A bioreactor was developed using a single and a dual biocatalyst configuration, which was capable of hydrolysing hydantoin and monosubstituted hydantoins to produce amino acids. To our knowledge this system is the first such dual biocatalyst system reported for the production of amino acids.
- Full Text:
- Date Issued: 2003
- Authors: Kirchmann, Shaun
- Date: 2003
- Subjects: Hydantoin Amino acids Hydrolysis
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3969 , http://hdl.handle.net/10962/d1004028
- Description: This thesis describes a series of investigations into the hydantoin-hydrolysing activity of bacterial strains RU-KM1 and RU-OR, which were previously isolated for their ability to hydrolyse hydantoins to amino acids. The main aim of the study was to develop biotransformations with potential application in the production of enantiomerically pure amino acids using a bioreactor based system utilising the hydantoin hydrolysing enzymes of the two isolated microorganisms. Different substituted hydantoins may be used as substrates by these enzymes for the production of a variety of amino acids. These are not only important for amino acid production, but they may be used for production of other industrially important compounds, such as semisynthetic penicillin/ampicillin, L-aspartame (sweetener), Fluvalinate (insecticide), Enalapril (ACE inhibitor). Thus, the ability of the above-mentioned strains to hydrolyse these substrates was investigated, with the view to utilizing the maximum potential of these biocatalysts. Hydantoin conversion involves a two-step hydrolysis reaction which yields, initially, an N-carbamylamino acid intermediate, and subsequently, an amino acid. The hydantoin-hydrolysing enzymes of a Pseudomonas sp. RU-KM1, and an Agrobacterium sp. RU-OR were characterised as whole cells and in a crude extract preparation, and reaction conditions for its biocatalytic application were optimised. The optimum conditions for conversion of hydantoin to glycine were found to be 1 hour at 40 °C, with conversion yields greater than 30 % achieved. The enzymes of RU-KM1 demonstrated considerable stability, retaining 80 % of their activity after storage for 2 weeks at 4 °C. The activities of the enzymes were increased by the addition of a detergent to the extraction medium, suggesting that the enzymes might be membrane-bound. The results of the determination of the metal-dependence of the hydantoinase and N-carbamoylase of RU-KM1 suggested that these enzymes required metal ions for activity, with metal ions such as Cu[superscript (2+)], Fe[superscript (2+)], and Co[superscript (2+)] resulting in no significant change in enzyme activity, however there was an activation of the enzymes when Mn[superscript (2+)] was added to the enzymes. The stereoselectivity of the enzymes was investigated, and the results suggested that the hydantoinase was D-selective, whereas the N-carbamoylase was shown to be L-selective by other researchers. The hydantoin substrate selectivity of RU-KM1 and RU-OR was investigated, and the organisms were shown to be able to hydrolyse all of the seven substrates tested. However, there was a difference in activity levels between crude extract preparations and whole cells, with crude extracts generally showing slightly lower activity than whole cells in RU-KM1, and the whole cells or RU-OR showing the lower activity than its crude extract. Some difference was also observed in the order of preference of substrates between whole cells and crude extracts. The preferred substrate for RU-KM1 whole cells was isopropylhydantoin, whereas the crude extract preparation preferentially hydrolysed p-hydroxyphenylhydantoin, achieving 57 % and 52 % conversions respectively. RU-OR whole cells preferred methylhydantoin where as the crude extract preferred isopropylhydantoin, and showed 49 % and 51 % conversions respectively. The enzymes were characterised in terms of their temperature and pH optima, inducer requirements, and product inhibition studies. The hydantoinase of RU-KM1 was shown to be inducible with low levels of hydantoin, and thermostable upto 75 °C with its optima between 60 and 70 °C. The N-carbamoylase was shown to have its optima at 50 °C. The addition of ATP (0.5 mM), DTT (1 mM) and a protease inhibitor (2 mg.mL[superscript (-1)]) all increased the hydantoinase activity of RU-KM1 crude extract, however they had very little effect on the N-carbamoylase activity. The hydantoinase enzyme from extracts of RU-KM1 was partially purified by development of cell disruption methods using mechanical and lysing enzymes, followed by precipitation and chromatographic resolution. The results obtained showed a hydantoinase enzyme of between 48 and 66 kDa. RU-KM1 was grown under fermentation conditions using different minimal media. The activity and yields under these conditions were low. Previous attempt to grow the organism in a rich medium had resulted in an increase in biomass but no hydantoinase activity. A rich medium was developed by carbon and nitrogen optimisation and yielded biomass up to 30 g.L[superscript (-1)] dry cell weight. The hydantoinase activity was restored by nitrogen starvation in stationary phase. This resulted in high biomass with increased activity. This data is currently in press. Crude extract and whole cells were immobilised on flat sheet membranes, hollow fibre membranes and in alginate beads. Low hydantoinase activity was measured in bioreactors using membranes in different configurations. A significant increase in hydantoinase activity was measured when the crude extract was immobilised in sodium alginate, as a result of stabilisation of the N-carbamoylase. Temperature and pH optima were unaffected by the immobilisation procedure, however the durability of the enzymes increased 2-fold. Different configurations of the bioreactor were investigated, as well as a hydroxyphenylhydantoin as an alternative substrate in this study. The bioreactors showed a near 95 % conversion of the hydantoin to glycine, and a 99 % conversion using HPG. In conclusion, the hydantoin-hydrolysing enzymes of RU-KM1 have been shown to be possibly membrane associated, which is a novel finding. This study has shown that the hydantoinase of RU-KM1 is D-stereoselective, with high temperature stability. A growth medium was developed for the rapid production of active biomass. A bioreactor was developed using a single and a dual biocatalyst configuration, which was capable of hydrolysing hydantoin and monosubstituted hydantoins to produce amino acids. To our knowledge this system is the first such dual biocatalyst system reported for the production of amino acids.
- Full Text:
- Date Issued: 2003
Binding and transcriptional activation by Uga3p, a zinc binuclear cluster protein of Saccharomyces cerevisiae redefining the UAS [subscript GABA] and the Uga3p binding site
- Authors: Idicula, Anu Mary
- Date: 2003
- Subjects: Saccharomyces cerevisiae GABA
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3933 , http://hdl.handle.net/10962/d1003992
- Description: Uga3p, a member of the zinc binuclear cluster transcription factor family, is required for [gamma]-aminobutyric acid-dependent transcription of the UGA genes in Saccharomyces cerevisiae. Crystallographic data of some of the protein-DNA complexes of this family reveal that members of this family bind to CGG triplets. A conserved 19-nucleotide activation element in certain UGA gene promoter regions contains a CCG-N4-CGG everted repeat, proposed to be the binding site of Uga3p, UAS[subscript GABA]. The spacer region (N4) between the CGG triplets has been suggested to be the specificity determinant for binding to UAS[subscript GABA]. The data available from the Saccharomyces genome database indicates that there are multiple repeats of -CCG-N4-CGG- regions within the genome. These transcription factors are involved in the activation of specific pathways and the question arises as to how their specificity of binding is determined. The aim of this study was to understand the binding characteristics of Uga3p to UAS[subscript GABA] and to determine the affinity and specificity of this interaction. In this study, full-length (tagged and untagged) and truncated (1-124 a.a.) Uga3p was produced in a heterologous expression system (E. coli). The interaction of Uga3p with UAS[subscript GABA] in Saccharomyces cerevisiae was characterized in terms of binding in vitro and the transcriptional activation of lacZ reporter genes in vivo. The Uga3p was capable of binding to these sites in vitro independent of exogenous GABA. Electrophoretic mobility shift assays (EMSA) of the full-length Uga3p with the wild type UAS[subscript GABA] sequences produced two distinct mobility complexes. The complexes formed in the EMSA of the full-length Uga3p were those specific to the interaction of the Uga3p to UAS[subscript GABA]. The truncated Uga3p(1-124 a.a.), which has the DNA-binding zinc cluster domain, the linker region and the putative coiled-coil domain was not functionally equivalent to the full-length protein with respect to binding in vitro because the EMSAs of the UAS[subscript GABA] with the truncated Uga3p produced indistinct complexes. EMSAs using mutant UAS[subscript GABA] sequences and heterologously-produced full-length Uga3p, demonstrated that UAS[subscript GABA] consists of two, independent Uga3p-binding sites. This work presents evidence that the two Uga3p molecules bound to UAS[subscript GABA] most likely interact with each other. Unlike other zinc cluster binding sites the Uga3p-binding site is an asymmetric site of 5’-SGCGGNWWT-3’ (S= G or C, W = A or T and N = no nucleotide or G or C). UAS[subscript GABA] is a palindrome containing the two asymmetric Uga3p-binding sites. The two-site consensus sequence required for the binding of Uga3p to the UAS[subscript GABA] is present upstream of UGA1 (region -387 to -370) and UGA4 (region -403 to -387). Furthermore, a single Uga3p-binding site was identified in the 5’ untranslated regions of UGA2 (region -219 to -211). GABA-dependent transcriptional activation by UAS[subscript GABA] in vivo could be directly correlated to a high affinity, specific interaction of two Uga3p molecules to this UAS. Binding with high affinity required the conserved sequences flanking the everted repeat. This study provided evidence that the binding pattern of Uga3p is novel compared to other zinc cluster motifs investigated, as the sequences flanking the everted repeat are important regions for recognition by Uga3p. The studies with the truncated Uga3p (1 –124 a.a.), also suggested that the regions C-terminal to the DNA-binding motif and putative coiled-coil area of this protein are important for Uga3p-specific interactions with UAS[subscript GABA]. Investigation of regions C-terminal to the zinc cluster, linker and putative coiledcoil revealed an eight-motif regulatory region similar to that in other zinc cluster proteins. This indicated that the regions C-terminal to these domains are important for the regulation and activity of these proteins. A putative seven repeat WD40-like motif was identified within this region. This putative domain has been speculated to be important for protein-protein interactions. Phosphorylation and dephosphorylation in other proteins of this class have been indicated to be important for the regulation of the activity of these proteins. The bioinformatic analysis of Uga3p revealed two possible cAMP/cGMP-dependent protein kinase phosphorylation sites, four putative protein kinase C phosphorylation motifs and four putative casein kinase II phosphorylation motifs. This study has contributed to the understanding of the nature of interactions between Uga3p and its specific UAS [subscript GABA] and how the regions flanking the everted repeat determine its specificity. The comparison of the nature of the binding of truncated and full-length Uga3p in vitro provided evidence for the role played by the full-length protein in determining this specific interaction. This evidence suggested that the in vitro binding evidence for other proteins of this family, using truncated peptides that carry the DNA-binding domain, might not reflect the true nature of interactions between the proteins of this class and their specific UASs in vivo.
