http://hdl.handle.net/10386/72 Thu, 20 Jun 2013 00:51:00 GMT 2013-06-20T00:51:00Z http://hdl.handle.net/10386/857 Title: Heterologous expression of a Mukwa (pterocarpus angolensis ) seed lectin (Pal) gene in Escherichia coli, Saccharomyces cerevisiae and Yarrowia lipolytica and construction of Pal recombinant vector for expression in Aspergillus niger Authors: Ngoepe, Mafora Gloria Abstract: Pterocarpus angolensis seed lectin (PAL), a 28 kDa non glycosylated protein, was initially successfully cloned and expressed in E. coli for ease of high protein production. It was discovered, however, as in similar studies that the recombinant PAL yield in E. coli is low and localized intracellularly. This makes extraction even more difficult because most of the protein is lost either when the cell undergoes lysing or when there is incomplete extraction. As a result of the low yields in E. coli, expression vectors were constructed for pal expression in S. cerevisiae, Y. lipolytica and A. niger. Colony PCR of S. cerevisiae transformants confirmed the presence of pal gene whilst sequencing revealed a 66% homology to native PAL. Expression of recombinant PAL in S. cerevisiae, which was expected to be intracellular, was doubtfully unsuccessful since no signal was detected following Western blot analysis. A pBARMTE1-pal expression vector was successfully constructed and could be used for expression studies in Aspergillus niger, however, it was not used in this study. A pal gene whose codons were optimized for Y. lipolytica was synthesized and successfully cloned and expressed in Y. lipolytica. Gene sequence alignment of native pal and the codon optimized pal showed 81% homology whilst the amino acid alignment showed 100% homology. A 31 kDa, recombinant PAL was successfully expressed in Y. lipolytica. The recombinant PAL was approximately 3 kDa larger than native PAL. It was established that this is due to glycosylation of the recombinant PAL. This recombinant protein was found to be more thermostable than native PAL since it demonstrated haemagglutination activity after 10 minutes of exposure in a boiling water bath and only lost activity after 2 hours of exposure to boiling. This study succeeded in producing a more stable extracellular recombinant PAL which demonstrated biochemical activity that was largely similar to that of native PAL but only differed in carbohydrate specificity and haemagglutinating strengths. Description: Thesis (M.Sc. (Microbiology)) --University of Limpopo, 2011 Sat, 01 Jan 2011 00:00:00 GMT http://hdl.handle.net/10386/857 2011-01-01T00:00:00Z http://hdl.handle.net/10386/805 Title: Screening, isolation and characterization of lectins extracted from mushrooms indegenous to Southern Africa Authors: Maribeng, Reagile Abstract: Lectins are among a large number of proteins produced by mushrooms. Mushroom lectins with important biological functions have been isolated and studied. However, none of the studies were reported on lectins isolated from mushrooms indigenous to southern Africa. A galactose-specific lectin from one of the common mushroom species in southern Africa, Schizophyllum commune, was isolated and characterized. Initially, twenty mushroom samples were collected and their crude extracts screened for lectin activities. Assays involved in the screening procedures included heamagglutination, carbohydrate inhibition, enzyme linked glycoprotein (ELGA) and various stability assays. Four different mushroom samples exhibited positive lectin activities with varying stabilities towards thermal treatment and susceptibility to proteolytic degradation. Further screening assays resulted in ZHR1 being selected for identification and purification of the lectin. This was due to its ability to agglutinate rabbit erythrocytes. In addition to its activity being destroyed after 3 hours of treatment with trypsin-NIPAAM conjugate, the activity of this lectin was also completely destroyed after an hour incubation in boiling water. In contrast to other mushroom extracts assayed, heamagglutination activity of the crude extract of ZHR1 was not inhibited by glycoproteins only but also by the sugars such as galactose, lactose and mannose. ZHR1 was identified as S. commune. S. commune lectin (ScL) was purified using affinity chromatography on a fetuin-agarose column and further purified using gel-filtration chromatography on Biogel P-100 column. ScL was characterized as a glycosylated, galactose-specific dimeric lectin with a