Faculty of Applied Sciences

Permanent URI for this communityhttp://ir-dev.dut.ac.za/handle/10321/5

Browse

Search Results

Now showing 1 - 10 of 19

Protein engineering of fungal xylanase
(2007) Stephens, Dawn Elizabeth
Protein engineering technologies, such as directed evolution and DNA recombination, are often used to modify enzymes on a genetic level for the creation of useful industrial catalysts. Pre-treatment of paper pulps with xylanases have been shown to decrease the amounts of toxic chlorine dioxide used to bleach pulp. This study was undertaken to improve the thermal and alkaline stabilities of the xylanase from the fungus Thermomyces lanuginosus using ep-PCR and DNA shuffling.
Molecular characterization of aflatoxigenic and non-aflatoxigenic aspergillus isolates
(2007) Mngadi, Phakamile Truth
For decades the genus Aspergillus (of fungi) has been classified based on morphological and growth criteria. Members of the Aspergillus section Flavi are economically valuable and methods of differentiating them are thus very important. Several molecular methods have been developed to distinguish these strains. Also, a number of biochemical and genetic studies have been used in order to provide a better means of classification (Lee et al., 2004). Aflatoxins, the most frequently studied mycotoxins, are produced by certain Aspergillus species/strains/isolates of fungi. The aflatoxin biosynthetic pathway studies have led to a number of discoveries. Several structural and regulatory genes (and their enzymes) involved in the biosynthesis of aflatoxins have been discovered and purified (Trail et al., 1995). Aflatoxin production and contamination of agricultural crops are major causes of economic losses in agriculture. Thus, better methods of characterization/differentiation are required for both aflatoxigenic and non-aflatoxigenic isolates. Molecular biology is one of the current tools used to differentiate between these isolates. Polymerase Chain Reaction (PCR)-based randomly amplified polymorphic DNA (RAPD) analysis has been used successfully in the analysis of DNA relatedness of species of fungi, bacteria, plants and animals. Dendograms which evaluate/assess the likeness between different isolates has also been used (Martinez et al., 2001). Restriction fragment length polymorphism (RFLP) analysis has been applied to a number of studies to detect differences between fungi and to establish relationships between them. Therefore, the scope of this study was to investigate RAPD analysis (with dendograms) and detection of RFLPs by hybridization as molecular methods that can distinctly differentiate or characterize the aflatoxigenic and non-aflatoxigenic Aspergillus isolates.
Molecular analyses of pure cultures of filamentous bacteria isolated from activated sludge
(2005) Naidoo, Dashika
The activated sludge process is the mostl used biological treatment process. Engineers and microbiologists are constantly seeking ways to improve process efficiency, which can be attributed to the increasing demand for fresh water supplies and proper environmental management. Since the inception of the activated sludge process, bulking and foaming have been major problems affecting its efficiency. Filamentous bacteria have been identified as the primary cause of bulking and foaming. Numerous attempts have been made to resolve this problem. Some of these attempts were effective as interim measures but failed as long term control strategies. The identification of filamentous bacteria and the study of their physiology have been hampered by the unreliability of conventional microbiological techniques. This is largely due to their morphological variations and inconsistent characteristics within different environments. To fully understand their role in promoting bulking and foaming, filamentous bacteria need to be characterized on a molecular level. The aim of this study was, therefore, to identify filamentous bacteria in pure culture with the purpose of validating these findings to the physiological traits of the pure cultures when they were isolated. Fourteen different filamentous cultures were used for this study. The cultures were identified using specific oligonucleotide probes via fluorescent in situ hybridisation and nucleotide sequencing. Prior to sequencing, an agarose gel and a denaturing gradient gel Electrophoresis profile were determined for each isolate. The various techniques were optimised specifically for the filamentous isolates. The isolates were identified as Gordonia amarae, Haliscomenobacter hydrossis, Acinetobacter sp./Type 1863, Type 021N, Thiothrix nivea, Sphaerotilus natans and Nocardioform organisms.
Production of enzymes for application on animal feeds
