Browsing by Author "Nepolo, Emmanuel"
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Item Isolation and characterization of starch, starch biosynthetic genes and protease inhibitors from Marama beab (Tylosema Esculentum)(2014) Nepolo, EmmanuelMarama bean (Tylosema esculentum) is a highly nutritious plant and is currently regarded as a prospective crop for the future in arid zone agri-ecologies of the world. Starch is a major storage component in higher plants and in marama bean it’s mostly found stored in the tuber. Starch is used in both food and non-food industries. Starch biosynthesis involves groups of committed enzymes. Aims of the present study were to determine the physicochemical and pasting properties of native marama bean starch isolate and characterize marama starch biosynthesis genes and detect serine protease inhibitor activities in green and mature marama seeds. The total starch content of marama bean tubers was determined by amyloglucosidase/α-amylase enzymatic digestion and amylose content by Concanavalin A precipitation. A complementary Deoxyribonucleic acid (cDNA) library was constructed from marama tuber for the screening and isolation of Soluble Starch Synthase I (SSSI) and a Polymersae Chain Reaction (PCR) based strategy was used to isolate Adenosine diphosphate-glucose pyrophosphorylase (AGPase) and Starch Branching Enzymes (SBEs) using degenerative primers designed at the conserved motif of corresponding cloned plant starch synthesizing genes. Detection of serine protease inhibitor activities in green and mature marama seeds was established using the reverse zymogram technique and fluorogenic substrate N-alpha-benzoyl-l-arginine 7-amido-4-methylcoumarin hydrochloride and cDNA clone encoding a serine protease gene from marama was isolated using newly developed degenerate PCR primers. Native marama starch content was 87.38 mg starch/gram fresh weight and the total amylose content was 35 %. Phosphate at the C-6 position determined as Glucose-6-Phosphate was 0.788 nmol G6P/mg. The starch granules were round to elliptical with smooth surfaces and their sizes ranged from 8 -20 μm. The pasting properties of pasting temperature, host paste, peak, final viscosity, breakdown and set back showed higher values for marama starch in contrast to commercial potato starch. A cDNA clone encoding a SSSI from T. esculentum was isolated and identified by cDNA screening. The cDNA clone is 684 bp in length and encodes 228 amino acid residues. Sequencing of cloned cDNA showed 100% identity with potato SSSI.The phylogenetic tree indicated the divergence of SSI in higher plants proceed in line with evolutions of monocots from dicots. AGPase and SBEs genomic clones from T.esculentum were isolated and their sequence features revealed. AGPase small and large subunit clones both showed 96% identities with Glycine max, while SBEI and SBEII clones showed 91 and 93% identities to Cicer arietinum and Medicago truncatula. Phylogenetic trees for both AGPase and SBEs clones exhibited higher sequence similarity to the AGPase and SBEs of dicotyledons. Reverse zymogram analysis revealed four putative serine inhibitor activities in mature seeds and not in green seeds. Fluorogenic substrate analysis showed significantly higher (P <0.05) trypsin activity in mature marama bean (2326±356 Fluorescence Units mg 1 protein) than green seeds (362±73 FU mg 1 protein).The analyses showed lower (P <0.05) serine protease activity in both cowpea (877±138 FU mg 1 protein) and soybean (381±36 FU mg 1 protein). A partial cDNA clone encoding a serine protease gene from marama bean showed 100% identity to trypsin inhibitor for cowpea. Physicochemical properties of marama starch revealed considerable high amylose content and pasting properties when compared to other tuberous starch producing crops. Molecular features of starch synthesizing genes in marama were similar to that of the same genes characterized in other plant species. Serine inhibitors activities in marama seed may as well be an indicative that marama seed may have potent serine protease inhibitors which could be utilized in pharmaceuticals and in human health. Physicochemical properties of marama starch would suggest that it could find applications in foods and non-food uses that require higher amylose content and high pasting properties. Isolation and characterization of marama starch biosynthetic genes provide theoretical foundation in understanding the structure and functions of SSSI, AGPase and SBEs. Serine protease inhibitor gene isolation and detection of serine protease inhibitor of marama seeds will broaden the pool for plant serine protease genes and might be an ideal choice for developing pharmaceutical products. Future studies need to establish the potential uses of marama starch, characterize marama starch biosynthetic gene structures, functions, regulation and expression. It would be interesting to study in the future any reason for the higher serine protease activity in mature seeds than in green seeds as established recently.This study has clearly contributed to starch biology by making known for the first time the physicochemical and functional properties of marama tuber starch. It also established for the first time the genetic signature of genes involved in starch biosynthesis of marama bean, which may confer unique properties to marama tuber starch. At the same time, the presence of serine protease inhibitors activities and serine protease inhibitor gene in marama seeds with possible application in pharamaceutical products were investigated for the first time making this investigation novel.Item Physicochemical and functional