molecular weight of approximately 32 and 33 kDa. ScL is a thermolabile lectin which loses its activity as early as 5 minutes after being incubated at 60°C. Anti-ScL antibodies, to be employed in screening for the presence of ScL in the protein extracts, were developed in the rabbits and their interaction with ScL was detected using the double immunodiffusion assays whereas their specificity towards the lectin was detected using Western blot. ScL is one of the first mushroom lectins to be isolated and studied in southern African region. If the lectin is found to be exhibiting important biological functions, ScL can be of commercial importance in the region. Description: Thesis (M.Sc. (Microbiology)) -- University of Limpopo, 2011 Sat, 01 Jan 2011 00:00:00 GMT http://hdl.handle.net/10386/805 2011-01-01T00:00:00Z http://hdl.handle.net/10386/748 Title: Metazoan parasites and health of selected cyprinids at Nwanedi-Luphephe dams Authors: Mbokane, Esau Mathews Abstract: The present MSc dissertation emanates from seasonal surveys conducted by the fish parasitological group of the Department of Biodiversity and Aquaculture Research Unit of the University of Limpopo, Turfloop Campus. The first part of the present study was aimed at investigating the metazoan parasites of three cyprinids occurring in the Nwanedi-Luphephe Dams. The main purpose of it was to determine temporal changes in the intensity of infestation in terms of prevalence, mean intensity and abundance of parasite species parasitizing the cyprinids studied over a two year period. Ecological parameters including species host-specificity, seasonality, and gender preference and host size versus species intensity are discussed for each parasite. Altogether 152 specimens were examined for parasites and a total of 2 432 metazoan parasites of ten species were recorded. At the sampling site, all three hosts co-occurred, however, a substantial proportion of Barbus radiatus was collected from the perennial stream feeding one of the twindams. Fish were sampled by means of gill nets and electrofishing or seine netting in accordance with the habitat conditions. Hosts were killed and organs investigated for metazoan parasites. After collection of parasites, standard methods for processing individual parasites were followed. The results obtained revealed the following groups of parasites; monogeneans (ectoparasites) included Dactylogyrus spinicirrus, D. afrolongicornis afrolongicornis, D. afrolongicornis alberti, Afrodiplozoon polycotyleus, Gyrodactylus sp., and Dogielius sp. (all recorded from the gills); Crustacea, Dolops ranarum was found from the mouth cavity, gills and skin of Labeobarbus marequensis. Of these, only two specialists, both monogeneans, were found on Barbus trimaculatus namely, D. afrolongicornis afrolongicornis and D. afrolongicornis alberti. Based on morphology of the haptoral hard parts, these two species were almost similar to each other than to D. spinicirrus. The appreciable difference between D. afrolongicornis afrolongicornis and D. afrolongicornis alberti was mainly in the shape of the marginal bar. Both D. spinicirrus and A. polycotyleus were widely distributed and recorded on the gills of all hosts during all seasons. Both species were recorded for the first time on B. radiatus. Also, D. spinicirrus was recorded for the first time on the gills of B. trimaculatus. Based on comparison with the original material, the species could be identified to species level. These analyses provided sufficient evidence for restoration of Afrodiplozoon polycotyleus as a valid taxon. The existence of two species, Gyrodactylus sp. and Dogielius sp. were recorded for the first time on B. radiatus in South Africa, and this possibly represents new species. The endoparasites included the following groups: digeneans- Diplostomulum metacercariae from the eyes of Lb. marequensis, Ornithodiplostomum sp. and black spot (grubs) were recorded from B. trimaculatus. The latter was also recorded in the muscle of B. radiatus. Unidentified digenean cysts were recovered from the gills and in the body cavity of both Lb. marequensis and B. trimaculatus; nematodes were represented by Contracaecum larvae in the body cavity of both Lb. marequensis and B. trimaculatus; cestodes were represented by gryporynchid larvae from the intestine of B. radiatus. The general high prevalence and intensities of ectoparasites recorded is an indication that the Nwanedi-Luphephe Dams has a biotic mechanism