(2007) Godana, Busiswa; Singh, Suren; Mitra, R.
Ruminants diets in most developing countries are based on fibrous feeds, mainly mature pastures and crop residues. These feeds are unbalanced and particularly deficient in protein, minerals and vitamins and are highly lignified with low digestibility. These characteristics result in low intake and productivity and only approximately 40% of energy in forage becomes available to the animal. Enzymes can be used as biological tool to enhance digestion through the action of cellulase, hemicellulase and lignase enzymes resulting in improved meat, milk and wool production. The development of feed additives holds great promise for the improvement of livestock growth and yield for both large commercial and smaller subsistence farmers. The aim of this study was to develop optimal media for the production of lignocellulolytic enzymes (laccases, xylanases, and cellulases) and to evaluate the application of these enzymes to improve the nutritional digestibility of high fibre feedstocks, such as veld grass.
Functional characterisation of heterotrophic denitrifying bacteria in wastewater treatment systems
(2005) Ramdhani, Nishani; Bux, Faizal
Atmospheric nitrogen pollution is on the increase and human activities are directly or indirectly responsible for the generation of the various nitrogen polluting compounds. This can lead to the two major problems of eutrophication and groundwater pollution. Therefore, the removal of nutrients such as nitrogen and phosphorus from wastewater is important. Nitrogen removal from wastewater is achieved by a combination of nitrification and denitrification. Thus, there is a need to identify and characterise heterotrophic denitrifying bacteria involved in denitrification in wastewater treatment systems. The aim of this study, therefore, was to characterise heterotrophic denitrifying bacteria through detailed biochemical and molecular analysis, to facilitate the understanding of their functional role in wastewater treatment systems. Drysdale (2001) isolated heterotrophic denitrifiers to obtain a culture collection of 179 isolates. This culture collection was used to screen for nitrate and nitrite reduction using the colorimetric biochemical nitrate reduction test. The isolates were thereafter Gram stained to assess their gram reaction, cellular and colonial morphology. Based on these results identical isolates were discarded and a culture collection of approximately 129 isolates remained. The genetic diversity of the culture collection was investigated by the analysis of polymerase chain reaction (PCR)-amplified 16S ribosomal DNA (rDNA) fragments on polyacrylamide gels using denaturing gradient gel electrophoresis (DGGE). Thus DNA fragments of the same length but different nucleotide sequences were effectively separated and microbial community profiles of eight predominant isolates were created. Batch experiments were conducted on these eight isolates, the results of which ultimately confirmed their characterisation and placed them into their four functional groups i.e. 3 isolates were incomplete denitrifiers, 2 isolates were true denitrifiers, 2 isolates were sequential denitrifiers and 1 isolate was an exclusive nitrite reducer.
Improvement of thermostability of a fungal xylanase using error-prone polymerase chain reaction (EpPCR)
(2007) Pillay, Sarveshni; Permaul, Kugen; Singh, Suren
Interest in xylanases from different microbial sources has increased markedly in the past decade, in part because of the application of these enzymes in a number of industries, the main area being the pulp and paper industry. While conventional methods will continue to be applied to enzyme production from micro-organisms, the application of recombinant DNA techniques is beginning to reveal important information on the molecular basis and this knowledge is now being applied both in the laboratory and commercially. In this study, a directed evolution strategy was used to select an enzyme variant with high thermostability. This study describes the use of error-prone PCR to modify the xylanase gene from Thermomyces lanuginosus DSM 5826, rendering it tolerant to temperatures in excess of 80°C. Mutagenesis comprised of different concentrations of nucleotides and manganese ions. The variants were generated in iterative steps and subsequent screening for the best mutant was evaluated using RBB-xylan agar plates. The optimum temperature for the activity of xylanases amongst all the enzyme variants was 72°C whilst the temperature optimum for the wild type enzyme was 70°C. Long term thermostability screening was therefore carried out at 80°C and 90°C. The screen yielded a variant which had a 38% improvement in thermostability compared to the wild type xylanase from pX3 (the unmutated gene). Successive rounds of error-prone PCR were carried out and in each round the progeny mutant displayed better thermostability than the parent. The most stable variant exhibited 71% residual activity after 90 minutes at 80˚C. Sequence analysis revealed four single amino acid residue changes that possibly enhanced their thermostabilities. This in vitro enzyme evolution technique therefore served as an effective tool in improving the thermostable property of this xylanase which is an important requirement in industry and has considerable potential for many industrial applications.
