The effect of combined vitamin E succinate and ascorbic acid supplementation on growth and cyclooxygenase expression in murine melanoma (BL6) cells
- Authors: Van Rooyen, Megan Lynne
- Date: 1999
- Subjects: Vitamin E , Vitamin C , Melanoma
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3982 , http://hdl.handle.net/10962/d1004041 , Vitamin E , Vitamin C , Melanoma
- Description: This thesis examines the effect of combined vitamin E succinate and Asc supplementation on the in vitro growth of a non-malignant monkey kidney (LLCMK) and a malignant melanoma (BL6) cell line, with nutritional concentration ranges of 5-20µg/ml and 25-50µg/ml respectively. Vitamin E and C are thought to interact synergistically to inhibit tumour cell growth by virtue of their antioxidant properties, whereby they quench free radicals and terminate lipid peroxidation. Furthermore vitamin E and C are thought to modulate the biosynthetic pathways in arachidonic acid metabolism at a number of different points. This may also offer a means of regulating tumour cell growth. It is well documented that vitamin E and C are distributed in the lipid and aqueous phases in the cell respectively. However, the cells need to obtain the vitamins from the environment in which they are found in order to exert a growth inhibitory effect. Supplementation of combined vitamin E succinate and Asc on BL6 and LLCMK cells resulted in a significant increase in LLCMK cell growth, and a significant decrease in cell growth was observed in BL6 cells. Vitamin E succinate in its esterified form cannot function as an antioxidant and requires the cleavage of the succinate to become an active antioxidant. The metabolism of vitamin E succinate to form free vitamin E in LLCMK and BL6 cells resulted in the cleavage of the succinate group from the vitamin E molecule in BL6 cells only, thus suggesting that an esterase may be present in BL6 cells. This would allow for a synergistic interaction between the two vitamins. The arachidonic acid cascade generates a family of bioactive lipids that modulate diverse physiological and pathological responses including tumour growth and promotion. The enzyme prostaglandin endoperoxide synthase (PGHS) or cyclooxygenase (Cox) is the key enzyme in the biosynthetic pathway leading to the formation of prostaglandins. Two enzyme isoforms of Cox have been identified, Cox 1 and Cox 2. Supplementation with vitamin E succinate and Asc at a combination 20:25µg/ml respectively resulted in a trend of increasing Cox activity over 12 hours suggesting that vitamin E and Asc have a stimulatory effect on Cox activity in BL6 cells. The inhibitors of Cox 2, dexamethasone, showed a decreasing trend in Cox activity at the 20:25µg/ml combination, while cycloheximide showed an initial stimulatory effect and then a gradual decrease in Cox activity. The elimination of the Cox activity by dexamethasone suggests that transcriptional regulation may be occurring in BL6 cells. We examined by Northern blot analysis whether combined supplementation of vitamin E succinate and Asc caused an elevation of Cox 2 RNA expression in BL6 cells. An inducible effect of Cox 2 was observed after 2 hours of supplementation with a combination of vitamin E succinate and Asc in BL6 cells, however the results are inconclusive and further studies are required to substantiate this finding.
- Full Text:
- Date Issued: 1999
The isolation and characterisation of thermostable hydantoinases from hydantoinase-producing bacteria
- Authors: Phehane, Vuyisile Ntosi
- Date: 1999
- Subjects: Hydantoin , Bacteria -- Physiology , Enzymes
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3998 , http://hdl.handle.net/10962/d1004058 , Hydantoin , Bacteria -- Physiology , Enzymes
- Description: In order to characterise thermostable hydantoin-hydrolysing enzymes from bacteria, locally-isolated thermophilic organisms were screened for the ability to convert hydantoin to N-carbamylglycine at 55°C using the hydantoinase enzyme. Cell disruption of a selected strain, RU-20-15, was conducted by French pressing to release enzyme from within the cell. In all of the experiments conducted, the amounts of product were low. In view of the low yields of products formed by the thermophiles, a previously-isolated Gram negative strain, RU-KM3L was selected from a number of mesophiles by screening for hydantoinase and carbamylase activity over a 40-55°C temperature range. Hydantoin conversion at 40°C using crude extract from pressed cells of this organism was similar to conversion at 50°C, and therefore subsequent assays were conducted at the higher temperature. The growth kinetics of RU-KM3L cells were studied and the enzyme activities of the extracts were compared in complete and chemically-defined media. The results suggested that the optimal time to harvest cells was at early stationary phase, when using complete medium for culture of cells; the specific activity of enzyme extracts produced by culture in complete medium was higher than that obtained in chemically-defined medium. 5-methylhydantoin was shown to be the preferred substrate for both the hydantoinase and carbamylase enzymes in the crude extract of RU-KM3L. The substrate specificity of the hydantoinase and carbamylase enzymes of the crude RU-KM3L extract was observed to be altered in the presence of increasing amounts of hydantoin, 5,5-dihydrouracil (DHU) and 5-thiouracil (TU) as inducers, showing selectivity for 5-methylhydantoin over hydantoin at inducer concentrations of 0.1 to 1%. A limiting effect on the hydrolysis of 5-methylhydantoin was observed when DHU and 5,5-dimethylhydantoin (DMH) were used as inducers, while the limiting effect on hydantoin specificity was observed when DHU and TU were used as inducers. The limiting effect was observed to be dependent upon the concentration of inducer, and was not observed when hydantoin was used as an inducer. The optimal time for assay of the hydantoinase enzyme in crude extract preparations at 50°C was observed to be 3h. Alkaline conditions were shown to be optimal for both the hydantoinase and carbamylase enzymes of RU-KM3L. Assay for enzyme activities of RU-KM3L extract in the presence of metal ions showed Mn²⁺ ions (and to a lesser extent, Co²⁺) to activate both the hydantoinase and carbamylase activities. Cu²⁺ ions were observed to inhibit the hydantoinase enzyme. In order to determine the location of the enzymes within the cell, cell debris from disrupted cells of RU-KM3L was removed by centrifugation. A decrease in enzyme activity in the supernatant was observed, and suggested association of the enzymes with the cell membrane. Ammonium sulfate fractionation experiments conducted on the crude extract provided further evidence for this result. Sonication of the crude enzyme extract was the only successful method for the releasing of membrane-associated enzyme. Of a number of strategies investigated, the use of sucrose at 50% (w/v) concentration was shown to preserve the hydantoinase and carbamylase enzyme activities during lyophilisation. Furthermore, assay for these enzyme activities showed the activities to be higher after lyophilisation in the presence of sucrose. However, sucrose did not increase the thermostability of lyophilised crude enzyme extracts. Water-miscible organic solvents at 1% concentration were shown to be inhibitory to the hydantoinase and carbamylase enzymes of RU-KM3L, and the inhibition was also observed to increase with increasing concentrations of these solvents. Hydantoinase activity in the presence of water-immiscible organic solvents was shown to increase with an increase in the hydrophobicity of these solvents, but the activity observed was not significantly higher than activity in the absence of solvent when hydantoin and 5-methylhydantoin were used as substrates. The possibility of reversing the hydantoinase enzyme reaction by water-immiscible organic solvents was investigated, and the results obtained suggested that the reaction could be reversed. It was thought that the partitioning of substrates or products into hydrophobic organic solvents could influence the reaction equilibrium, but the partitioning observed was not sufficient to affect reaction rates. Peptide synthesis was shown to have occurred in small amounts when the hydantoinase reaction was carried out in the presence of water-immiscible organic solvents. In conclusion, the hydantoin-hydrolyzing enzyme activity of a crude extract preparation from the bacterial strain RU-KM3L was characterised at elevated temperatures, and in the presence of watermiscible and -immiscible organic solvents.