which might have enabled it to sustain the growth rate of ectoparasite intra-population. There was no correlation between either fish length or condition factor and the number of parasites. The study indicated that the abundance of monogeneans is partly influenced by season and that of endoparasites was principally governed by the presence of intermediate hosts and definitive hosts. The second part of this dissertation dealt with the health status of Lb. marequensis. Fish health was assessed using condition-related indices including condition factor and a modified Health Assessment Index (HAI) and the associated Parasite Index (PI). The HAI was performed to determine and examine any macroscopic abnormalities regarding external features and internal organs. The purpose of combining the two indices was to use the infestation of the metazoan parasites found on and/or in Lb. marequensis to determine whether or not the environment they live in was healthy. Both indices together with the condition factor provided relatively simple and rapid indications of how well fish were coping in their environment. The HAI score varied amongst the four sampling seasons. The highest individual mean value was 63 in winter, followed by a score of 50 in autumn, while the lowest were 42 and 33 in summer and spring respectively. To authenticate the HAI and PI data, certain water quality variables were measured and are discussed in detail in this dissertation. The Nwanedi-Luphephe Dams are generally believed to have good water quality. This was supported in this study; conditions assessed in fish using the aforementioned indices did not differ greatly between seasons, nor did the conditions deviate appreciably from normality. The HAI values were low overall which signifies a healthy fish profile for the system. The present investigation showed the existence of differences in the occurrence of individual parasite to be linked to water temperature changes. Thus, seasonal changes do influence parasite developmental stages to a certain degree. Tested heavy and trace metals were within the permissible limits as provided by the Department of Water Affairs and Tourism (DWAF, 1996). Description: Thesis (M.SC. (Aquaculture)) --University of Limpopo, 2011 Sat, 01 Jan 2011 00:00:00 GMT http://hdl.handle.net/10386/748 2011-01-01T00:00:00Z http://hdl.handle.net/10386/524 Title: Screening, isolation and purification of bioactive compounds with antibacterial activity against mycobacterium smegmatis Authors: Mmushi, Tshepo Joseph Abstract: The leaves of fifteen plant species were collected from the Lowveld Botanical Garden in Nelspruit, Mpumalanga Province, South Africa. The collection was based on a list of plants and their ethnopharmacological information provided by the Phytomedicine Programme at the University of Pretoria. The dried leaves of the plants were powdered and extracted using hexane, dichloromethane, acetone and methanol. The extracts were screened for antibacterial activity against Mycobacterium smegmatis and Rhodococcus erythropolis. The acetone extract of Milletia stulhimannii was the most active, showing activity against Mycobacterium smegmatis and Rhodococcus erythropolis with MIC values 0.13 and 0.08 mg/ml, respectively. Acetone extracts for all plants had the lowest MIC values ranging between 0.11-1.25 mg/ml and 0.08-1.25 mg/ml for M. smegmatis and R. erythropolis, respectively. Milletia stulhimannii, Albizia gummifera, Xanthocercis zambesiaca and Barringtonia racemosa extracts have shown the greatest potential for anti-tubercolosis agents. These were all active against M. smegmatis with an average MIC value of acetone extracts of 0.13 mg/ml. Apodytes dimidiata was selected for the isolation of active compounds since its activity on qualitative antibacterial activity assays was highly prominent on TLC plates in comparison to the other plant extracts. Two compounds were isolated from A. dimidiata but after purification, their MICs were above 2.5 mg/ml indicating a possible loss of activity during purification. The preliminary NMR spectra analysis suggested that the compounds were a long fatty acid and a triterpene. Future work is required to elucidate the chemical structures of the latter compounds and to test the activity of these compounds against Mycobacterium tuberculosis. Description: Thesis (M.Sc. (Microbiology)) --University of Limpopo, 2011 Sat, 01 Jan 2011 00:00:00 GMT http://hdl.handle.net/10386/524 2011-01-01T00:00:00Z