- Full Text:
- Date Issued: 2003
- Authors: Idicula, Anu Mary
- Date: 2003
- Subjects: Saccharomyces cerevisiae GABA
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3933 , http://hdl.handle.net/10962/d1003992
- Description: Uga3p, a member of the zinc binuclear cluster transcription factor family, is required for [gamma]-aminobutyric acid-dependent transcription of the UGA genes in Saccharomyces cerevisiae. Crystallographic data of some of the protein-DNA complexes of this family reveal that members of this family bind to CGG triplets. A conserved 19-nucleotide activation element in certain UGA gene promoter regions contains a CCG-N4-CGG everted repeat, proposed to be the binding site of Uga3p, UAS[subscript GABA]. The spacer region (N4) between the CGG triplets has been suggested to be the specificity determinant for binding to UAS[subscript GABA]. The data available from the Saccharomyces genome database indicates that there are multiple repeats of -CCG-N4-CGG- regions within the genome. These transcription factors are involved in the activation of specific pathways and the question arises as to how their specificity of binding is determined. The aim of this study was to understand the binding characteristics of Uga3p to UAS[subscript GABA] and to determine the affinity and specificity of this interaction. In this study, full-length (tagged and untagged) and truncated (1-124 a.a.) Uga3p was produced in a heterologous expression system (E. coli). The interaction of Uga3p with UAS[subscript GABA] in Saccharomyces cerevisiae was characterized in terms of binding in vitro and the transcriptional activation of lacZ reporter genes in vivo. The Uga3p was capable of binding to these sites in vitro independent of exogenous GABA. Electrophoretic mobility shift assays (EMSA) of the full-length Uga3p with the wild type UAS[subscript GABA] sequences produced two distinct mobility complexes. The complexes formed in the EMSA of the full-length Uga3p were those specific to the interaction of the Uga3p to UAS[subscript GABA]. The truncated Uga3p(1-124 a.a.), which has the DNA-binding zinc cluster domain, the linker region and the putative coiled-coil domain was not functionally equivalent to the full-length protein with respect to binding in vitro because the EMSAs of the UAS[subscript GABA] with the truncated Uga3p produced indistinct complexes. EMSAs using mutant UAS[subscript GABA] sequences and heterologously-produced full-length Uga3p, demonstrated that UAS[subscript GABA] consists of two, independent Uga3p-binding sites. This work presents evidence that the two Uga3p molecules bound to UAS[subscript GABA] most likely interact with each other. Unlike other zinc cluster binding sites the Uga3p-binding site is an asymmetric site of 5’-SGCGGNWWT-3’ (S= G or C, W = A or T and N = no nucleotide or G or C). UAS[subscript GABA] is a palindrome containing the two asymmetric Uga3p-binding sites. The two-site consensus sequence required for the binding of Uga3p to the UAS[subscript GABA] is present upstream of UGA1 (region -387 to -370) and UGA4 (region -403 to -387). Furthermore, a single Uga3p-binding site was identified in the 5’ untranslated regions of UGA2 (region -219 to -211). GABA-dependent transcriptional activation by UAS[subscript GABA] in vivo could be directly correlated to a high affinity, specific interaction of two Uga3p molecules to this UAS. Binding with high affinity required the conserved sequences flanking the everted repeat. This study provided evidence that the binding pattern of Uga3p is novel compared to other zinc cluster motifs investigated, as the sequences flanking the everted repeat are important regions for recognition by Uga3p. The studies with the truncated Uga3p (1 –124 a.a.), also suggested that the regions C-terminal to the DNA-binding motif and putative coiled-coil area of this protein are important for Uga3p-specific interactions with UAS[subscript GABA]. Investigation of regions C-terminal to the zinc cluster, linker and putative coiledcoil revealed an eight-motif regulatory region similar to that in other zinc cluster proteins. This indicated that the regions C-terminal to these domains are important for the regulation and activity of these proteins. A putative seven repeat WD40-like motif was identified within this region. This putative domain has been speculated to be important for protein-protein interactions. Phosphorylation and dephosphorylation in other proteins of this class have been indicated to be important for the regulation of the activity of these proteins. The bioinformatic analysis of Uga3p revealed two possible cAMP/cGMP-dependent protein kinase phosphorylation sites, four putative protein kinase C phosphorylation motifs and four putative casein kinase II phosphorylation motifs. This study has contributed to the understanding of the nature of interactions between Uga3p and its specific UAS [subscript GABA] and how the regions flanking the everted repeat determine its specificity. The comparison of the nature of the binding of truncated and full-length Uga3p in vitro provided evidence for the role played by the full-length protein in determining this specific interaction. This evidence suggested that the in vitro binding evidence for other proteins of this family, using truncated peptides that carry the DNA-binding domain, might not reflect the true nature of interactions between the proteins of this class and their specific UASs in vivo.
- Full Text:
- Date Issued: 2003
Development of integrated algal ponding systems in the treatment of wine distillery wastewaters
- Authors: Dekker, Leendert Gideon
- Date: 2003
- Subjects: Sewage -- Purification -- Anaerobic treatment Wine and wine making -- Waste disposal -- South Africa
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4062 , http://hdl.handle.net/10962/d1004530
- Description: In South Africa, wastewater disposal in the wine and distilling industry is undergoing a profound transformation as a result of fundamental changes in regulations and license requirements. To deal with this problem conventional Waste Stabilisation Ponding systems have been used by the industry together with irrigation and evaporation disposal practises. Although effective in the evaporation and containment disposal functions, these pond systems are generally not properly designed and/or managed, resulting in overloading and, at times, the generation of seriously offensive odour problems. Preliminary studies on the feasibility of utilising the Advanced Integrated Wastewater Ponding System as a core treatment technology in winery wastewater treatment were conducted. Results indicated that specific problems had to be addressed before successful ponding treatment could be achieved. This research programme undertook an investigation of the performance of a demonstration ponding system treating household sewage, which formed the basis of the research due to limited experience reported on ponds treating wine industry wastewaters. Malfunctions identified were in correlation with the preliminary winery waste ponding survey, which included unstable fermentation pit functions and inadequate nutrient removal. Retrofitting the fermentation pit with a nylon net across the rising water column resulted in improved retention of active anaerobic sludge, especially during periods of system start-up and/or organic overloading. An investigation into nutrient removal utilising algal biomass provided a valuable contribution towards development of an independent nutrient removal system. Harvested algal biomass was passively manipulated to release polysaccharides under anoxic conditions, with subsequent use as a carbon source by denitrifying organisms. Following denitrification, the still viable algal cells were introduced into a High Rate Algal Pond raceway for photosynthetically produced alkalinity. This high pH environment resulted in induced calcium phosphate mineral formation and subsequent precipitation, as well as effective ammonia stripping from the water. Based on the novel positive research outcomes a decision was made to proceed to the construction of a pilot-scale integrated ponding system treating wastewater from a wine lees factory. The system linked the Anaerobic Baffle Reactor, for pre-treatment, with the improved Advanced Integrated Wastewater Ponding System. The potential of this system has shown that a Waste Stabilisation Ponding system can be engineered to treat wine industry wastewaters and thereby effectively reduce the organic and nutrient loads, by using low-cost retrofitted upgrading unit operations. Valuable algal biomass may also be recovered as a by-product of the treatment process.