- Full Text:
- Date Issued: 1999
The removal of toxic heavy metals from aqueous solutions by algal extracellular polysaccharides
- Authors: Selepe, Mamaropeng Marcus
- Date: 1999
- Subjects: Heavy metals -- Absorption and adsorption , Copper , Lead , Algae -- Biotechnology , Polysaccharides -- Biotechnology
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3994 , http://hdl.handle.net/10962/d1004054 , Heavy metals -- Absorption and adsorption , Copper , Lead , Algae -- Biotechnology , Polysaccharides -- Biotechnology
- Description: This study investigated the possible use of algal extracellular polysaccharide as a biosorbent for removal of heavy metals (copper and lead) from aqueous solutions as a means of bioremediation for metal containing effluents. This biopolymer has good biosorbent properties and a potential to provide a cost effective, selective and efficient purification system. A variety of environmental conditions induce the production of extracellular polysaccharides in algae. The production of exopolysaccharides by Dunaliella cultures was induced by nitrogen deficient conditions. A high ratio of carbon to nitrogen source considerably enhanced the polysaccharide release. Purified extracellular polysaccharide samples exhibited a monosaccharide composition consisting of the following sugars: xylose, arabinose, 2-0-methyl mannose, mannose, glucose and galactose. The relative abundance (%) of these sugars were calculated relative to xylose. The major sugar constituent was 2-0-methyl mannose, which was present at approximately 160% relative to xylose. The percentage relative abundance of other sugars was as follows: 18.8; 86.8; 85.3 and 22.3% for arabinose; mannose; glucose and galactose respectively. The identity of the various constituents were confirmed by mass spectrometry. The ability of Dunaliella exopolysaccharides to accumulate metals was investigated. The following parameters were studied because they affect metal uptake: solution pH, biomass concentration, temperature, time and metal concentration. The uptake of both copper and lead were pH dependent. However, metal uptake was not significantly affected by temperature. Kinetic studies showed that Dunaliella extracellular polysaccharides exhibit good bioremediation properties. Metal uptake was rapid. In addition, the exopolysaccharide has good metal binding capacity with an uptake capacity for lead of 80 mg/g from a solution containing initial lead concentration of approximately 40 mg/l. Competition studies revealed that the presence of a second metal in solution inhibits uptake of the other metal compared to uptake in single metal solution of that particular metal. The presence of lead inhibited the uptake of copper from approximately 65% in single metal solution to 10% in binary metal solution. The presence of copper also inhibited lead uptake, though not to the same extent. Higher concentrations of lead could not completely prevent removal of copper from solution and visa versa. The same was true for lead which could not be displaced by a four-fold concentration of copper. Instead, a certain percentage of copper was always removed showing that lead did not compete with copper for these binding sites. In conclusion it appears that, copper and lead bind to different sites on Dunaliella exopolysaccharides and that they exhibit selective or preferential removal of lead.
- Full Text:
- Date Issued: 1999
The role of vitamin E succinate in regulation of growth and cyclooxygenase expression in B16 murine melanoma cells
- Authors: Van der Merwe, Adele Shanette
- Date: 1999
- Subjects: Melanoma , Vitamin E
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3988 , http://hdl.handle.net/10962/d1004047 , Melanoma , Vitamin E
- Description: This study was undertaken to determine the effects and possible mechanism of action of vitamin E succinate supplementation on B16 murine melanoma cell growth in vitro. Studies revealed that supplementation of 5, 7 and 10µg/ml of this vitamin significantly inhibited growth of B16 cells. Non-malignant LLCMK cells supplemented with the same concentrations of vitamin E succinate resulted in similar inhibition of cell growth. The actual mechanism by which vitamin E succinate inhibits B16 cell growth is unclear, though there has been much speculation about its possible role as an antioxidant. Vitamin E succinate is not a physiological antioxidant and for this ester to behave as an antioxidant, cleavage of the ester bond must occur, releasing the antioxidant vitamin E part of the molecule. To determine whether the observed inhibitory effects on B16 cell growth were due to the intact vitamin E succinate or the vitamin E cleavage product, cleavage studies were undertaken. Results from these studies revealed that in B16 cells vitamin E succinate cleavage did not occur suggesting that the observed inhibitory effects of vitamin E succinate on B16 cells were due to the intact compound. In contrast vitamin E succinate cleavage was shown to occur in LLCMK cells, suggesting that these cells may contain an esterase capable of liberating succinic acid and vitamin E. Further studies focussed on the possible role of vitamin E succinate in regulation of cyclooxygenase activity in B16 cells as vitamin E succinate was found to effect the activity of various enzymes involved in the arachidonic acid cascade, notably cyclooxygenase, the rate-limiting enzyme in prostaglandin synthesis. Time course studies were used to determine when the cyclooxygenase protein was being produced, thus allowing an estimation of when the gene was being 'switched on'. These studies revealed that vitamin E succinate does not significantly effect cyclooxygenase activity in B16 cells over a period of 2 to 12 hours as compared to the OE control cultures. Further studies using RNA techniques investigated whether vitamin E succinate was having an effect on cyclooxygenase activity at a molecular level. These investigations were unsuccessful for the 6 day supplementation for a number of possible reasons, the main reason being RNA stability. Subsequent studies revealed an increase in COX mRNA after 2 hours, suggesting that the gene was 'switched on' soon after supplementation with vitamin E succinate, and further increases in COX mRNA were observed after 8 to 12 hours. The molecular studies were, however, inconclusive. Previous studies suggested that vitamin E succinate was indirectly causing growth inhibition of B16 cells via regulation of cyclooxygenase activity, however, this study does not support these findings and it would seem unlikely that regulation of cyclooxygenase expression in B16 cells by vitamin E succinate has a role to play in the mechanism by which vitamin E succinate inhibits growth in B16 cells.
