Master of Science (M.Sc.) Theses and Dissertations

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    Purification and characterisation of 3-mercaptopyruvate sulfurtransferase from the liver of fruit bat (eidolon helvum)
    (Department of Biochemistry and Molecular Biology, Obafemi Awolowo University, Ile Ife, Nigeria., 2024) Alalade, Foluso Ruth.
    This study isolated and purified 3-mercaptopyruvate sulfutransferase (3-MST) from the liver of fruit bat (Eidolon Helvum). It also determined the physicochemical properties of 3-MST from E.helvum. These were with a view to providing information on the physicochemical properties of the enzyme. Eidolon helvum (fruit bat) were collected from the Botanical Garden, Obafemi Awolowo University, Ile-Ife, Nigeria and identified at the department of Zoology, O.A.U, Ile-Ife. The E.helvum were transported to the Enzymology Laboratory, Department of Biochemistry and Molecular Biology, Obafemi Awolowo University, Ile-Ife, Nigeria and the liver were immediately excised stored in the refrigerator until use. The liver was allowed to thaw weighed and homogenized using 3 volumes of homogenization buffer (0.1M phosphate buffer, pH 7.4). The homogenate was centrifuged at 10,000 rpm for 15 minutes at 4 ºC to remove cell debris. The supernatant was collected and assayed for 3-MST activity and protein concentration was determined following standard methods. Purification of the enzyme from Eidolon helvum was carried out using ammonium sulphate precipitation, ion exchange chromatography on CM Sephadex C-50 cation exchanger and Sephadex G-100 gel filtration chromatography. The native molecular weight of the purified 3- mercaptopyruvate sulfurtransferase was determined using Sephadex G-100 while the subunits molecular weight of the purified 3-mercaptopyruvate sulfurtransferase was determined using SDS PAGE. The purified enzyme was further characterized by determining the effects of pH and temperature (optimum and stability). The kinetic parameters, substrate specificity (using sulphur compounds), effects of inhibitors and effects of metals was also determined. 3-Mercaptopyruvate sulfurtransferase from the liver of E. helvum was purified with a specific activity of 11.08 U/mg, purification fold of 8.21 and % yield of 46.92. The native molecular weight and subunit molecular weight were 36.24 kda and 35.72 kda respectively. The apparent Km and Vmax values for potassium cyanide and mercaptoethanol were 8.60 ± 0.17 mM, 96.65 ± 0.56 MU/ml/min and 7.77 ± 0.42 mM, 48.02 ± 0.38 MU/ml/min respectively. The optimum temperature and pH were 70 ºC and 7.0 respectively. Substrate specificity study involving the use of other sulphur compounds revealed that 3-MST from the liver of E. helvum showed high preference for mercaptoetanol and sodium thiosulphate. Metal salts (SnCl2, HgCl2, MnCl2, NaCl, MgCl2 and KCl) showed different degrees of inhibition of 3-MST from the liver of E. helvum. At 70 ºC, 3-MST from the liver of Eidolon helvum retained 66.67 % of its activity for 40 minutes. Urea and EDTA inhibited the activity of the 3-MST while DPPD did not affect the activity of the enzyme. The study established the presence of Mercaptopyruvate sulfurtransferase (a cyanide detoxifying enzyme) in the liver of Eidolon helvum. It further showed the physicochemical properties of 3-MST in the liver of Eidolon helvum. This could possibly be responsible for the mechanism of survival of the Eidolon helvum through cyanide detoxification in their environment.
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    Comparative Assessment of Plant-Based Extracts and Synthetic Insecticides on the Growth, Yeild and Proximate Composition of Cowpea (Vigna Unguiculata L. Walp)
    (Department of Institute ofr Ecology and Environmental Studies, Faculty of Science, Obafemi Awolowo University., 2022) FEYINSOLAMI, Ebunoluwa adebayo
    This study assessed the effect of plant-based and synthetic insecticides application on the growth response and yield of cowpea. It also investigated the effects of the insecticides application on the biomass, grain yield and proximate composition of cowpea grains produced. These were with a view to providing information on the use of plant-based extracts on quantity and quality of cowpea grains. The experiment was conducted on a vacant land measuring 11.75 m x 7.50 m behind the Institute of Ecology and Environmental Studies, Obafemi Awolowo University, Ile-Ife. Viable seeds of Ife-brown cultivar of cowpea were purchased from the Institute of Agricultural Research and Training, Ibadan. The experimental location was cleared manually two times using a cutlass and hand-held hoe. The experiment consisted of four treatments which were: extracts of three plant-based (Azadirachta indica, Tithonia diversifolia, Chromolaena odorata) and cypermethrin that served as control. The extracts of fresh shoots of A. indica, T. diversifolia, and C. odorata were separately prepared using standard method. The experiment was made up of 12 plots, each measuring 1.5 m x 2.0 m and the plots were arranged in a randomized complete block design. Four seeds per stand were sown using 50 cm x 30 cm spacing and seedlings thinned to 2 stands per hole at 2 weeks after sowing (WAS). The plots were weeded at 3 and 6 WAS. The cowpea stands were sprayed with plant-based and cypermethrin using the rates 100 g/L/plot and 15 mL/L/plot respectively at 5, 6, 7 and 8 WAS. Growth parameters such as plant height, number of leaves and stem girth were measured bi-weekly from 2 to 8 WAS and extent of leaf damage at 7 and 8 WAS. Tagged cowpea stands were carefully uprooted at 10 WAS to determine the total biomass yield. Cowpea pods were harvested when the pods turned yellow at 10 WAS and threshed. Proximate composition of cowpea grains (crude protein, ash, fibre, carbohydrate, fat and dry matter), pre- xv and post-cropped soil analyses were carried out using standard methods. Data obtained were subjected to analysis of variance and their treatment means were separated using Tukey’s multiple comparison test at p < 0.05. The results showed that pH of the pre-cropped soil was 6.94 and soil texture was loamy sand. Organic carbon and total nitrogen were 0.64 and 5.82 g/kg, respectively and these values reduced to between 13 and 30% across the treatment plots for the post-cropped soil. The growth parameters; height (cm), number of leaves and stem girth (cm) at 6 WAS were: 23.06 ± 0.86, 55.29 ± 4.59 and 1.58 ± 0.05 for A. indica; 24.15 ± 0.75,57.58 ± 3.94 and 1.69 ± 0.06 for T. diversifolia; 21.76 ± 0.68, 48.38 ± 2.15 and 1.55 ± 0.05 for C. odorata; and 21.99 ± 1.18, 45.26 ± 3.45 and 1.46 ± 0.07 for cypermethrin, respectively. Also, the grain yield of cowpea with cypermethrin, 1.08 t/ha was significantly (p < 0.05) higher than the best plant-based insecticide, T. diversifolia. Cowpea grains obtained with T. diversifolia and C. odorata had comparable and high values of crude protein (29.7%, 28.3%) and fibre (6.1%, 6.5%) respectively. The study concluded that T. diversifolia compared favourably with cypermethrin, in terms of grain yield of cowpea, whereas T. diversifolia and C.