- Full Text:
- Date Issued: 2003
- Authors: Dekker, Leendert Gideon
- Date: 2003
- Subjects: Sewage -- Purification -- Anaerobic treatment Wine and wine making -- Waste disposal -- South Africa
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4062 , http://hdl.handle.net/10962/d1004530
- Description: In South Africa, wastewater disposal in the wine and distilling industry is undergoing a profound transformation as a result of fundamental changes in regulations and license requirements. To deal with this problem conventional Waste Stabilisation Ponding systems have been used by the industry together with irrigation and evaporation disposal practises. Although effective in the evaporation and containment disposal functions, these pond systems are generally not properly designed and/or managed, resulting in overloading and, at times, the generation of seriously offensive odour problems. Preliminary studies on the feasibility of utilising the Advanced Integrated Wastewater Ponding System as a core treatment technology in winery wastewater treatment were conducted. Results indicated that specific problems had to be addressed before successful ponding treatment could be achieved. This research programme undertook an investigation of the performance of a demonstration ponding system treating household sewage, which formed the basis of the research due to limited experience reported on ponds treating wine industry wastewaters. Malfunctions identified were in correlation with the preliminary winery waste ponding survey, which included unstable fermentation pit functions and inadequate nutrient removal. Retrofitting the fermentation pit with a nylon net across the rising water column resulted in improved retention of active anaerobic sludge, especially during periods of system start-up and/or organic overloading. An investigation into nutrient removal utilising algal biomass provided a valuable contribution towards development of an independent nutrient removal system. Harvested algal biomass was passively manipulated to release polysaccharides under anoxic conditions, with subsequent use as a carbon source by denitrifying organisms. Following denitrification, the still viable algal cells were introduced into a High Rate Algal Pond raceway for photosynthetically produced alkalinity. This high pH environment resulted in induced calcium phosphate mineral formation and subsequent precipitation, as well as effective ammonia stripping from the water. Based on the novel positive research outcomes a decision was made to proceed to the construction of a pilot-scale integrated ponding system treating wastewater from a wine lees factory. The system linked the Anaerobic Baffle Reactor, for pre-treatment, with the improved Advanced Integrated Wastewater Ponding System. The potential of this system has shown that a Waste Stabilisation Ponding system can be engineered to treat wine industry wastewaters and thereby effectively reduce the organic and nutrient loads, by using low-cost retrofitted upgrading unit operations. Valuable algal biomass may also be recovered as a by-product of the treatment process.
- Full Text:
- Date Issued: 2003
Isolation of antigenic peptides of Cowdria ruminantium and their encoding genes using a genome-derived phage display library
- Authors: Fehrsen, Jeanni
- Date: 2003
- Subjects: Bacteriophages -- Genetics Ruminants -- Diseases Heartwater
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3920 , http://hdl.handle.net/10962/d1003979
- Description: The development of new and effective vaccines and immunodiagnostic reagents requires the characterisation of antigenically relevant proteins and their interactions with the products of the immune system. Phage display technology was investigated as a means of elucidating some of the antigenic properties of the rickettsial parasite, Cowdria ruminantium (Cowdria). Randomly fragmented gene-derived libraries have been useful in elucidating viral and other epitopes, but only limited work has been done with entire genomes. A phage display library expressing a repertoire of Cowdria peptides was constructed. It was sufficiently large to represent the organism's genome, but lacked phages displaying peptides coded for by genes containing a Pvu II restriction enzyme site, including the one coding for the major antigenic protein 1 (MAP1). This was considered advantageous since MAP1 is immunodominant and has already been well characterised. Affinity selection with antibodies against Cowdria proteins other than MAP1 allowed several antibody-reactive peptides to be isolated. These selected sequences were placed in the context of the genome by screening a lambda bacteriophage library and by comparison with Cowdria DNA sequences. Apart from showing that antigenic mimics were present in the phage display library, six open reading frames encoding putative Cowdria proteins were identified. All had similarities to, or motifs in common with, membrane proteins and are thus likely to be exposed to the host's humoral immune system. Some of the proteins identified were larger than the antigens used to elicit the antibodies used for selection, probably as a result of the presence of cross-reactive epitopes. Despite limitations experienced when extending a fragmented-gene approach for epitope location to genomes, it was possible to identify an antigenic region on MAP1 by comparison with selected mimics. In addition, binding peptide sequences were identified with two monoclonal antibodies that had been raised against non-Cowdria antigens. An epitope on the VP7 protein of bluetongue virus was identified and peptides were found that reacted with a monoclonal antibody directed against malignant catarrhal fever virus. Thus, apart from being able to identify several potentially important Cowdria epitopes and genes, the fragmented-genome library holds promise as a universal reagent for identifying useful mimics.
- Full Text:
- Date Issued: 2003
- Authors: Fehrsen, Jeanni
- Date: 2003
- Subjects: Bacteriophages -- Genetics Ruminants -- Diseases Heartwater
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3920 , http://hdl.handle.net/10962/d1003979
- Description: The development of new and effective vaccines and immunodiagnostic reagents requires the characterisation of antigenically relevant proteins and their interactions with the products of the immune system. Phage display technology was investigated as a means of elucidating some of the antigenic properties of the rickettsial parasite, Cowdria ruminantium (Cowdria). Randomly fragmented gene-derived libraries have been useful in elucidating viral and other epitopes, but only limited work has been done with entire genomes. A phage display library expressing a repertoire of Cowdria peptides was constructed. It was sufficiently large to represent the organism's genome, but lacked phages displaying peptides coded for by genes containing a Pvu II restriction enzyme site, including the one coding for the major antigenic protein 1 (MAP1). This was considered advantageous since MAP1 is immunodominant and has already been well characterised. Affinity selection with antibodies against Cowdria proteins other than MAP1 allowed several antibody-reactive peptides to be isolated. These selected sequences were placed in the context of the genome by screening a lambda bacteriophage library and by comparison with Cowdria DNA sequences. Apart from showing that antigenic mimics were present in the phage display library, six open reading frames encoding putative Cowdria proteins were identified. All had similarities to, or motifs in common with, membrane proteins and are thus likely to be exposed to the host's humoral immune system. Some of the proteins identified were larger than the antigens used to elicit the antibodies used for selection, probably as a result of the presence of cross-reactive epitopes. Despite limitations experienced when extending a fragmented-gene approach for epitope location to genomes, it was possible to identify an antigenic region on MAP1 by comparison with selected mimics. In addition, binding peptide sequences were identified with two monoclonal antibodies that had been raised against non-Cowdria antigens. An epitope on the VP7 protein of bluetongue virus was identified and peptides were found that reacted with a monoclonal antibody directed against malignant catarrhal fever virus. Thus, apart from being able to identify several potentially important Cowdria epitopes and genes, the fragmented-genome library holds promise as a universal reagent for identifying useful mimics.
- Full Text:
- Date Issued: 2003
Metallophthalocyanines as photocatalysts for transformation of chlorophenols and self-assembled monolayers for electrochemical detection of thiols and cyanides
- Authors: Ozoemena, Kenneth Ikechukwu
- Date: 2003
- Subjects: Electrochemistry Cyanides Thiols Chlorophenols Photocatalysis
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4089 , http://hdl.handle.net/10962/d1007709
- Description: Photochemical properties of sulphonated phthalocyanine complexes of aluminium, zinc, tin and silicon, and octa-carboxyphthalocyanine complexes of aluminium and zinc have been investigated. These water-soluble metallophthalocyanine (MPc) complexes, especially the sulphonated aluminium and zinc phthalocyanines, were found to be good photosensitisers for the transformation of the toxic mono-, tri- and penta-chlorophenols in aqueous solutions. The efficiency of MPc sensitiser towards photo-transformation of chlorophenols depends on its effectiveness to generate singlet oxygen as well as its photostability. Octa-substituted thiol-derivatised phthalocyanine complexes of cobalt, iron and zinc were synthesized and their spectral and electrochemical properties investigated. The photochemical properties ofthe zinc phthalocyanine complexes in non-aqueous solutions were comparable to those in literature. Ultrathin films of the octasubstituted thiol-derivatised phthalocyanine complexes of cobalt, iron and zinc were, for the first time, immobilized onto gold electrodes using the self-assembling technique. Surface electrochemistry indicates that the ultrathin films are surface-confined self-assembled monolayer (SAM) species. Gold electrodes modified with the redox-active SAMs of cobalt and iron phthalocyanine complexes proved to be potential electrochemical sensors for the detection of thiols (L-cysteine, homocysteine and penicillamine) and thiocyanate in aqueous solutions (pH 4). The limits of detection for the thiols and thiocyanate were in the range of ∼ 10⁻⁷ and 10⁻⁶ mol dm⁻³, respectively. The modification process was reproducible and the modified electrodes showed good stability and, if stored in pH 4 buffer solutions, could be used for the analysis of thiols and thiocyanate for about a month without the need for recalibration. Etching of gold marred electrochemical detection of cyanide with the MPc-SAM-modified gold electrodes. Interestingly, however, kinetic and equilibria studies revealed strong interaction of octabutylthiophthalocyaninatoiron (II), FeOBTPc, with cyanide in both DMF and DMSO solutions.