- Full Text:
- Date Issued: 1999
An investigation into the biological treatment of platinum refinery effluent
- Authors: Smith, Roland Paul
- Date: 200u
- Subjects: Uncatalogued
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/193464 , vital:45334
- Description: This Review and project will discuss and demonstrate the use made of Biotechnology in the production and reduction of metals. It will look at how and why metal binding takes place, known platinum group metal speciation will be included. Examples of how to improve metal binding efficiency will be discussed by stimulating ligand activity by polarisation. Various biotechnical options available, with emphasis placed on the use of the aquatic fern and algae will be given as examples of biological treatment of heavy metals in particular the aquatic fern Azolla. The method of standard preparation and the use of Inductively Coupled Plasma Emission Spectrophotometer (ICP) used for analytical analysis will be included so that consideration can be given to the collection of analytical data in the provision of evidence to support or provide a conclusion. The outcome of the test work utilising the aquatic plant Azolla has proven that it can be used to remediate platinum refinery effluent. This process can offer an alternative to the classical chemical method normally used, which is economically viable and environmentally friendly in comparison to the common methods of refinery effluent treatment. , Thesis (MSc) -- Faculty of Science, Biochemistry, Microbiology and Biotechnology, 200u
- Full Text:
- Date Issued: 200u
An investigation into dopamine-melatonin interactions in the rat Corpus striatum and pineal gland: a possible pineal-striatal axis
- Authors: Boyd, Clinton Shane
- Date: 2000
- Subjects: Pineal gland -- Research Melatonin Dopamine -- Physiological effect Dopamine Brain chemistry Rats -- Physiology
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3906 , http://hdl.handle.net/10962/d1003965
- Description: Dysfunction of central dopaminergic systems has been implicated in neuroendocrine, neurodegenerative and psychiatric disorders. Monoamine oxidase and catechol-Omethyltransferase represent the key catabolic enzymes of dopamine, terminating neurotransmission following synaptic release of this catecholamine. Thus, both enzymes have been associated with the pathology of dopaminergic systems and represent therapeutic targets elf enormous clinical importance. Some neuroendocrine and circadian effects of melatonin have been attributed to an antidopamimetic effect of this pineal hormone in the hypothalamus and pituitary. Furthermore, both melatonin and dopamine modulate the behavioural output of the mesencephalic dopaminergic pathways of the basal ganglia, including movement disorders. However, the biochemical basis for the tonic inhibitory effect of melatonin in the nigro-striatal pathway has been poorly delineated. Thus, this study determined whether melatonin influences dopaminergic function in the corpus striatum of the Wistar rat by modulating monoamine oxidase and catecholO- methyltransferase activity. Reciprocally, the putative existence of an intrapineal dopaminergic system was investigated by determining the effect of selective dopaminergic agents, R-( -)apomorphine, haloperidol and dopamine, on indole metabolism of the pineal gland. The akinetic state of drug-induced catalepsy was employed as an animal model of Parkinson's disease to probe the neurotransmitter systems involved in the behavioural effects of melatonin. Indole metabolism was a reliable indicator of state-dependent metabolic fluxes in pineal gland function. These included a robust diurnal and seasonal variation in N-acetylserotonin and melatonin biosynthesis, and photoperiod- and drug-induced alterations of Inftabolism. The predominant changes could be attributed to an effect on serotonin N-acetyltransferase activity and/or the melatoninl5-methoxytryptophol ratio. Pineal 5-methoxyindole biosynthesis was determined primarily by the bioavailability of the corresponding 5-hydroxyindole and its affinity for hydroxyindole-O-methyltransferase. Evidence was found for the negative feedback or paracrine control of pineal indole metabolism by melatonin. A high inter-individual variability was observed in the biosynthesis of N-acetylserotonin and melatonin biosynthesis, and the weight of the pineal glands. Accordingly, the rats could be classified as either high or low capacity producers of these two indoles. R-(-)-apomorphine and dopamine in vitro, but not acute haloperidol in vivo, had dose- and phase-dependent effects on pineal indole metabolism. The predominant effect was a suppression of the scotophase-dependent induction ofN-acetylserotonin and melatonin biosynthesis by dopamine and R-( -)-apomorphine. It is postulated that these agonists inhibited nocturnal N-acetyltransferase activity via postsynaptic pineal D2 or D2-like receptors. The observed modulatory nature of the intrapineal dopaminergic system suggests that dopamine may be involved in the long-term regulation of pineal indole biosynthesis. Several lines of evidence are presented that the activity of striatal monoamine oxidase A and catechol-O-methyltransferase, represented predominantly by the soluble isoform, is statedependent and regulated in vivo by endogenous melatonin. Firstly, both enzymes showed a daynight variation in activity. Secondly, acute and subchronic administration and photoperiod manipulation studies indicated that both exogenous and endogenous melatonin inhibited each enzyme in a chronotypic fashion, with a more robust effect against catechol- -methyltransferase. The intensity of the in vivo effects was critically dependent on the dose, duration, route and the phase-timing of administration during the light dark cycle, and the length of the exposure to constant light. Melatonin in vitro had no effect on basal or Mg2+ -induced catechol-Omethyltransferase activity. Thus, it is proposed that the in vivo effects of the hormone can be attributed to a time-dependent change in the amount of active molecules of this enzyme. In contrast, melatonin and numerous other endogenous indolic compounds were found to be reversible inhibitors of striatal monoamine oxidase A in vitro. Structure-activity modeling revealed that the 5-methoxy moiety on the indole nucleus and substitution of the free primary amine of these compounds were the principal determinants of the potency and time-dependency of inhibition. Thus melatonin most likely has a direct inhibitory effect in vivo at the level of the active site of monoamine oxidase A. Exogenous melatonin alone had no cataleptogenic potential whereas a variety of behavioural responses were observed following intraperitoneal administration of y-hydroxybutyrate. The latter responses were state-dependent with day-night variations in intensity. Furthermore, yhydroxybutyrate stimulated melatonin biosynthesis during the photophase both in vitro and in vivo. These results point to a possible involvement of melatonin in the behavioural and neurochemical effects of y-hydroxybutyrate. Thus the general conclusion is that dopamine and melatonin display functional antagonism at the level of the pineal gland and corpus striatum of the Wistar rats. Therefore melatonin may be an important homeostatic modulator of dopaminergic neurotransmission throu~out the central nervous system. Furthermore, the putative existence of a functional pineal-striatal axis would greatly strengthen the argument for a holistic concept of brain homeostasis. The ability of endogenous melatonin to regulate monoamine oxidase A and catechol-O-methyltransferase may represent an alternative strategy for the treatment of disorders associated with these enzymes.
- Full Text:
- Date Issued: 2000
Characterization of amide bond hydrolysis in novel hydantoinase-producing bacteria
- Authors: Skepu, Zoleka G
- Date: 2000
- Subjects: Amides , Hydrolysis , Hydantoin , Imides
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3911 , http://hdl.handle.net/10962/d1003970 , Amides , Hydrolysis , Hydantoin , Imides