Elucidation of the microbial community structure within a laboratory scale activated sludge process using molecular techniques
(2006) Padayachee, Pamela; Bux, Faizal
The microbial community present in a laboratory-scale modified Ludzack-Ettinger activated sludge system was investigated using a combination of novel molecular techniques. The parent system was investigated for a duration of one year and samples were taken at regular intervals to determine the profile and structure of the microbial community present within the anoxic and aerobic zones of the MLE system. The combination of molecular techniques included fluorescent in situ hybridisation (FISH) and denaturing gradient gel electrophoresis (DGGE). FISH was performed using oligonucleotide probes, which were complementary to conserved regions of the rRNA for the alpha, beta and gamma subclasses of the gram negative family Proteobacteria as well as a group-specific HGC oligonucleotide probe as a representative of the gram positive actinomycetes branch. The total eubacteria present was determined using the EUB oligonucleotide probes, EUB388, EUB388-II and EUB388-III. The DGGE analysis of PCR-amplified 16S rDNA gene segments was used to examine the microbial community profile in the anoxic and aerobic zones. The profile for each of the zones revealed a number of consistent bands throughout the duration of the laboratory-scale process. However, the profiles obtained suggested that a diverse microbial community existed within the aerobic and anoxic zones. The bands also indicated the presence of dominant and less dominant species of bacteria. Hybridisations obtained from the FISH analyses indicated that the alpha and gamma subclasses were predominant within the anoxic zone and the aerobic zone showed a dominance of the beta subclass of Proteobacteria. The steady state behaviour of the MLE system was confirmed with the results obtained from COD, TKN, nitrates and OUR analytical tests. COD and nitrogen mass balances were conducted to confirm the acceptance of the results obtained for each batch as an indication of the system performance for the MLE model. Nitrogen mass balances indicated an upset in the nitrogen levels for batches two and seven.
The essential oil from Cymbopogon validus
(2007) Naidoo, Nelisha; Odhav, Bharti; Baijnath, Himansu
The chemical and biological properties of the essential oil from Cymbopogon validus were investigated. Hydro-distillation was used to extract the oil from C. validus, the flower-heads, leaves, culms and rhizomes. The percentage oil yields obtained from the plant organs varied from 0.05 to 1.23%, with the greatest concentration found in the flower-heads and rhizomes, 1.23 and 1.12% respectively. A sensory evaluation of the oil revealed that the essential oil was slightly murky, pale yellow in colour, had a strong turpentine-like smell and remained liquid at room temperature. The oxidative stability of C. validus oil was evaluated by determining its Rancimat induction period (negative), peroxide value (60.56 meq/kg), iodine value (84.55), percentage free fatty acids (0.19%) and percentage cholesterol (3.03%). These results indicated that the oil was highly susceptible to oxidation. Chromatographic profiles of the oils from C. validus, as well as the plant organs were generated using gas chromatography-mass spectroscopy (GC-MS). Predominant compounds present in the oil included alpha-Cubebene, Camphene, Geraniol, Limonene, Myrcene, Palmitic acid and Sabinene. C. validus essential oil was also investigated for its antimicrobial (disk diffusion), antioxidant (1, 1-Diphenyl-1-Picrylhydrazyl (DPPH) assay), anti-inflammatory (5-lipoxygenase assay), anti-mosquito properties (insecticidal, larvicidal and repellency assays) and toxicity profile (Brine shrimp and Ames assays). The oil showed poor antimicrobial activity and inhibited the growth of only Gram positive bacteria with a minimum inhibitory concentration (MIC) of 0.0625 (vol/vol) for Bacillus, Micrococcus and Staphylococcus species. The oil also exhibited excellent antioxidant activity, scavenging more than 80% of DPPH free