- Full Text:
- Date Issued: 2003
- Authors: Ozoemena, Kenneth Ikechukwu
- Date: 2003
- Subjects: Electrochemistry Cyanides Thiols Chlorophenols Photocatalysis
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4089 , http://hdl.handle.net/10962/d1007709
- Description: Photochemical properties of sulphonated phthalocyanine complexes of aluminium, zinc, tin and silicon, and octa-carboxyphthalocyanine complexes of aluminium and zinc have been investigated. These water-soluble metallophthalocyanine (MPc) complexes, especially the sulphonated aluminium and zinc phthalocyanines, were found to be good photosensitisers for the transformation of the toxic mono-, tri- and penta-chlorophenols in aqueous solutions. The efficiency of MPc sensitiser towards photo-transformation of chlorophenols depends on its effectiveness to generate singlet oxygen as well as its photostability. Octa-substituted thiol-derivatised phthalocyanine complexes of cobalt, iron and zinc were synthesized and their spectral and electrochemical properties investigated. The photochemical properties ofthe zinc phthalocyanine complexes in non-aqueous solutions were comparable to those in literature. Ultrathin films of the octasubstituted thiol-derivatised phthalocyanine complexes of cobalt, iron and zinc were, for the first time, immobilized onto gold electrodes using the self-assembling technique. Surface electrochemistry indicates that the ultrathin films are surface-confined self-assembled monolayer (SAM) species. Gold electrodes modified with the redox-active SAMs of cobalt and iron phthalocyanine complexes proved to be potential electrochemical sensors for the detection of thiols (L-cysteine, homocysteine and penicillamine) and thiocyanate in aqueous solutions (pH 4). The limits of detection for the thiols and thiocyanate were in the range of ∼ 10⁻⁷ and 10⁻⁶ mol dm⁻³, respectively. The modification process was reproducible and the modified electrodes showed good stability and, if stored in pH 4 buffer solutions, could be used for the analysis of thiols and thiocyanate for about a month without the need for recalibration. Etching of gold marred electrochemical detection of cyanide with the MPc-SAM-modified gold electrodes. Interestingly, however, kinetic and equilibria studies revealed strong interaction of octabutylthiophthalocyaninatoiron (II), FeOBTPc, with cyanide in both DMF and DMSO solutions.
- Full Text:
- Date Issued: 2003
Molecular characterization of the tetratricopeptide repeat-mediated interactions of murine stress-inducible protein 1 with major heat shock proteins
- Authors: Odunuga, Odutayo Odutola
- Date: 2003
- Subjects: Plants -- Effect of stress on Proteins -- Purification Electrophoresis Heat shock proteins
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4091 , http://hdl.handle.net/10962/d1007724
- Description: Murine stress-inducible protein 1 (mSTI1) is a co-chaperone that is homologous with the human heat shock protein 70 (Hsp70)/heat shock protein 90 (Hsp90)-organizing protein (Hop). The two proteins are homologues of the highly conserved stress-inducible protein 1 (STI1) family of co-chaperones. The STI1 proteins interact directly and simultaneously at some stage, with Hsp70 and Hsp90 in the formation of the hetero-multi-chaperone complexes that facilitate the folding of signal transducing kinases and functional maturation of steroid hormone receptors. The interactions of mSTI1 with both Hsp70 and Hsp90 is mediated by a versatile structural protein-protein interaction motif, the tetratricopeptide repeat (TPR). The TPR motif is a degenerate 34-amino acid sequence a-helical structural motif found in a significant number of functionally unrelated proteins. This study was aimed at characterizing the structural and functional determinants in the TPR domains of mSTI1 responsible for binding to and discriminating between Hsp70 and Hsp90. Guided by data from Hop's crystal structures and amino acid sequence alignment analyses, various biochemical techniques were used to both qualitatively and quantitatively characterize the contacts necessary for the N-terminal TPR domain (TPR1) of mSTI1 to bind to the C-terminal EEVD motif of heat shock cognate protein 70 (Hsc70) and to discriminate between Hsc70 and Hsp90. Substitutions in the first TPR motif of Lys⁸ or Asn¹² did not affect binding of mSTI1 to Hsc70, while double substitution of these residues abrogated binding. A substitution in the second TPR motif of Asn⁴³ lowered but did not abrogate binding. Similarly, a deletion in the second TPR motif coupled with a substitution of Lys⁸ or Asn¹² reduced but did not abrogate binding. Steady state fluorescence and circular dichroism spectroscopies revealed that the double substitution of Lys⁸ and Asn¹² resulted in perturbations of inter-domain interactions in mSTl1. Together these results suggest that mSTI1-Hsc70 interaction requires a network of electrostatic interactions not only between charged residues in the TPR1 domain of mSTI1 and the EEVD motif of Hsc70, but also outside the TPR1 domain. It is proposed that the electrostatic interactions in the first TPR motif collectively made by Lys⁸ and Asn¹² define part of the minimum interactions required for successful mSTI1-Hsc70 interaction. In the first central TPR domain (TPR1A), single substitution of Lys³°¹ was sufficient to abrogate the mSTI1-Hsp90 interaction. Using a truncated derivative of mSTI1 incapable of binding to Hsp90, residues predicted by crystallographic data to determine Hsp70 binding specificity were substituted in the TPR1 domain. The modified protein had reduced binding to Hsc70, but showed significant binding capacity for Hsp90. In contrast, topologically equivalent substitutions on a truncated derivative of mSTI1 incapable of binding to Hsc70 did not confer Hsc70 specificity on the TPR2A domain. These data suggest that binding of Hsc70 to the TPR1 domain is more specific than binding of Hsp90 to the TPR2A domain. In addition, residues C-terminal of helix A in the second TPR motif of mSTI1 were shown to be important in determining specific binding to Hsc70. Binding assays using surface plasmon resonance spectroscopy showed that the affinities of binding of mSTI1 to Hsc70 and Hsp90 were 2 μM and 1.5 μM respectively. Preliminary in vivo studies revealed differences in the dynamics of binding of endogenous and exogenous recombinant mSTI1 with Hsc70 and Hsp90. The outcome of this study poses serious implications for the mechanisms of mSTI1 interactions with Hsc70 and Hsp90 in the cell.
- Full Text:
- Date Issued: 2003
- Authors: Odunuga, Odutayo Odutola
- Date: 2003
- Subjects: Plants -- Effect of stress on Proteins -- Purification Electrophoresis Heat shock proteins
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4091 , http://hdl.handle.net/10962/d1007724
- Description: Murine stress-inducible protein 1 (mSTI1) is a co-chaperone that is homologous with the human heat shock protein 70 (Hsp70)/heat shock protein 90 (Hsp90)-organizing protein (Hop). The two proteins are homologues of the highly conserved stress-inducible protein 1 (STI1) family of co-chaperones. The STI1 proteins interact directly and simultaneously at some stage, with Hsp70 and Hsp90 in the formation of the hetero-multi-chaperone complexes that facilitate the folding of signal transducing kinases and functional maturation of steroid hormone receptors. The interactions of mSTI1 with both Hsp70 and Hsp90 is mediated by a versatile structural protein-protein interaction motif, the tetratricopeptide repeat (TPR). The TPR motif is a degenerate 34-amino acid sequence a-helical structural motif found in a significant number of functionally unrelated proteins. This study was aimed at characterizing the structural and functional determinants in the TPR domains of mSTI1 responsible for binding to and discriminating between Hsp70 and Hsp90. Guided by data from Hop's crystal structures and amino acid sequence alignment analyses, various biochemical techniques were used to both qualitatively and quantitatively characterize the contacts necessary for the N-terminal TPR domain (TPR1) of mSTI1 to bind to the C-terminal EEVD motif of heat shock cognate protein 70 (Hsc70) and to discriminate between Hsc70 and Hsp90. Substitutions in the first TPR motif of Lys⁸ or Asn¹² did not affect binding of mSTI1 to Hsc70, while double substitution of these residues abrogated binding. A substitution in the second TPR motif of Asn⁴³ lowered but did not abrogate binding. Similarly, a deletion in the second TPR motif coupled with a substitution of Lys⁸ or Asn¹² reduced but did not abrogate binding. Steady state fluorescence and circular dichroism spectroscopies revealed that the double substitution of Lys⁸ and Asn¹² resulted in perturbations of inter-domain interactions in mSTl1. Together these results suggest that mSTI1-Hsc70 interaction requires a network of electrostatic interactions not only between charged residues in the TPR1 domain of mSTI1 and the EEVD motif of Hsc70, but also outside the TPR1 domain. It is proposed that the electrostatic interactions in the first TPR motif collectively made by Lys⁸ and Asn¹² define part of the minimum interactions required for successful mSTI1-Hsc70 interaction. In the first central TPR domain (TPR1A), single substitution of Lys³°¹ was sufficient to abrogate the mSTI1-Hsp90 interaction. Using a truncated derivative of mSTI1 incapable of binding to Hsp90, residues predicted by crystallographic data to determine Hsp70 binding specificity were substituted in the TPR1 domain. The modified protein had reduced binding to Hsc70, but showed significant binding capacity for Hsp90. In contrast, topologically equivalent substitutions on a truncated derivative of mSTI1 incapable of binding to Hsc70 did not confer Hsc70 specificity on the TPR2A domain. These data suggest that binding of Hsc70 to the TPR1 domain is more specific than binding of Hsp90 to the TPR2A domain. In addition, residues C-terminal of helix A in the second TPR motif of mSTI1 were shown to be important in determining specific binding to Hsc70. Binding assays using surface plasmon resonance spectroscopy showed that the affinities of binding of mSTI1 to Hsc70 and Hsp90 were 2 μM and 1.5 μM respectively. Preliminary in vivo studies revealed differences in the dynamics of binding of endogenous and exogenous recombinant mSTI1 with Hsc70 and Hsp90. The outcome of this study poses serious implications for the mechanisms of mSTI1 interactions with Hsc70 and Hsp90 in the cell.