- Description: This thesis describes a series of investigations into the amide bond-hydrolyzing activity of bacterial strains RU-KM1, RU-KM3L, RU-KM3S, and RU-OR, which were previously isolated for their ability to hydrolyze 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 and related compounds. Several compounds may be used as substrates by biocatalysts for the production of amino acids, such as hydantoins, amino nitriles, and amides. These compounds are not only important for amino acid production, but they may be used for production of other industrially important compounds, such as 2- arylpropionic acids, which are non-steroidal anti-inflammatory drugs. Thus, the ability of the above-mentioned strains to hydrolyze these substrates was investigated, with the view to utilizing the maximum potential of these biocatalysts. The compounds used as substrates in the investigation are all essentially amides. Thus, the ability of the strains to hydrolyze imides, hydantoins, and amides, was investigated. In particular, imides have a structure which is very similar to that of hydantoins, and thus it was an objective of the study to determine whether these strains could hydrolyze imides. Imidehydrolyzing activity has only recently been discovered in microorganisms. Hydantoin conversion involves a two-step hydrolysis reaction which yields, initially, an Ncarbamylamino acid intermediate, and subsequently, an "-amino acid. The hydantoinhydrolyzing enzymes of a Pseudomonas putida strain, RU-KM3S, were characterized in a crude extract preparation and reaction conditions for its biocatalytic application were optimized. The optimum conditions for conversion of 5-methylhydantoin were found to be 3 hours at 40°C, with conversion yields greater than 50% achieved. The enzymes of RU-KM3S demonstrated considerable stability, retaining 80% of their activity after incubation at 40°C for 3 hours. 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-carbamylase of RUKM3S suggested that these enzymes required metal ions for activity, with metal ions such as Mg²⁺, Mn²⁺, Zn²⁺, and Co²⁺ resulting in activation of the enzymes. However, Cu²⁺ and Fe²⁺ caused inactivation of these enzymes. The stereoselectivity of the enzymes was investigated, and the results suggested that the hydantoinase was non-selective, whereas the N-carbamylase was L-selective. The hydantoin substrate selectivity of RU-KM3S was compared to that of three other hydantoinase-producing bacteria, RU-KM1, RU-KM3L, and RU-OR. The four strains were able to hydrolyze 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 higher activity than whole cells, except in the case of RU-KM1. 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 hydrolyzed p-hydroxyphenylhydantoin. RU-KM3L whole cells achieved a higher conversion yield with isobutylhydantoin, whereas the crude extract achieved a higher yield with 5-t-butylhydantoin. RU-KM3S whole cells and crude extract preferentially hydrolyzed 5-n-butylhydantoin, although the yield was greater with the crude extract. The highest conversion yields were observed with RU-KM3S crude extract, with conversion yields of 71.6% and 100% for n-butylhydantoin and phydroxyphenylhydantoin, respectively.The ability of RU-KM1, RU-KM3L, and RU-KM3S to hydrolyze nitriles, initially to amides and subsequently to carboxylic acids, was investigated. These strains were demonstrated to be unable to utilize acrylonitrile, propionitrile and benzonitrile as nitrogen sources, but were able to hydrolyze acrylonitrile, propionitrile and acetonitrile, in resting cell reactions. Nitrile hydrolysis was demonstrated to be inducible in all three strains, and the enzyme system responsible for nitrile hydrolysis was proposed to be a nitrile hydratase-amidase system. Amidase activity in the four bacterial strains was investigated. The ability of RU-KM1, RUKM3L, RU-KM3S, and RU-OR to utilize amides as a nitrogen source was investigated, and the results showed that propionamide was a good nitrogen source for all four of the strains. Amide-hydrolyzing activity, by resting cells, was shown to be inducible by propionamide in all four strains. RU-KM3S demonstrated superior amide-hydrolyzing ability in that it hydrolyzed propionamide, acetamide, and acrylamide to a greater extent than the other strains. Resting cells of RU-KM1 and RU-OR were demonstrated to have the ability to hydrolyze the imide substrate, succinimide, and this imidase activity was found to be inducible. These strains were also able to utilize this imide as the sole source of nitrogen for growth, which is a novel finding, as to date, bacteria have only be reported to utilize imides as a carbon source. The identity of the enzyme system responsible for succinimide hydrolysis is not yet clear. In conclusion, the hydantoin-hydrolyzing enzymes of RU-KM3S have been shown to be possibly membrane associated, which is a novel finding that has also been proposed in three other hydantoinase-producing strains in our laboratory. This study has shown that the Ncarbamylase of RU-KM3S is L-stereoselective, which, to our knowledge, is the first report of an L-stereospecific N-carbamylase in a Pseudomonas putida. Publication of these findings is already in progress. This is the first report on the study of imide hydrolysis in either an Agrobacterium tumefaciens or a Pseudomonas sp., and publications reporting these results are in preparation.
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- Date Issued: 2000
Enzymes with biocatalytic potential from Sorghum bicolor
- Authors: Nganwa, Patience Jennifer Kengyeya
- Date: 2000
- Subjects: Enzymes , Sorghum
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3908 , http://hdl.handle.net/10962/d1003967 , Enzymes , Sorghum
- Description: Sorghum is a staple food in the semi-arid tropics of Asia and Africa, sustaining the lives of the poorest rural people. This project set out to improve the potential economic value of Sorghum bicolor as a crop. The task was undertaken by screening for selected enzymes in the plant that would have a potential market for use in industrial applications and in biotransformations, specifically proteases, polyphenol oxidases and peroxidases. Asurveywas conducted using standard enzyme assays and crude plant extracts, to determine whether the selected enzymes were present. Grain tissue did not appear to have significant protease or polyphenoloxidase activity, but high levels of peroxidases were detected, withthe young grain extracts showing more activity(4.63U/mL)thanripegrain extracts (0.62 U/mL). Leaf tissue extracts contained low levels of protease activity, a considerable amount of polyphenol oxidase (0.127 U/mL), and peroxidase (4.7 U/mL) activities comparable with that found in grain tissue. Root tissue extract was found to contain the highest levels of peroxidase activity (7.8 U/mL) compared to the other extracts. Therefore, sorghum peroxidase from the root was isolated, purified, characterized and applied to biotransformation reactions. Different sorghum strains,withvaryinggraincolour, (Zimbabwe - bronze, Seredo - brown and Epurpur - cream/white) were investigated for the presence of polyphenol oxidase and peroxidase activities. Results of spectrophotometric analysis showed that the enzymes did not appear to be strain specific. However, gel electrophoresis analysis revealed differences in band patterns among the strains. Partial purification of sorghum root peroxidase was achieved after centrifugation, extraction with polyvinylpolypyrrolidone (PVPP), ultrafiltration, and hydrophobic chromatography with phenyl Sepharose, followed by polyacrylamidegelelectrophoresis (PAGE). The specific activity of the 5-fold purified enzyme was found to be 122.3 U/mg. After PAGE analysis, two bands with molecular weights of approximately 30 000 and 40 000 were detected, which compares well with horse radish peroxidase (HRP) which has a molecular weight of approximately 44 000. The colour intensity of the bands in the activity gels indicated that sorghum root peroxidase had apparently higher levels of peroxidase activity than commercial horseradish peroxidase (HRP). Characterizationexperiments revealed that sorghumroot peroxidase is active over a broad temperature range and remains active at temperatures up to 100°C. It also has a broad substrate range. The optimum pH of the enzyme was found to be pH 5 - 6. Under standardized assay conditions, the optimal substrate concentration, using o-dianisidine as substrate, was 50 mM, and the optimal H2O2 concentration under these conditions was found to be 100 mM. Sorghum root peroxidase was applied in a preliminary investigation into the oxidative biotransformationof a number of aromatic compounds. The products obtained were comparable withthose whenthe compounds are reacted with HRP which is the most commonly used commercial peroxidase and has been extensively studied. However, HRP is relatively costly, and the use of peroxidase from sorghum roots as an alternative source, appears to be promising. A patent has been provisionally registered, covering application of sorghum root peroxidase for biotransformations.