radicals and possesses anti-inflammatory activity (IC50=190 ppm). C. validus oil showed good adulticidal activity (53.7% mortality) and excellent larvicidal (100% mortality) and repellent activity (100% repellency) against Anopheles arabiensis mosquitoes. At high concentrations, the oil was toxic to brine shrimp larvae. However, when diluted it was safe and the minimum inhibitory concentration was 0.0001(vol/vol). The absence of revertant colonies at all essential oil concentrations in the Ames test suggest that the oil is not mutagenic. These results lead the way for exploiting C. validus oil as a multi-functional agent that has antibacterial, anti-oxidant, anti-inflammatory, and anti-mosquito properties.
Comparative analysis of genetically modified maize by implementation of a half-seed extraction technique
(2007) Pienaar, Fernando; Permaul, Kugen
The development of transgenic plants resulted in the need to utilize the various molecular methods (e.g., ELISA, real - time PCR etc.) for the detection or analysis of the presence or absence of a specific trait in a particular plant (Bt in this study). The overall aim of this study was to optimize a half – seed extraction technique as part of a laboratory protocol for transgenic maize plants and to explore the possibility of using the following molecular techniques: horizontal isoelectric focusing, real - time PCR and ELISA, as methods for detection of the Bt trait for incorporation into the half – seed extraction protocol.
Chemoprotective action of natural products on cultured human epithelial cells exposed to aflatoxin B1
(2005) Reddy, Lalini; Odhav, Bharti
Previous studies indicate that a mutation in the non-oncogenic p53 gene is epidemiologically linked to human HCC (Ozturk, 1991; Chan et al., 2003). Hsu et al. (1991) found this link in Chinese, South African and Asian patients and Hollstein et al. (1993) found the same gene mutation in Taiwanese patients. The incidence of these aberrations is reported to be about 20- 50% in HCC’s (Kishimoto et al., 1997). There is sufficient evidence to indicate that carotenoids in addition to their well known antioxidant properties (Paiva and Russel, 1999), also affect intercellular communication, immune responses, neoplastic transformations and growth control, and cellular levels of enzymes that detoxify carcinogens (Zhang et al., 1991; Brockman et al., 1992; Pryor et al., 2000). To date studies carried out have used the rat (Foote et al., 1970; Gradelet et al., 1998) and the mule duckling model (Cheng et al., 2001) to show the protective effect of these carotenoids against AFB1 exposure. Of the well known carotenoids, lycopene and beta- carotene occur in abundance in fruits and vegetables and are safe for human consumption. Aflatoxin B1 frequently induces mutations of the p53 gene which is linked to HCC. Although there is much evidence from epidemiological studies linking the beneficial aspects of carotenoids to the prevention of cancer, the cellular and molecular mechanisms need to be understood in order to implement large scale intervention strategies to prevent AFB1 induced carcinoma. The use of chemical or dietary interventions to alter the susceptibility of humans to the actions of carcinogens and to block, retard or reverse carcinogenesis is an emerging chemoprotective strategy for disease prevention (Abdulla and Gruber, 2000; Kensler et al., 2003; Bingham and Riboli, 2004). Chemoprotection by natural products involves maintaining cellular integrity, preventing DNA alterations, activation of p53 suppressor protein and apoptosis. The aim of this study was thus to investigate the cellular and molecular mechanisms by which beta-carotene and lycopene may prevent the AFB1-induced toxic changes in human hepatocytes. In order to achieve this aim, the following objectives were set out: i. To optimise an in vitro system for the evaluation of AFB1 damage to cultured hepatocytes. ii. To determine the biochemical protection offered by beta-carotene and lycopene to AFB1-exposed hepatocytes, by measuring the mitochondrial activity, cell viability and ROS levels using appropriate enzyme assays and flow cytometry. iii. To determine the cellular protection offered by beta-carotene and lycopene to AFB1-exposed hepatocytes, by studying the morphological changes at the structural and ultrastructural levels using phase contrast light and electron microscopy respectively. iv. To determine the molecular protection offered by beta-carotene and lycopene to AFB1-exposed hepatocytes, by detecting apoptotic bodies as genomic markers and measuring the levels of p53 protein and AFB1-N7-guanine adducts produced.

Faculty of Applied Sciences

Browse

Filters

Settings

Sort By

Results per page

Search Results