- Full Text:
- Date Issued: 2003
Over-expression, purification and biochemical characterisation of trypanosomal heat shock protein
- Authors: Edkins, Adrienne Lesley
- Date: 2003
- Subjects: Heat shock proteins , Heat shock proteins -- Structure activity relationships
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4106 , http://hdl.handle.net/10962/d1011736 , Heat shock proteins , Heat shock proteins -- Structure activity relationships
- Description: The molecular chaperone process of assisted protein folding, characteristic of members of the Heat Shock Protein 70 kDa (Hsp70) and Heat Shock Protein 40kDa (Hsp40) families, is essential for cytoprotection in stressful cellular conditions. Examples of such conditions are heat shock or invasion by pathogens. The Hsp70/Hsp40 process of assisted protein folding is dependent on ATP (governed by the intrinsic ATPase activity of Hsp70) and the ability of molecular chaperones to recognise and bind non-native protein conformations. Here, we analyse and attempt to characterise the molecular chaperone activity of an inducible, cytoplasmic Hsp70 (TcHsp70) from Trypanosoma cruzi and its interactions with its potential partner Hsp40s, Tcj 1, Tcj2, Tcj3 and Tcj4. A bioinformatic analyses of the primary sequences of the trypanosomal proteins revealed that they all contained the canonical domains that define other members of the Hsp70 and Hsp40 family. Tcj2 and Tcj4 showed deviations from the consensus sequence in their substrate binding regions, which may have implications for their substrate binding specificities. TcHsp70, Tcj 1, Tcj2, Tcj3 and Tcj4 were over-expressed recombinantly as 6xHis-tag fusion proteins in Escherichia coli. His-TcHsp70, Tcjl-His and His-Tcj2 were successfully purified by Nickel-affinity chromatography for functional analyses to assess the molecular chaperone activity of His-TcHsp70 in terms of its ATPase activity and substrate binding ability. The basal ATPase activity of His-TcHsp70 was determined as 40 nmol Pi/min/mg, significantly higher than that reported for other Hsp70s. This basal ATPase activity was stimulated to a maximal level of 60 nmol Pi/min/mg in the presence of His-Tcj2 and a model non-native substrate, reduced carboxymethylated αx-lactalbumin (RCMLA). Using native polyacrylamide gel electrophoresis and Western analysis, His-TcHsp70 was shown to form discrete complexes when in the presence of Tcj 1- His, His-Tcj2 and/or RCMLA. These complexes potentially represent His-TcHsp70 - RCMLA or His-TcHsp70 - Tcj interactions, that may be indicative of chaperone activity. In vivo complementation assays showed that Tcj2, but not Tcj3, was able to overcome the temperature sensitivity of the ydjJ mutant Saccharomyces cerevisiae strain JJ160, suggesting that Tcj2 may be functionally equivalent to the yeast Hsp40 Ydj1.
- Full Text:
- Date Issued: 2003
- Authors: Edkins, Adrienne Lesley
- Date: 2003
- Subjects: Heat shock proteins , Heat shock proteins -- Structure activity relationships
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4106 , http://hdl.handle.net/10962/d1011736 , Heat shock proteins , Heat shock proteins -- Structure activity relationships
- Description: The molecular chaperone process of assisted protein folding, characteristic of members of the Heat Shock Protein 70 kDa (Hsp70) and Heat Shock Protein 40kDa (Hsp40) families, is essential for cytoprotection in stressful cellular conditions. Examples of such conditions are heat shock or invasion by pathogens. The Hsp70/Hsp40 process of assisted protein folding is dependent on ATP (governed by the intrinsic ATPase activity of Hsp70) and the ability of molecular chaperones to recognise and bind non-native protein conformations. Here, we analyse and attempt to characterise the molecular chaperone activity of an inducible, cytoplasmic Hsp70 (TcHsp70) from Trypanosoma cruzi and its interactions with its potential partner Hsp40s, Tcj 1, Tcj2, Tcj3 and Tcj4. A bioinformatic analyses of the primary sequences of the trypanosomal proteins revealed that they all contained the canonical domains that define other members of the Hsp70 and Hsp40 family. Tcj2 and Tcj4 showed deviations from the consensus sequence in their substrate binding regions, which may have implications for their substrate binding specificities. TcHsp70, Tcj 1, Tcj2, Tcj3 and Tcj4 were over-expressed recombinantly as 6xHis-tag fusion proteins in Escherichia coli. His-TcHsp70, Tcjl-His and His-Tcj2 were successfully purified by Nickel-affinity chromatography for functional analyses to assess the molecular chaperone activity of His-TcHsp70 in terms of its ATPase activity and substrate binding ability. The basal ATPase activity of His-TcHsp70 was determined as 40 nmol Pi/min/mg, significantly higher than that reported for other Hsp70s. This basal ATPase activity was stimulated to a maximal level of 60 nmol Pi/min/mg in the presence of His-Tcj2 and a model non-native substrate, reduced carboxymethylated αx-lactalbumin (RCMLA). Using native polyacrylamide gel electrophoresis and Western analysis, His-TcHsp70 was shown to form discrete complexes when in the presence of Tcj 1- His, His-Tcj2 and/or RCMLA. These complexes potentially represent His-TcHsp70 - RCMLA or His-TcHsp70 - Tcj interactions, that may be indicative of chaperone activity. In vivo complementation assays showed that Tcj2, but not Tcj3, was able to overcome the temperature sensitivity of the ydjJ mutant Saccharomyces cerevisiae strain JJ160, suggesting that Tcj2 may be functionally equivalent to the yeast Hsp40 Ydj1.
- Full Text:
- Date Issued: 2003
The bioaccumulation of platinum (IV) from aqueous solution using sulphate reducing bacteria: role of a hydrogenase enzyme
- Authors: Rashamuse, Konanani Justice
- Date: 2003
- Subjects: Sulfur bacteria , Bioremediation , Enzymes -- Metabolism , Platinum , Platinum compounds , Reduction (Chemistry) , Hydrogenation
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4003 , http://hdl.handle.net/10962/d1004063 , Sulfur bacteria , Bioremediation , Enzymes -- Metabolism , Platinum , Platinum compounds , Reduction (Chemistry) , Hydrogenation
- Description: The enzymatic reduction of a high-valence form of metals to a low-valence reduced form has been proposed as a strategy to treat water contaminated with a range of metals and radionuclides. Metal reduction by sulphate reducing bacteria (SRB) is carried out either chemically (involving reduction by hydrogen sulphide) or enzymatically (involving redox enzymes such as the hydrogenases). While reduction of metal ions by hydrogen sulphide is well known, the enzymatic mechanism for metal reduction is poorly understood. The aims of this study were to investigate the role of SRB in facilitating platinum removal, and to investigate the role of a hydrogenase in platinum reduction in vitro. In order to avoid precipitation of platinum as platinum sulphide, a resting (non-growing) mixed SRB culture was used. The maximum initial concentration of platinum (IV), which SRB can effectively remove from solution was shown to be 50 mg.l⁻¹. Electron donor studies showed high platinum (IV) uptake in the presence of hydrogen, suggesting that platinum (IV) uptake from solution by SRB requires careful optimization with respect to the correct electron donor. Transmission electron microscopy (TEM) and energy dispersive X-ray (EDX) analysis indicated that platinum was being precipitated in the periplasm, a major area of hydrogenase activity in SRB. Purification of the hydrogenase by ammonium sulphate precipitation (65%), Toyopearl-Super Q 650S ion exchange and Sephacry 1 S-100 size exclusion chromatography revealed that the hydrogenase was monomeric with a molecular weight of 58 KDa, when analyzed by 12% SDS-PAGE. The purified hydrogenase showed optimal temperature and pH at 35°C and 7.5 respectively, and a poor thermal stability. In vitro investigation of platinum reduction by purified hydrogenase from mixed SRB culture showed that hydrogenase reduces platinum only in the presence of hydrogen. Major platinum (IV) reduction was observed when hydrogenase was incubated with cytochrome C₃ (physiological electron carrier in vivo) under hydrogen. The same observations were also noted with industrial effluent. Collectively these findings suggest that in vitro platinum reduction is mediated by hydrogenase with a concerted action of cytochrome C₃ required to shuttle the electron from hydrogenase.