- Full Text:
- Date Issued: 2000
Genetic variation within and between some rare and common taxa of Cape Proteaceae and the implications for their conservation
- Authors: Brown, Susan Ann
- Date: 2000
- Subjects: Proteaceae -- South Africa Nature conservation -- South Africa Plant conservation -- South Africa
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3905 , http://hdl.handle.net/10962/d1003964
- Full Text:
- Date Issued: 2000
Serotonin-melatonin interactions in acetaminophen and N,N-dimethylformamide toxicity
- Authors: Anoopkumar-Dukie, Shailendra
- Date: 2000
- Subjects: Serotonin , Acetaminophen , Melatonin
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3898 , http://hdl.handle.net/10962/d1003957 , Serotonin , Acetaminophen , Melatonin
- Description: Acetaminophen and N,N-dimethylformamide (DMF) are compounds which are extremely toxic to the liver. Acetaminophen is a drug which is well known for its analgesic and antipyretic properties. However, the abuse potential of this agent as a non-narcotic analgesic in alcoholics is well known. It is also the leading cause of overdose in England. DMF toxicity results mainly from occupational exposure. At present there are no known reports of an antidote for DMF poisoning, while N-acetylcysteine, the antidote for acetaminophen poisoning, is known to produce adverse effects. The present study evaluates the potential of melatonin as an antidote for acetaminophen and DMF poisoning. This study also investigates the mechanism underlying acetaminophen addiction and abuse. Initial studies involved in vitro techniques in an attempt to remove the complexities of organ interactions. The photodegradation studies, using ultraviolet (UV) light, revealed that melatonin accelerates the rate of acetaminophen degradation in the presence of air, and reduces the rate of degradation in the presence of nitrogen. This study also revealed that melatonin is rapidly degraded in the presence of air, following UV irradiation. The effect of DMF on hydroxyl radical generation was also determined. DMF was shown to act as a free radical scavenger, rather that a generator of free radicals. The in vitro studies were followed by lipid peroxidation determination. DMF (0.4ml/kg and 0.8ml/kg) did not produce any significant increases in lipid peroxidation in the liver. Three different doses of acetaminophen (30mg/kg, 100mg/kg, and 500mg/kg) were administered to rats for seven days. Acetaminophen (500mg/kg) was shown to significantly increase (p<0.05) lipid peroxidation in the liver. Melatonin (2.5mg/kg) was not able to significantly reduce the damage. The lower doses of acetaminophen (30mg/kg and 100mg/kg) did not increase lipid peroxidation. Electron microscopy studies showed that DMF adversely affects the liver, and in particular, the endoplasmic reticulum. Co administration of melatonin (2.5mg/kg) was able to reduce the damage. Further experiments need to be performed before an accurate assessment can be made on the ability of melatonin as an antidote for DMF and acetaminophen poisoning. Several experiments were done in an attempt to uncover the biochemical mechanism underlying acetaminophen addiction and abuse. The first experiment targeted the liver enzyme tryptophan-2,3-dioxygenase (TDO). This enzyme is the major determinant of tryptophan levels in vivo. Acetaminophen administration (100mg/kg for three hours) was shown to significantly inhibit (p<0.05) the activity of TDO, indicating increased peripheral levels of tryptophan. This experiment was followed up with determination of brain serotonin and pineal melatonin. Brain serotonin was determined using the ELISA technique. Melatonin was estimated using this technique as well as with pineal organ culture. Acetaminophen administration (100mg/kg for three hours) significantly increased (p<0.05) brain serotonin levels. Using organ culture where exogenous (3H) tryptophan is metabolised to (3H) melatonin, acetaminophen (100mg/kg for three hours) was shown to significantly increase (p<0.05) pineal melatonin concentrations. However, the ELISA technique did not reveal any changes in endogenous pineal melatonin levels. The final experiment was the determination of urinary 5-hydroxyindole acetic acid (5- HIAA), the major metabolite of serotonin, following acetaminophen administration (100mg/kg for three hours). Acetaminophen was shown to significantly reduce 5-HIAA levels (p<0.05) suggesting reduced catabolism of serotonin. The findings of this study indicate that acetaminophen mimics the actions of an antidepressant. This compelling finding has important clinical implications, and needs to be examined further.
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- Date Issued: 2000
Spirulina as a bioremediation agent : interaction with metals and involvement of carbonic anhydrase
- Authors: Payne, Rosemary Anne
- Date: 2000
- Subjects: Spirulina , Bioremediation , Carbonic anhydrase
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3909 , http://hdl.handle.net/10962/d1003968 , Spirulina , Bioremediation , Carbonic anhydrase
- Description: Heavy metal contamination from mining and other industrial operations is becoming an increasing problem with regards to the depleting water resources in South Africa. This study involved the investigation of the use of an algal biomass as a possible alternative to the traditional chemical means of removing these metals. When the toxic effects of metals were investigated, Spirulina was found to have a threshold level of about 30 μM for copper, zinc and lead. Copper and zinc appeared to have a direct effect on the photosynthetic pathway, thereby causing a rapid decline in cell growth. Lead on the other hand seemed to affect surface properties and hence took longer to cause deterioration in growth. Although relatively low concentrations of metal may have a toxic effect on the cyanobacterium, Spirulina may have potential as a precipitation agent. The role of Spirulina in the precipitation of heavy metals appears to be through its ability to maintain a high pH in the surrounding medium, possibly through the enzyme carbonic anhydrase. Subsequent studies therefore focused on the assay and isolation of this enzyme. Two different radiotracer assays, in which carbonic anhydrase converts radiolabelled bicarbonate to carbon dioxide, were investigated, but were found to have several problems. Results were insensitive and could not be reproduced. The standard Wilbur-Anderson method subsequently investigated also proved to be insensitive with a tremendous degree of variability. Although not quantitative, SDS-PAGE proved to be the most reliable method of detection, and was therefore used in subsequent procedures. Chlamydomonas reinhardtii was the subject of initial enzyme isolation studies as these procedures are well documented. Although the published protocols proved unsuccessful, affinity chromatography of a membrane stock solution from Chlamydomonas reinhardtii yielded two relatively pure protein bands. These bands were presumed to represent two subunits of carbonic anhydrase, although Western blot analysis would be required to confirm their identity. Purification of carbonic anhydrase from Spirulina, however, proved unsuccessful and results obtained were very inconclusive. Hence, further analysis of Spirulina is required. The possibility of cloning CA from a genomic library was also considered, but suitable primers could not be designed from the aligned sequences.
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- Date Issued: 2000
Studies of the population structure and generic diversity of domesticated and "wild" ostriches (Struthio camelus)
- Authors: Bezuidenhout, Cornelius Carlos
- Date: 2000
- Subjects: Ostriches Ostriches -- Genetics Ostriches -- South Africa
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3907 , http://hdl.handle.net/10962/d1003966
- Description: DNA sequencing and restriction fragment length polymorphism analysis (RFLP) of polymerase chain reaction (PCR) amplified mitochondrial DNA fragments, and random amplified polymorphic DNA sequence (RAPD) analysis were techniques evaluated in this study for applicability in the investigation various aspects of genetic diversity within the ostrich (Struthio camelus). The genetic aspects that were investigated were (i) relationships between ostrich subspecies, (ii) genetic variability between and within domesticated populations of southern African ostriches (Struthio camelus australis), (iii) linking egg production in domesticated ostriches to RAPD profiles, and (iv) determining the zygosity of twin ostriches. In the first part of this study DNA sequencing and the polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP) methods were evaluated for resolving genetic differences in the small mtDNA fragments ofthe ostrich. DNA sequencing ofPCR amplified 450 bp 12S rRNA gene fragments of representatives from the southern African population ostrich (S.c. australis) did not reveal any differences between the populatiohs from different geographical areas, representing ostrich lineages with different breeding histories. The PCRRFLP analysis ofmtDNA fragments (450 bp 12S rRNA gene fragment and 550 bp D-loop region) also did not reveal any genetic variability between the domesticated s.,c. australis populations included in this study. PCR-RFLP analysis of a 450 bp 12S rRNA gene fragment, however, showed differences between the subspecies s.c. australis and s.c. molybdophanes. The proportion of shared fragments (F) between these two subspecies was 0.286 and nucleotide sequence divergence estimated at 8.9 %. Divergence time between these two subspecies was estimated at 4.5 million years ago. The data presented from this study are comparable to the data from a previous study in which the entire mitochondrial genome and a larger number of restriction enzymes were used. The PCR-RFLP method thus demonstrated its usefulness for genetic studies of ostriches at thesubspecies level. The sequences used in this study could not reveal any markers that were useful for genetic studies of ostriches at the population level. In the second part of the study the RAPD method was evaluated for application in the genetic studies of ostriches. RAPD profiles, based on three RAPD primers, revealed differences between three subspecies of ostriches and indicated relationships between these subspecies that are consistent with observations from other studies. The numerical analysis of pooled and individual primer data demonstrated that the subspecies s.c. australis is more closely related to s.c. massaicus than to s.c. molybdophanes. RAPD marker differences between s.c. molybdophanes on the one hand, and s.c. massaicus and s.c. australis on the other is also consistent with observations from studies that proposed separate specie~ status for s.c. molybdophanes. RAPD analysis by five primers revealed geographic variation between s.c. australis populations. The clustering patterns observed in the dendrograms and Neighbour Joining Trees generated by computer programs showed trends of separating ostric1;t populations into geographical groups, possibly reflecting their different breeding histories. In the RAPD profiles of the inbred population, band-sharing was generally greater than in the outbreeding group. RAPD analysis thus showed that it may be a useful method in the population studies of domesticated S. c. australis. RAPDs also generated data that grouped ostriches according to trends in egg production capabilities. Analysis ofRAPD profiles by computer software showed a Neighbour Joining Tree and a dendrogram that predominantly grouped ostriches into clusters associated with either good or poor egg production. Evidence supporting the suitability of RAPDs as a tool in breeding programmes of ostriches was thus provided by this study. RAPDs also provided data, demonstrating that two sets of ostrich twins were non-identical twins. It was demonstrated by this study that RAPDs analysis may be a useful technique for applying to (1) systematic (2) population (3) breeding and (4) twin studies of ostriches (Struthio camelus).