characteristics of starch extracted from Marama bean tuber (Tylosema esculentum Burchell A. Schreiber)(University of Namibia, 2015) Nepolo, Emmanuel; Llyod, J.R.; Chimwamurombe, Percy M.Marama bean (Tylosema esculentum Burchell A. Schreiber) is a highly nutritious plant and is currently regarded as a prospective crop for the future in arid zone agri-ecologies of the world. Starch is a major storage component in higher plants and it is used in both food and non-food industries. The marama bean plant is a creeper with stems that lie prostrate on the ground in several directions up to six metres long in length which spread from a tuberous root below the ground. The tuber has a reddish brown bark and it can weigh about 1 kg and up to more than 10 kg. The plant produce attractive bright yellow flowers along the stems, each with erect petals and stamens, and are followed by marama fruits or bean. Mature marama bean cotyledons are white to cream in colour, encapsulated in hard, woody seed coats, reddish to brownish black in colour. Until recently, the basic knowledge of the physicochemical and functional properties of marama tuber starch is has not been yet reported. The present study reports for the first time the physicochemical and functional properties of marama tuber starch and makes a possible provision for a new starch source. Native marama starch content was 87.38 mg starch/gram fresh weight and the total amylose content was 35%. Phosphate at the C-6 position determined as Glucose-6-Phosphate was 0.788 nmol G6P/mg. The starch granules were round to elliptical with smooth surfaces and their sizes ranged from 8 -20 μm. The pasting properties of pasting temperature, host paste, peak, final viscosity, breakdown and set back showed higher values for marama starch in contrast to commercial potato starch. This study has clearly contributed to starch biology by making known for the first time the physicochemical and functional properties of marama tuber starch. It is hoped that by further exploring the potential of marama starch as a raw material, it can be applied in various applications in both industry and food processing that will produce high valued products.Item Study on indigenous medicinal knowledge in Caprivi region(2011) Du Preez, Iwanette C.; Nepolo, Emmanuel; Siyengwa, Riina; Shapi, Martin K.; Cheikhyoussef, Ahmad; Mumbengegwi, Davis R.SUMMARY: Indigenous knowledge is defined as the uniqe knowledge in a given culture, which is passed on orally from generation to generation (Mapaure & Hatuikulipi, 2007; Dan et al., 2010). This indigenous knowldege is regarded as valuable and considered as the local people’s capital (Mapaure and Hatuikulipi, 2008; Cheikhyoussef et al, 2011), and if not preserved, it may be lost forever to society. The study on Indigenous Medicinal Knowledge in Caprivi region was done in collaboration with the Directorate of Research Science and Technology (DRST) of the Ministry of Education, and was done as a continuing research work for the national survey on Traditional Healing in Namibia. The traditional medical systems are generally based on the uses of natural and local products which are commonly related to the people’s perspective on the world and life (Toledo et al. 2009). Traditional African medicine is 'the sum total of practices, measures, ingredients and procedures of all kinds whether material or not, which from time immemorial has enabled the African to guard against diseases, to alleviate his/her suffering and to cure him/herself' (Busia, 2005). The traditional healing practices in Namibia have been studied by a number of researchers (Shapi et al, 2009; Dan et al, 2010). The aim of this national survey will be to create a database based on the indigenous knowledge of medicinal plants used by traditonal healers and/or knowledge holders to treat a variety of ailments and diseases, as well as for cultural beliefs and practices. As such, the overall aim of this fieldtrip was to interview traditonal healers in the Caprivi region and retrieve as much information as possible with regarding to the uses of medicinal plants, and to collect voucher specimens to be deposited with the National Botanical Research Institute (NBRI), Windhoek, Namibia.Item Subjective well-being, psychological well-being and anxiety symptoms of medical and pharmacy students in Namibia(University of Namibia, 2017) Perstling, Martina; Nepolo, Emmanuel; Udjombala, N.; Nyarango, Peter; Karuaihe, M.; Hunter, Christian J.Medical and pharmacy students at the School of Medicine of Namibia underwent a survey investigating their level of mental well-being and anxiety symptoms. Not only were the two constructs, subjective well-being and psychological well-being positively inter- related but also correlated inversely with anxiety symptoms. Environmental mastery in the psychological well-being scale was negatively associated with anxiety (coefficient: -0.17, 95% CI: [-0.27,-0.7], p-value = 0.001). Supportive qualitative data brought some environmental challenges forward that were typical for medical and pharmacy students as these professions are fundamentally emotionally challenging, in addition to a taxing curriculum, work overload and time constraints influencing well-being negatively. Supplementary information contributed towards additional challenges which deviate from the common hardships of studying high profile professions such as medicine and pharmacy. Firstly, most of the students needed to migrate to the capital city to study, finding themselves in a new and unfamiliar environment; and, secondly poverty influenced the well-being of the students in addition to physical safety issues.