- Full Text:
- Date Issued: 2003
- Authors: Rashamuse, Konanani Justice
- Date: 2003
- Subjects: Sulfur bacteria , Bioremediation , Enzymes -- Metabolism , Platinum , Platinum compounds , Reduction (Chemistry) , Hydrogenation
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4003 , http://hdl.handle.net/10962/d1004063 , Sulfur bacteria , Bioremediation , Enzymes -- Metabolism , Platinum , Platinum compounds , Reduction (Chemistry) , Hydrogenation
- Description: The enzymatic reduction of a high-valence form of metals to a low-valence reduced form has been proposed as a strategy to treat water contaminated with a range of metals and radionuclides. Metal reduction by sulphate reducing bacteria (SRB) is carried out either chemically (involving reduction by hydrogen sulphide) or enzymatically (involving redox enzymes such as the hydrogenases). While reduction of metal ions by hydrogen sulphide is well known, the enzymatic mechanism for metal reduction is poorly understood. The aims of this study were to investigate the role of SRB in facilitating platinum removal, and to investigate the role of a hydrogenase in platinum reduction in vitro. In order to avoid precipitation of platinum as platinum sulphide, a resting (non-growing) mixed SRB culture was used. The maximum initial concentration of platinum (IV), which SRB can effectively remove from solution was shown to be 50 mg.l⁻¹. Electron donor studies showed high platinum (IV) uptake in the presence of hydrogen, suggesting that platinum (IV) uptake from solution by SRB requires careful optimization with respect to the correct electron donor. Transmission electron microscopy (TEM) and energy dispersive X-ray (EDX) analysis indicated that platinum was being precipitated in the periplasm, a major area of hydrogenase activity in SRB. Purification of the hydrogenase by ammonium sulphate precipitation (65%), Toyopearl-Super Q 650S ion exchange and Sephacry 1 S-100 size exclusion chromatography revealed that the hydrogenase was monomeric with a molecular weight of 58 KDa, when analyzed by 12% SDS-PAGE. The purified hydrogenase showed optimal temperature and pH at 35°C and 7.5 respectively, and a poor thermal stability. In vitro investigation of platinum reduction by purified hydrogenase from mixed SRB culture showed that hydrogenase reduces platinum only in the presence of hydrogen. Major platinum (IV) reduction was observed when hydrogenase was incubated with cytochrome C₃ (physiological electron carrier in vivo) under hydrogen. The same observations were also noted with industrial effluent. Collectively these findings suggest that in vitro platinum reduction is mediated by hydrogenase with a concerted action of cytochrome C₃ required to shuttle the electron from hydrogenase.
- Full Text:
- Date Issued: 2003
The development and evaluation of Cryptophlebia Leucotreta granulovirus (CrleGV) as a biological control agent for the management of false codling moth, Cryptophlebia Leucotreta, on citrus
- Authors: Moore, Sean Douglas
- Date: 2003
- Subjects: Cryptophlebia leucotreta Cryptophlebia leucotreta -- Control Pests -- Biological control Citrus -- Diseases and pests
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3942 , http://hdl.handle.net/10962/d1004001
- Description: A granulovirus isolated from Cryptophlebia leucotreta larvae was shown through restriction endonuclease analysis to be a novel strain (CrleGV-SA). No more than one isolate could be identified from a laboratory culture of C. leucotreta. However, a preliminary examination of restricted DNA profiles of isolates from different geographical regions indicated some minor differences. In surface dose bioassays on artificial diet, LC50 and LC90 values with neonate larvae were estimated to be 4.095 x 103 OBs/ml and 1.185 x 105 OBs/ml respectively. LT50 and LT90 values with neonate larvae were estimated to be 4 days 22 h and 7 days 8 h, respectively. Detached fruit (navel orange) bioassays with neonate larvae indicated that virus concentrations that are likely to be effective in the field range from 1.08 x 107 to 3.819 x 1010 OBs/ml. In surface dose bioassays with fifth instar larvae LC50 and LC90 values were estimated to be 2.678 x 107 OBs/ml and 9.118 x 109 OBs/ml respectively. LT50 and LT90 values were estimated to be 7 days 17 h and 9 days 8 h, respectively. A new artificial diet for mass rearing the host was developed. Microbial contamination of diet was significantly reduced by adding nipagin and sorbic acid to the diet and by surface sterilising C. leucotreta eggs with Sporekill. Almost 20 % more eggs were produced from moths reared on the new diet compared to moths reared on the old diet. A further 9 % improvement in egg production and a reduction in the labour required to produce eggs, was made with the development of a new oviposition cage attached to the moth eclosion box. Virus was mass produced in fifth instar C. leucotreta larvae by surface inoculating diet with the LC90. When 300 individuals were placed onto inoculated diet, 56 % of them were recovered six to 11 days later as infected larvae. Mean larval equivalents was 1.158 x 1011 OBs/larva. When larvae and diet were harvested together, highest yields of virus were achieved at eight days after inoculation. Microbial contamination in semi-purified preparations of CrleGV ranged from 176211 to 433594 (OB:CFU ratio). Half-life of CrleGV in the field was estimated to be less than 1 day on the northern aspect of trees and between 3 - 6 days on the southern aspect. Original activity remaining (OAR) of the virus dropped below 50 % after 5 days on the northern aspect of trees and was still at 69 % on the southern aspect of trees after 3 weeks. In field trials, CrleGV reduced C. leucotreta infestation of navel oranges by up to 60 % for a period of 39 days. CrleGV in combination with augmentation of the C. leucotreta egg parasitoid, Trichogrammatoidea cryptophlebiae, reduced infestation by 70 %. The integration of CrleGV into an integrated pest management (IPM) system for the management of C. leucotreta on citrus is proposed.
- Full Text:
- Date Issued: 2003
- Authors: Moore, Sean Douglas
- Date: 2003
- Subjects: Cryptophlebia leucotreta Cryptophlebia leucotreta -- Control Pests -- Biological control Citrus -- Diseases and pests
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3942 , http://hdl.handle.net/10962/d1004001
- Description: A granulovirus isolated from Cryptophlebia leucotreta larvae was shown through restriction endonuclease analysis to be a novel strain (CrleGV-SA). No more than one isolate could be identified from a laboratory culture of C. leucotreta. However, a preliminary examination of restricted DNA profiles of isolates from different geographical regions indicated some minor differences. In surface dose bioassays on artificial diet, LC50 and LC90 values with neonate larvae were estimated to be 4.095 x 103 OBs/ml and 1.185 x 105 OBs/ml respectively. LT50 and LT90 values with neonate larvae were estimated to be 4 days 22 h and 7 days 8 h, respectively. Detached fruit (navel orange) bioassays with neonate larvae indicated that virus concentrations that are likely to be effective in the field range from 1.08 x 107 to 3.819 x 1010 OBs/ml. In surface dose bioassays with fifth instar larvae LC50 and LC90 values were estimated to be 2.678 x 107 OBs/ml and 9.118 x 109 OBs/ml respectively. LT50 and LT90 values were estimated to be 7 days 17 h and 9 days 8 h, respectively. A new artificial diet for mass rearing the host was developed. Microbial contamination of diet was significantly reduced by adding nipagin and sorbic acid to the diet and by surface sterilising C. leucotreta eggs with Sporekill. Almost 20 % more eggs were produced from moths reared on the new diet compared to moths reared on the old diet. A further 9 % improvement in egg production and a reduction in the labour required to produce eggs, was made with the development of a new oviposition cage attached to the moth eclosion box. Virus was mass produced in fifth instar C. leucotreta larvae by surface inoculating diet with the LC90. When 300 individuals were placed onto inoculated diet, 56 % of them were recovered six to 11 days later as infected larvae. Mean larval equivalents was 1.158 x 1011 OBs/larva. When larvae and diet were harvested together, highest yields of virus were achieved at eight days after inoculation. Microbial contamination in semi-purified preparations of CrleGV ranged from 176211 to 433594 (OB:CFU ratio). Half-life of CrleGV in the field was estimated to be less than 1 day on the northern aspect of trees and between 3 - 6 days on the southern aspect. Original activity remaining (OAR) of the virus dropped below 50 % after 5 days on the northern aspect of trees and was still at 69 % on the southern aspect of trees after 3 weeks. In field trials, CrleGV reduced C. leucotreta infestation of navel oranges by up to 60 % for a period of 39 days. CrleGV in combination with augmentation of the C. leucotreta egg parasitoid, Trichogrammatoidea cryptophlebiae, reduced infestation by 70 %. The integration of CrleGV into an integrated pest management (IPM) system for the management of C. leucotreta on citrus is proposed.
- Full Text:
- Date Issued: 2003
The development of a baculovirus expression system for the production of Helicoverpa armigera stunt virus capsids for use in the encapsidation of foreign molecules
- Mosisili, Kekeletso Mpho Thakane
- Authors: Mosisili, Kekeletso Mpho Thakane
- Date: 2003
- Subjects: Helicoverpa armigera , Fall armyworm , Baculoviruses , Insects -- Viruses , RNA -- Analysis
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4088 , http://hdl.handle.net/10962/d1007700 , Helicoverpa armigera , Fall armyworm , Baculoviruses , Insects -- Viruses , RNA -- Analysis
- Description: The capsid protein of Helicoverpa armigera stunt virus (HaSV) a T=4 insect virus was expressed in Spodoptera frugiperda 9 cells using a baculovirus vector. When the insect cells were infected at a high MOl the expressed coat protein assembled into virus-like particles (VLPs) that spontaneously underwent maturation and were morphologically indistinguishable from wild-type HaSV. The VLPs were electron dense when viewed under EM and encapsidated their coat protein mRNA. When Sf9 cells were infected at a low multiplicity of infection (MOl) the expressed capsid protein assembled into procapsids that did not spontaneously undergo maturation. These procapsids underwent autoproteolytic maturation cleavage when they were treated with an acidic buffer. The procapsids were used in the encapsidation of a FITC labelled peptide. The peptide encapsidating VLPs showed an increase in their buoyant density that was not collaborated by an increase in the concentration of the FITC labelled peptide detected when these samples were compared to control samples with similar buoyant densities.