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- Date Issued: 2000
Sulphide-enhanced hydrolysis of primary sewage sludge : implications for the bioremediation of sulphate-enriched wastewaters
- Authors: Whittington-Jones, Kevin John
- Date: 2000
- Subjects: Bioremediation Sewage sludge Hydrolysis
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3910 , http://hdl.handle.net/10962/d1003969
- Description: The potential application of sulphate reducing bacteria for the bioremediation of acid mine drainage has already been recognised, and offers significant financial advantages over conventional chemical treatment approaches. Although the technology has been demonstrated successfully on both small- and large-scale, it’s extensive implementation has been constrained by the provision of suitable and cost effective electron donor and carbon sources. Primary sewage sludge is readily available in large quantities, but the slow rate of solubilization and low yield of soluble products do not apparently favour its use for this application. A number of pre-treatment steps have been introduced in an attempt to improve the yield and rates under methanogenic conditions. However, although early work suggested that degradation of lignocellulose and proteins may be more rapid under sulphate reducing conditions, the fate of primary sewage sludge under these conditions has been ignored. It was proposed that by combining the hydrolysis of primary sewage sludge and biological sulphate reduction, in a settling sludge bed, both processes would be enhanced. The aim of this study was to test this hypothesis on laboratory- and pilot-scale, and attempt to elucidate the underlying mechanism involved. The solubilization of primary sewage sludge was enhanced in the presence of sulphate reduction in continuous laboratory-scale reactors. Particulate matter accumulated in the bed of non-sulphidogenic systems, but not in sulphidogenic ones. This was attributed to increased solubilization and the smaller average floc size in the latter. Solubilization occurred within the settling sludge bed of the reactors, and offered a possible explanation for the better performance of the multiple- over single-stage reactor. A pilot-scale Falling Sludge Bed Reactor was constructed at Grootvlei Gold Mine, Springs, South Africa, and resulted in the solubilization of more than 70% of the influent primary sewage sludge. The system was also found to be highly resilient to severe perturbations, and returned rapidly to steady-state. Flask studies revealed that the hydrolysis of both proteins and complex carbohydrates was accelerated in the presence of biological sulphate reduction or sulphide. A study of the enzymology of sludge digestion revealed that sulphate reduction had little direct effect on the activity of the hydrolytic enzymes, but that reactor design was critical in the prevention of washout of these enzymes. Finally, a descriptive model was developed to explain the enhanced hydrolysis of primary sewage sludge. The model incorporated the effect of sulphidogenesis on floc fracture and reflocculation, and likely implications for mass transfer limitations.
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- Date Issued: 2000
The effect of 6-Methoxy-2-Benzoxazolinone (6-MBOA) on indoleamine regulation and its possible role in depression
- Authors: Tanda, Sindiswa Eunice
- Date: 2000
- Subjects: Tryptophan -- Physiological effect , Tryptophan -- Therapeutic use , Antidepressants
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3912 , http://hdl.handle.net/10962/d1003971 , Tryptophan -- Physiological effect , Tryptophan -- Therapeutic use , Antidepressants
- Description: Tryptophan is an essential amino acid that is obtained from the diet. Approximately 98 % of ingested tryptophan is metabolized by the enzyme tryptophan 2,3-dioxygenase (TDO). The metabolism of tryptophan by TDO is an important determinant of tryptophan bioavailability to the brain for serotonin (5-HT) biosynthesis, an essential amine in affective disorders such as depression. Studies done on circadian rhythmicity of the enzyme activity have shown that, TDO activity is high during the scoto-phase (dark-phase), which is attributable to the de novo enzyme synthesis that occurs during this phase. 6-Methoxy-2-benzoxazolinontr-(6-MBOA), a structural analogue of melatonin (aMT) was shown to inhibit TDO activity in both the photo-phase (light-phase) and the scoto-phase with greater potency during the light-phase. Further studies were directed at demonstrating the effects of 6-MBOA on the brain tryptophan hydroxylase (TH) activity, which is a rate limiting enzyme in 5-HT biosynthesis and subsequently on 5-HT levels. The findings showed that, 6-MBOA induces TH activity with a concomitant rise in brain 5-HT levels. The blockade of 5-HT re-uptake into the presynaptic neuron leads to an increase in 5-HT available for the stimulatory action of 5-HT receptors. An attempt to establish whether the administration of 6-MBOA would block the binding of 5-HT to receptors on the synaptosomal membrane showed that 6-MBO A only inhibits the binding of 5 -HT at specific concentrations. In view of the positive effects imposed by 6-MBOA on brain 5-HT levels, urinary 5-hydroxyindole acetic acid (5-HIAA) excretion was measured before and after treatment with 6-MBOA. 5-HIAA excretion was found to be significantly increased after 6-MBOA treatment. Extensive research on the biosynthesis of pineal metabolites has been conducted in the past two decades. The pineal metabolites are synthesized from the precursor tryptophan. In order to obtain an overall picture of the effect of6-MBOA on pineal indole metabolism, an organ culture technique was employed. The results obtained showed that although 6-MBOA administration to rats caused a significant increase in aMT production, there was an insignificant increase in NAS production. This is an immediate precursor of aMT. Other pineal indoles were not affected at all by 6-MBOA administration. Furthermore, the production of pineal NAS and aMT showed an inter-individual variation with some animals producing very high, some very low and some produced average levels of these two metabolites in both photo and scoto-phase experiments. A study undertaken to investigate the circadian rhythm in endogenous aMT production using the competitive ELISA technique showed a clear pattern with high levels of aMT produced during the dark-phase and low levels ofaMT produced during the light-phase. Furthermore, the administration of6-MBOA to rats lead to a significant rise in endogenous aMT production.