- Full Text:
- Date Issued: 2003
- Authors: Mosisili, Kekeletso Mpho Thakane
- Date: 2003
- Subjects: Helicoverpa armigera , Fall armyworm , Baculoviruses , Insects -- Viruses , RNA -- Analysis
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4088 , http://hdl.handle.net/10962/d1007700 , Helicoverpa armigera , Fall armyworm , Baculoviruses , Insects -- Viruses , RNA -- Analysis
- Description: The capsid protein of Helicoverpa armigera stunt virus (HaSV) a T=4 insect virus was expressed in Spodoptera frugiperda 9 cells using a baculovirus vector. When the insect cells were infected at a high MOl the expressed coat protein assembled into virus-like particles (VLPs) that spontaneously underwent maturation and were morphologically indistinguishable from wild-type HaSV. The VLPs were electron dense when viewed under EM and encapsidated their coat protein mRNA. When Sf9 cells were infected at a low multiplicity of infection (MOl) the expressed capsid protein assembled into procapsids that did not spontaneously undergo maturation. These procapsids underwent autoproteolytic maturation cleavage when they were treated with an acidic buffer. The procapsids were used in the encapsidation of a FITC labelled peptide. The peptide encapsidating VLPs showed an increase in their buoyant density that was not collaborated by an increase in the concentration of the FITC labelled peptide detected when these samples were compared to control samples with similar buoyant densities.
- Full Text:
- Date Issued: 2003
The establishment of a virus free laboratory colony of Cryptophlebia leucotreta (False Codling Moth) and characterisation of Cryptophlebia leucotreta Granulovirus (CrleGV) genes
- Authors: Ludewig, Michael Hans
- Date: 2003
- Subjects: Cryptophlebia leucotreta , Cryptophlebia leucotreta -- Control , Pests -- Biological control , DNA viruses , Agricultural pests -- Biological control , Baculoviruses
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3957 , http://hdl.handle.net/10962/d1004016 , Cryptophlebia leucotreta , Cryptophlebia leucotreta -- Control , Pests -- Biological control , DNA viruses , Agricultural pests -- Biological control , Baculoviruses
- Description: Cryptophlebia leucotreta is an economically important agricultural pest throughout Sub-Saharan Africa. CrleGV has been considered as an alternative to chemical control of this pest due to its host specificity and innocuous nature towards vertebrates. A CrleGV free laboratory colony of C. leucotreta would be useful for the isolation of genotypically pure strains of the CrleGV and for virulence comparisons between isolates. It is preferable to have a full characterisation of CrleGV prior to its registration and release into the environment as a biopesticide. A laboratory colony of C. leucotreta, set up at Rhodes University, containing a low level of infection indicated that CrleGV is vertically transmitted. To establish a virus free laboratory colony of C. leucotreta, a solution of 3.5% sodium hypochlorite and 1% Tween 20 was used to surface decontaminate C. leucotreta eggs for removal of transovum CrleGV from the laboratory colony. No apparent infection by CrleGV was induced by subjecting larvae to stress. PCR of DNA extracted from larvae using CTAB failed to detect virus in the laboratory colony. This detection protocol was able to detect down to 60 fg (480 genome copies of CrleGV). The possibility of low-level virus remaining in the colony requires monitoring of genotypic purity of virus manipulated in the colony. Sequencing of Bam HI/KpnI fragments produced a preliminary sequence of the granulin region of CrleGV. This preliminary sequence supports the trend that the gene organisation of the granulin region of the granuloviruses infecting the family Tortricidae is conserved.
- Full Text:
- Date Issued: 2003
- Authors: Ludewig, Michael Hans
- Date: 2003
- Subjects: Cryptophlebia leucotreta , Cryptophlebia leucotreta -- Control , Pests -- Biological control , DNA viruses , Agricultural pests -- Biological control , Baculoviruses
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3957 , http://hdl.handle.net/10962/d1004016 , Cryptophlebia leucotreta , Cryptophlebia leucotreta -- Control , Pests -- Biological control , DNA viruses , Agricultural pests -- Biological control , Baculoviruses
- Description: Cryptophlebia leucotreta is an economically important agricultural pest throughout Sub-Saharan Africa. CrleGV has been considered as an alternative to chemical control of this pest due to its host specificity and innocuous nature towards vertebrates. A CrleGV free laboratory colony of C. leucotreta would be useful for the isolation of genotypically pure strains of the CrleGV and for virulence comparisons between isolates. It is preferable to have a full characterisation of CrleGV prior to its registration and release into the environment as a biopesticide. A laboratory colony of C. leucotreta, set up at Rhodes University, containing a low level of infection indicated that CrleGV is vertically transmitted. To establish a virus free laboratory colony of C. leucotreta, a solution of 3.5% sodium hypochlorite and 1% Tween 20 was used to surface decontaminate C. leucotreta eggs for removal of transovum CrleGV from the laboratory colony. No apparent infection by CrleGV was induced by subjecting larvae to stress. PCR of DNA extracted from larvae using CTAB failed to detect virus in the laboratory colony. This detection protocol was able to detect down to 60 fg (480 genome copies of CrleGV). The possibility of low-level virus remaining in the colony requires monitoring of genotypic purity of virus manipulated in the colony. Sequencing of Bam HI/KpnI fragments produced a preliminary sequence of the granulin region of CrleGV. This preliminary sequence supports the trend that the gene organisation of the granulin region of the granuloviruses infecting the family Tortricidae is conserved.
- Full Text:
- Date Issued: 2003
The hydrolysis of primary sewage sludge under biosulphidogenic conditions
- Molwantwa, Jennifer Balatedi
- Authors: Molwantwa, Jennifer Balatedi
- Date: 2003
- Subjects: Sewage sludge , Hydrolysis , Sewage -- Purification -- Activated sludge process
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3961 , http://hdl.handle.net/10962/d1004020 , Sewage sludge , Hydrolysis , Sewage -- Purification -- Activated sludge process
- Description: The potential for using readily available and cost-effective complex carbon sources such as primary sewage sludge for a range of environmental remediation processes, including biological sulphate reduction, biological nutrient removal and the bioremediation of acid mine drainage, has been constrained by the slow rate of solubilization and low yield of soluble products, which drive the above mentioned processes. Previous work conducted by the Environmental Biotechnology Group at Rhodes University indicated that the degradation of primary sewage sludge was enhanced under sulphate reducing conditions. This was proven in both laboratory and pilot-scale (Reciprocating Sludge Bed Reactor) systems, where the particulate matter accumulated in the sludge bed and the molecules in smaller flocs were rapidly solubilized. The current study was aimed at investigating in more detail the factors that govern the enhanced hydrolysis under sulphate reducing conditions, and to develop a descriptive model to explain the underlying mechanism involved. The solubilization of primary sewage sludge under sulphate reducing conditions was conducted in controlled flask studies and previously reported findings of enhanced hydrolysis were confirmed. The maximum percentage solubilization obtained in this study was 31% and 63% for the methanogenic and sulphidogenic systems respectively, and this was achieved over a period of 10 days. A rate of reducing sugar production and complex molecule breakdown of 51 mg. L⁻¹.hr⁻¹ and 167 mg.L⁻¹.hr⁻¹ was observed for the methanogenic and sulphidogenic systems respectively. The flask studies revealed that during hydrolysis of primary sewage sludge under sulphidogenic conditions there was enhanced production of soluble products, specifically carbohydrates (reducing sugars) and volatile fatty acids, compared to methanogenic conditions. The rate at which these products were utilized was also found to be more rapid under sulphidogenic as compared to methanogenic conditions. A study of the distribution of volatile fatty acids indicated that acetate was utilized preferentially in the methanogenic system, and that propionate, butyrate and valerate accumulated with time. The converse was found to occur in the sulphidogenic system. The descriptive model developed from the results of this study was based on the fact that a consortium of bacteria, composed of hydrolytic, acidogenic and acetogenic species, carries out the solubilization of complex carbon sources. Furthermore, it is essential that equilibrium between product formation and utilization is maintained, and that accumulation of soluble end products impacts negatively on the rate of the hydrolysis step. It is therefore proposed that the relatively poor utilization of VFA and reducing sugars in the methanogenic system activates a negative feedback inhibition on the hydrolytic and/ or acidogenic step. This inhibition is reduced in the sulphidogenic system where the utilization of end products is higher.