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- Date Issued: 2000
The measurement of genetic diversity in mycobacterium tuberculosis using random amplified polymorphic DNA profiling
- Authors: Richner, Sharon M
- Date: 2000
- Subjects: Tuberculosis -- History -- 20th century , Tuberculosis -- Africa, Southern , Tuberculosis -- Treatment , Tuberculosis -- Africa , Tuberculosis -- Prevention , Tuberculosis -- Pathogenesis , Mycobacterium tuberculosis
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4008 , http://hdl.handle.net/10962/d1004068 , Tuberculosis -- History -- 20th century , Tuberculosis -- Africa, Southern , Tuberculosis -- Treatment , Tuberculosis -- Africa , Tuberculosis -- Prevention , Tuberculosis -- Pathogenesis , Mycobacterium tuberculosis
- Description: Mycobacterium tuberculosis has caused a resurgence in pulmonary disease in both developed and developing countries in recent times, particularly amongst people infected with the human immunodeficiency virus. The disease has assumed epidemic proportions in South Africa and in the Eastern Cape Province in particular. Of further concern is the isolation of increasing numbers of multiply drug resistant strains. Knowledge of the genetic capability of this organism is essential for the successful development of novel antibiotics and vaccines in an attempt to bring the global pandemic under control. Measurement of the genetic diversity of the organism may significantly contribute to such knowledge, and is of vital importance in monitoring epidemics and in improving treatment and control of the disease. This will entail answering a number of questions related to the degree of genetic diversity amongst strains, to the difference between urban and rural strains, and between drug resistant and drug sensitive strains, and to the geographical distribution of strains. In order to establish such baseline information, RAPD profiling of a large population of isolates from the western and central regions of the Eastern Cape Province was undertaken. A smaller number of drug resistant strains from a small area of KwaZulu-Natal were also analysed, with a view to establishing the genetic difference between strains from the two provinces. Cluster analysis, analysis of molecular variance and Geographical Information Systems technology were used to analyse the RAPD profiles generated. An unexpectedly high degree of genetic diversity was detected in strains from both provinces. While no correlation was seen between genetic diversity and either urban-rural situation or geographical location, a small degree of population structure could be correlated with drug resistance in the Eastern Cape. Furthermore, a significant degree of population structure was detected between strains from the two provinces, although this was still within the parameters for conspecific populations. Future work is necessary to further characterise strains from rural areas of both provinces, as well as from the eastern region of the Eastern Cape in an attempt to pinpoint the cause of the separation of the provincial populations.
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- Date Issued: 2000
The nature and control of organic compounds in soda ash evaporate production
- Authors: Masemola, Patricia Mmoniemang
- Date: 2000
- Subjects: Organic compounds , Biotic communities , Sua Pan Soda Ash Project -- Botswana
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3902 , http://hdl.handle.net/10962/d1003961 , Organic compounds , Biotic communities , Sua Pan Soda Ash Project -- Botswana
- Description: Solar evaporite systems are man-managed ecosystems which are highly vulnerable to biological,physical and chemical disturbances. The problems encountered in such systems are in many cases found to be associated with the microbial ecology and the design of the system. This project focussed on investigating the nature of organic compounds contaminating soda ash produced at a solar evaporite production system located at Sua Pan in Botswana. Several years after the plant was commissioned, problems, including accumulation of total organic carbon (TOC) and discolouration of the soda ash product were encountered. The salt produced also retained high moisture content and was coloured pink. These phenomena impacted severely on the economic performance of the enterprise. This study was aimed at determining the origin and fate of these organic compounds within the system in order to elucidate the nature of the problem and also to conceptualise a remediation strategy suitable to reducing its impact. This was achieved by analysis of both dialysed and solvent extracts of the influent brine (well-brine), brine in the ponds (T-brine) and the bicarbonate filter cake. Although complete identification of the organic compounds isolated was not undertaken in this study, spectroscopic analysis of compounds isolated, by UV, IR, NMR and MS, strongly indicated that fulvic acids, a component of the influent well-brine organics, contribute to the organic contamination of the final product. Part of this component, however, is degraded during the ponding process. It was shown that an extracellular polysaccharide (EPS) produced by Dunaliella. spp., which proliferates in the evaporation ponds, contributes in a major way to the accumulation of TOC in the system. This was demonstrated by relating the sugar profile of carbohydrates isolated from the pond brine and final product, being arabinose, xylose, 2-o-methyl hexose, mannose, glucose and galactose. Studies reported show that EPS production was enhanced when algal cultures were exposed to stress conditions of high illumination, increasing salinity and temperature, and nitrogen limitation. Studies undertaken for the development of a remediation process for this system have shown that nutrient stripping and bacterial systems could be applied to deal with the dissolved TOC fraction, whereas adsorption systems could deal with the particulate fractions. Algal systems showed most potential for the removal of nutrients in the influent well-brine compared to chemical processes.Complete removal of ammonium and phosphorus removal efficiencies of pproximately 50% were achieved in an unoptimised pilot-scale Dunaliella-based HRAP. While similar effects were demonstrated for chemical processes, some economic constraints were noted. The potential of halophilic bacterial systems for the degradation of organic compounds in brine was also demonstrated. The limitations on the performance of such systems, associated with the low metabolic diversity, and poor immobilisation of physico-chemical processes were found to have a very low impact on the dissolved TOC fraction of the brine, the removal of the particulate material was found to result in a 35% TOC reduction in the final soda ash product and the production of a white final product.halobacteria, however, were noted. Although physico-chemical processes were found to have a very low impact on the dissolved TOC fraction of the brine, the removal of the particulate material was found to result in a 35% TOC reduction in the final soda ash product and the production of a white final product. Apart from a description of the microbial ecology of the ponds and the identification of major contributions to the TOC of the final product, a number of remediation strategies were evaluated and are described. These include chemical and biological stripping of nutrients sustaining microbial TOC production in the ponds, and also biological and physico-chemical processes for their removal once formed. Future studies to undertake the further development of these proposals has been described
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- Date Issued: 2000
The structure and microbiology of floating sulphide oxidising biofilms
- Authors: Gilfillan, Joanne Criseyde
- Date: 2000
- Subjects: Biofilms , Sulfides
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3903 , http://hdl.handle.net/10962/d1003962 , Biofilms , Sulfides
- Description: Floating sulphur biofilms are observed as surface layers in numerous aquatic sulphide-rich environments, and apparently play an important role in the cycling of sulphur in its various oxidation states. In addition to the conversion of sulphide to sulphur and/or sulphate species, it has been suspected that subsequent reduction back to sulphide may occur within the floating sulphur biofi1m in organic-rich environments. The use of sulphur biofilms for the harvesting of elemental sulphur from wastewater treatment systems has also been suggested. There is, however, little documented information in the literature on the structure of floating sulphur biofilms, or the microbial species responsible for their occurrence. In this study, floating sulphur biofilms were generated in a continuous flow baflle reactor and their structure was examined using scanning electron microscopy. It was found that they occur as layered structures with morphologically distinct bacterial forms present in different layers of the biofilm. The biofilpl structure was also found to be dynamic, with structural changes observed as feed conditions were altered. An enriched culture derived from the biofi1m demonstrated rates of sulphide oxidation comparable to values reported in the literature for liquid culture systems. The microbiology of the biofi1m was studied using traditional plate culture techniques and analysis ofrRNA genes. Identification of plate culture isolates as representatives of the biofi1m community proved to be limited, leading to a PeR-based cloning approach. The majority of the organisms present in the sulphur biofi1m were classified as species in the genus ~eudomonas, and a number of other bacterial species whose sulphide oxidising capacity has been noted previously. Surprisingly, only 2% of the clone library consisted of Thiobacillus spp., and no sulphate reducing bacteria were identified in the biofilm at all. These results indicate that in organic sulphide-rich environments facultative chemolithoheterotrophic bacterial forms predominate in floating sulphur biofilms, and that the complete biological cycling of sulphur may not occur in these systems.
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- Date Issued: 2000
A study of carbonate-rich brines from Sua Pan to characterize organic contaminants in the soda ash process
- Authors: Joseph, Manjusha
- Date: 2001
- Subjects: Sua Pan Soda Ash Project -- Botswana , Sodium carbonate -- Research , Biotic communities , Organic compounds
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4031 , http://hdl.handle.net/10962/d1004091 , Sua Pan Soda Ash Project -- Botswana , Sodium carbonate -- Research , Biotic communities , Organic compounds
- Description: Botswana Ash (Pty) Ltd which is situated in Sua Pan, north east Bostwana, is one of Africa's largest suppliers of salt and soda ash. For a number of years, the company has been experiencing problems which have resulted in the final soda ash product being contaminated and discoloured. The problems experienced at Sua Pan have been reported also to occur in other salt works all over the world. It has been suggested that contamination in many salt works could be possibly be due to the microbial activity by halophilic algae and bacteria that grow in the solar ponds. This study was undertaken to investigate the nature of the contaminating organic compounds present in the brine, to identify the compounds, and to establish how these components vary during the various stages of the soda ash processing. For this study, two sets of brine samples were used; the first set was collected before the summer rains and the second set was collected after the summer rains. Solid bicarbonate and soda ash samples were also used. Extractions, desalting, UV and HPLC analysis and oxidative biotransformations using four enzymes, were used for developing profiles and characterizing the brine components. From these studies, we were able to confirm that the components of the brine are organic in nature. A thorough study of one of the compounds isolated,from solid bicarbonate and soda ash was conducted using UV, HPLC, IR, NMR, HPLC-MS, GC-MS and TLC. The results of these analyses, show that the. isolated compound was benzyl butyl phthalate which is generally regarded to be humic in nature. This compound was found to be present in all the brine samples collected after the summer rains including the well brine, suggesting this compound occurs naturally and is not formed during the processing.
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- Date Issued: 2001
Assembly of full-length cDNA, and heterologous expression, of Nudaurelia B virus RNA
- Authors: Luke, Gary Joseph
- Date: 2001
- Subjects: Imbrasia cytherea , RNA , Viruses , DNA
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3913 , http://hdl.handle.net/10962/d1003972 , Imbrasia cytherea , RNA , Viruses , DNA
- Description: Nudaurelia beta virus (NβV) is a monopartite genome virus belonging to the family Tetraviridae. Its host range has been found to be limited to a single insect order, the Lepidoptera (moths and butterflies). The single-stranded positive-sense RNA genome consists of 6625 nucleotides containing two open reading frames (ORFs). The 5' proximal ORF of 5778 nucleotides encodes a protein of 215 kDa containing three functional domains characteristic of RNA-dependent RNA polymerase. The 3' proximal ORF, of 1836 nucleotides, encodes the 66 kDa capsid precursor protein and overlaps the replicase gene by more than 99% and is in the +1 reading frame relative to the replicase reading frame. The full-length cDNA construct of the NβV genome was assembled using a homologous overlapping PCR linking method. The starting material consisted of seven overlapping pieces that were constructed for sequencing. Due to the degradation of the full-length RNA obtained from virus extracted from field-collected Nudaurelia cytherea capensis larvae other alternative methods needed to be applied. Sub-cloning using restriction enzyme sites also required an alternative method being used, due to the abundance of restriction sites of the same type in the NβV genome. This led to the use of a method similar to "DNA Shuffling" where overlapping pieces were connected using a modified PCR protocol. After the construction of the NβV genome, the full-length PCR product was cloned and checked for large insertion and deletions that could have resulted from the PCR amplification. The heterologous expression of the NβV capsid protein linked to a fusion protein (Glutathione S-transferase) in E.coli, confirmed the authenticity of the prescribed capsid gene ORF. The expression showed that the virus protein was subjected to protease digestion in DH5α E.coli, suggesting that the protein was insoluble in the cell cytoplasm. The capsid gene expression in a modified E.coli strain, Epicurian Coli BL21-CodonPlus (DE3)-RIL, resulted in high levels of the correct molecular weight protein with minimal degradation. The modified strain was designed for over-expression of eukaryotic protein with lowered protease activity. The above results have opened the way for further research that would yield valuable insight into the molecular biology and replication strategy of the NβV in cell cultures.
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- Date Issued: 2001
Enzymology of activated sewage sludge during anaerobic treatment of wastewaters : identification, characterisation, isolation and partial purification of proteases
- Authors: Tshivhunge, Azwiedziswi Sylvia
- Date: 2001
- Subjects: Sewage sludge , Sewage sludge -- Environmental aspects , Sewage sludge digestion , Anaerobic bacteria
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4012 , http://hdl.handle.net/10962/d1004072 , Sewage sludge , Sewage sludge -- Environmental aspects , Sewage sludge digestion , Anaerobic bacteria
- Description: During anaerobic digestion bacteria inside the digester require a carbon source for their growth and metabolism, sewage sludge was used as a carbon source in this study. The COD content was used to measure the disappearance of the substrate. COD content was reduced by 48.3% and 49% in the methanogenic and sulphidogenic bioreactors, respectively, while sulphate concentration was reduced by 40%, producing 70mg/L of hydrogen sulphide as the end product over the first 5-7 days. Sulphate (which is used as a terminal electron acceptor of sulphur reducing bacteria) has little or no effect on the sulphidogenic and methanogenic proteases. Sulphite and sulphide (the intermediate and end product of sulphate reduction) increased protease activity by 20% and 40%-80%, respectively. Maximum protease activity occurred on day 21 in the methanogenic reactor and on day 9 in the sulphidogenic reactor. The absorbance, which indicates the level of amino acid increased to 2 and 9 for methanogenic and sulphidogenic bioreactors, respectively. Proteases that were active during anaerobic digestion were associated with the pellet (organic particulate matter) of the sewage. These enzymes have an optimum activity at pH 10 and at temperature of 50°C. The proteases that were active at pH 5 and 7, had optimum temperatures at 30°C and 60°C, respectively. Due to their association with organic particulate matter, these enzymes were stable at their optimum temperatures for at least five hours at their respective pH. Inhibition by PMSF, TPCK and 1.10-phenanthroline suggested that proteases inside the anaerobic digester are a mixture of cysteine, serine and metalloproteases. At pH 5, however, EDTA appeared to enhance protease activity by 368% (three-fold). Acetic acid decreased protease activity by 21%, while both propionic and butyric acid at 200 mg/L cause total inhibition of protease activity while these acids at higher pH (where they exist as their corresponding salts) exerted little effect. Copper, iron and zinc inhibited protease activity by 85% at pH 5 with concentrations ranging between 200 and 600 mg/L. On the other hand, nickel, showed an increase in protease activity of nearly 250%. At pH 7 and 10, copper had no effect on protease activity while iron, nickel and zinc inhibited these enzymes by 20-40%. Proteases at pH 7 were extracted from the pellet by sonication, releasing 50% of the total enzymes into the solution. The enzymes were precipitated by ammonium sulphate precipitation, and further purified by ion exchange chromatography and gel filtration. Ion exchange chromatography revealed that most of the enzymes that hydrolyse proteins are negatively charged while gel filtration showed that their molecular weight is approximately 500 kDa.
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- Date Issued: 2001