- Full Text:
- Date Issued: 2003
- Authors: Molwantwa, Jennifer Balatedi
- Date: 2003
- Subjects: Sewage sludge , Hydrolysis , Sewage -- Purification -- Activated sludge process
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3961 , http://hdl.handle.net/10962/d1004020 , Sewage sludge , Hydrolysis , Sewage -- Purification -- Activated sludge process
- Description: The potential for using readily available and cost-effective complex carbon sources such as primary sewage sludge for a range of environmental remediation processes, including biological sulphate reduction, biological nutrient removal and the bioremediation of acid mine drainage, has been constrained by the slow rate of solubilization and low yield of soluble products, which drive the above mentioned processes. Previous work conducted by the Environmental Biotechnology Group at Rhodes University indicated that the degradation of primary sewage sludge was enhanced under sulphate reducing conditions. This was proven in both laboratory and pilot-scale (Reciprocating Sludge Bed Reactor) systems, where the particulate matter accumulated in the sludge bed and the molecules in smaller flocs were rapidly solubilized. The current study was aimed at investigating in more detail the factors that govern the enhanced hydrolysis under sulphate reducing conditions, and to develop a descriptive model to explain the underlying mechanism involved. The solubilization of primary sewage sludge under sulphate reducing conditions was conducted in controlled flask studies and previously reported findings of enhanced hydrolysis were confirmed. The maximum percentage solubilization obtained in this study was 31% and 63% for the methanogenic and sulphidogenic systems respectively, and this was achieved over a period of 10 days. A rate of reducing sugar production and complex molecule breakdown of 51 mg. L⁻¹.hr⁻¹ and 167 mg.L⁻¹.hr⁻¹ was observed for the methanogenic and sulphidogenic systems respectively. The flask studies revealed that during hydrolysis of primary sewage sludge under sulphidogenic conditions there was enhanced production of soluble products, specifically carbohydrates (reducing sugars) and volatile fatty acids, compared to methanogenic conditions. The rate at which these products were utilized was also found to be more rapid under sulphidogenic as compared to methanogenic conditions. A study of the distribution of volatile fatty acids indicated that acetate was utilized preferentially in the methanogenic system, and that propionate, butyrate and valerate accumulated with time. The converse was found to occur in the sulphidogenic system. The descriptive model developed from the results of this study was based on the fact that a consortium of bacteria, composed of hydrolytic, acidogenic and acetogenic species, carries out the solubilization of complex carbon sources. Furthermore, it is essential that equilibrium between product formation and utilization is maintained, and that accumulation of soluble end products impacts negatively on the rate of the hydrolysis step. It is therefore proposed that the relatively poor utilization of VFA and reducing sugars in the methanogenic system activates a negative feedback inhibition on the hydrolytic and/ or acidogenic step. This inhibition is reduced in the sulphidogenic system where the utilization of end products is higher.
- Full Text:
- Date Issued: 2003
The phosphorylation and nuclear localization of the co-chaperone murine stress-inducible protein 1
- Authors: Longshaw, Victoria Mary
- Date: 2003
- Subjects: Phosphorylation Proteins Heat shock proteins
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3979 , http://hdl.handle.net/10962/d1004038
- Description: The co-chaperone murine stress-inducible protein 1 (mSTI1), a heat shock protein 70 (Hsp70)/ heat shock protein 90 (Hsp90) organizing protein (Hop) homologue, mediates the assembly of the Hsp70/Hsp90 chaperone heterocomplex. mSTI1 is phosphorylated in vitro by cell cycle kinases, proximal to a putative nuclear localization signal (NLS), substantiating a predicted CKII-cdc2-NLS (CcN) motif at position 189-239. Stable transfectants of NIH 3T3 fibroblasts that expressed mSTI1-EGFP, NLSmSTI1-EGFP and EGFP, were prepared. Fluorescence microscopy revealed mSTI1 was cytoplasmically localized, and that this localization was not affected by the fusion of mSTI1 with the EGFP moiety. NLSmSTI1-EGFP was targeted to the nucleus compared to EGFP, suggesting that the NLSmSTI1 was a functional NLS. The localization of mSTI1 was determined under normal and heat shock conditions, inhibition of nuclear export (leptomycin B), inhibition of CKII 5,6-dichlorobenzimidazole riboside, DRB), inhibition of cdc2 kinase (olomoucine), and G1/S phase arrest (hydroxyurea). mSTI1-EGFP and mSTI1 were excluded from the nucleus in the majority of resting cells, but accumulated in the nucleus following leptomycin B treatment, implying that mSTI1 possibly undergoes a functional import process, and export via the chromosomal region maintenance 1 (CRM-1)-mediated export pathway. Hydroxyurea and olomoucine (but not DRB or heat shock) treatment increased the proportion of cells in which mSTI1-EGFP exhibited cytoplasmic and nuclear localization. 2D gel electrophoresis detected three endogenous mSTI1 isoforms, which changed following hydroxyurea treatment. Furthermore, point inactivation and mimicking of phosphorylatable residues in mSTI1 altered the translocation of the protein and the isoform composition. Modification of mSTI1 at S189 and T198 decreased the number of isoforms of mSTI1-EGFP, suggesting that the protein is modified at these sites in vivo. The removal of the in vitro cdc2 kinase site at T198 promoted a nuclear localization during G1/S phase arrest. Therefore active cdc2 kinase, but not CKII, may be required for cytoplasmic localization of mSTI1. The CKII site appears to have no regulatory role under heat shock conditions or during the cell cycle. In vitro phosphorylation studies on untagged mSTI1 further supported the prediction that S189 is the only site recognised by CKII. The cdc2 kinase site at T198, however, although the major site, was not the only site phosphorylated in vitro. However, mSTI1 and cdc2 kinase did not interact in a detectable stable complex. Bioinformatic analysis of mSTI1 revealed NLS residues were conserved in STI1 proteins, and the NLS and TPR2A motifs were in close proximity. This may have mechanistic implications for the formation of the Hsp90-mSTI1 heterocomplex. The cytoplasmic or nuclear localization of mSTI1 is predicted to be the result of a dynamic equilibrium between nuclear import and nuclear export, the fulcrum of which may be shifted under different cell cycle conditions. These data provide the first evidence of regulated nuclear import/export of a major Hsp70/Hsp90 co-chaperone, and the regulation of this nuclear import by cell cycle status and cell cycle kinases.
- Full Text:
- Date Issued: 2003
- Authors: Longshaw, Victoria Mary
- Date: 2003
- Subjects: Phosphorylation Proteins Heat shock proteins
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3979 , http://hdl.handle.net/10962/d1004038
- Description: The co-chaperone murine stress-inducible protein 1 (mSTI1), a heat shock protein 70 (Hsp70)/ heat shock protein 90 (Hsp90) organizing protein (Hop) homologue, mediates the assembly of the Hsp70/Hsp90 chaperone heterocomplex. mSTI1 is phosphorylated in vitro by cell cycle kinases, proximal to a putative nuclear localization signal (NLS), substantiating a predicted CKII-cdc2-NLS (CcN) motif at position 189-239. Stable transfectants of NIH 3T3 fibroblasts that expressed mSTI1-EGFP, NLSmSTI1-EGFP and EGFP, were prepared. Fluorescence microscopy revealed mSTI1 was cytoplasmically localized, and that this localization was not affected by the fusion of mSTI1 with the EGFP moiety. NLSmSTI1-EGFP was targeted to the nucleus compared to EGFP, suggesting that the NLSmSTI1 was a functional NLS. The localization of mSTI1 was determined under normal and heat shock conditions, inhibition of nuclear export (leptomycin B), inhibition of CKII 5,6-dichlorobenzimidazole riboside, DRB), inhibition of cdc2 kinase (olomoucine), and G1/S phase arrest (hydroxyurea). mSTI1-EGFP and mSTI1 were excluded from the nucleus in the majority of resting cells, but accumulated in the nucleus following leptomycin B treatment, implying that mSTI1 possibly undergoes a functional import process, and export via the chromosomal region maintenance 1 (CRM-1)-mediated export pathway. Hydroxyurea and olomoucine (but not DRB or heat shock) treatment increased the proportion of cells in which mSTI1-EGFP exhibited cytoplasmic and nuclear localization. 2D gel electrophoresis detected three endogenous mSTI1 isoforms, which changed following hydroxyurea treatment. Furthermore, point inactivation and mimicking of phosphorylatable residues in mSTI1 altered the translocation of the protein and the isoform composition. Modification of mSTI1 at S189 and T198 decreased the number of isoforms of mSTI1-EGFP, suggesting that the protein is modified at these sites in vivo. The removal of the in vitro cdc2 kinase site at T198 promoted a nuclear localization during G1/S phase arrest. Therefore active cdc2 kinase, but not CKII, may be required for cytoplasmic localization of mSTI1. The CKII site appears to have no regulatory role under heat shock conditions or during the cell cycle. In vitro phosphorylation studies on untagged mSTI1 further supported the prediction that S189 is the only site recognised by CKII. The cdc2 kinase site at T198, however, although the major site, was not the only site phosphorylated in vitro. However, mSTI1 and cdc2 kinase did not interact in a detectable stable complex. Bioinformatic analysis of mSTI1 revealed NLS residues were conserved in STI1 proteins, and the NLS and TPR2A motifs were in close proximity. This may have mechanistic implications for the formation of the Hsp90-mSTI1 heterocomplex. The cytoplasmic or nuclear localization of mSTI1 is predicted to be the result of a dynamic equilibrium between nuclear import and nuclear export, the fulcrum of which may be shifted under different cell cycle conditions. These data provide the first evidence of regulated nuclear import/export of a major Hsp70/Hsp90 co-chaperone, and the regulation of this nuclear import by cell cycle status and cell cycle kinases.
- Full Text:
- Date Issued: 2003