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    A study of distributed lag model using koyck and almon techniques
    (Department of Mathematics, Falculty of Science, Obafemi Awolowo University, Ile-Ife., 2024) Toyin, Kayode Ajetunmobi.
    This study evaluated the effects of the choice of optimal truncation lag-length, determined the effects of multicollinearity and choice of approximating polynomial, and compared the efficiency of Almon and Koyck estimators. These were with a view to providing information on the effects of multicollinearity, choice of optimal truncation lag-length and choice of approximating polynomial on parameter estimation of Koyck and Almon distributed lag models. The study conducted a comprehensive time series analysis using monthly exchange rates (Naira-Dollar, Naira-Pound and Naira-Euro) and Inflation rate data from Central Bank of Nigeria over the period of January 2004 and April 2021 for Koyck Distributed Lag Model and Almon Distributed Lag Model. R Software Package was used to analyze the data for both models, while varying the order of approximating polynomials (k) and lags (q) with the condition that, degree of polynomial is less that number of lags. The research work employed standard procedure such as statistical significance, information criteria to determine the op timal truncation lag-length required for the implementation of distributed lag models. For the measure of multicollinearity in distributed lag models, Variance inflation factor was also used to determine severity of multicollinearity in the distributed lag models. The efficiency of Koyck and Almon estimators were compared using standard error The results of the study showed that Koyck estimator is preferable to Almon estimator due to lower standard error of Koyck estimators which indicate better precision of estimates. However, Almon estimators are preferable to Koyck estimators given the higher values of variance inflation factors (VIF) of Koyck estimator. To avoid problem of multicollinearity, the Almon estimator is the better choice. The study concluded that polynomial of order 2 is the best approximating polynomial for Almon distributed lag model and that Koyck estimators have higher precision in parameter estimation than Almon estimators.
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    Open Access
    Molecular characterization of antibiotic resistance in escherichin coil and klebsiella pneumoniae from selected livestock farms in Osun State, Nigeria.
    (Department of Microbiology, Faculty of Science, Obafemi Awolowo University, 2022) SULAYMAN Taofiq Adewale
    This study isolated Escherichia coli and Klebsiella pneumoniae from rectal and faecal samples of some selected livestock farms in Osun State, Nigeria, investigated the antibiotic susceptibility characteristics and the multiple antibiotic resistance index (MARI) phenotype properties of the confirmed isolates, as well as determined the occurrence of some clinically important resistant genes in the isolates. These were with a view to providing information for the mapping of antimicrobial resistance in the agroecosystem. A total of 197 samples were collected from apparently healthy cattle (13 faecal), pigs (61 rectal), and poultry (123 faecal) from 12 selected livestock farms within Osun State. Rectal swabs were collected by gently inserting moistened sterile swab sticks into the rectum of the animals while fresh faecal droppings were scooped with sterile swab sticks. E. coli and K. pneumoniae were presumptively isolated based on cultural characteristics. The identity of the presumptive isolates was confirmed using polymerase chain reaction detection of the uidA (E. coli) and ITS (K. pneumoniae). Antibiotic susceptibility of the isolates was determined using the disk diffusion method based on the interpretative standard described by the Clinical Laboratory Standard Institute. The presence of genes encoding clinically significant resistance determinants was also determined using polymerase chain reaction for the isolates. A total of 238 presumptive E. coli isolates and 123 presumptive Klebsiella pneumoniae isolates were recovered from the collected samples. Out of the presumptive isolates, 207 (87%) and 66 (53.7%) were confirmed as E. coli and K. pneumoniae, respectively, using polymerase chain reaction. However, 32 (48.5%) non-repetitive K. pneumoniae isolates and 90 (43.5%) non-repetitive E. coli isolates were selected for further analysis. For the E. coli isolates, a high resistance rate was observed against tetracycline (81.1%), ciprofloxacin (40%) and ampicillin (36.7%). In the K. pneumoniae isolates, high resistance rates were observed against ampicillin (78.1%), ciprofloxacin (65.6%) and tetracycline (50%). The multiple antibiotic resistancepattern of the isolates revealed resistance against at least three different antibiotics, while the multiple antibiotic resistance indices ranged from 0.3 to 0.8. Genes encoding beta-lactamases: blaTEM (83.3%), blaOXA-1-LIKE (37.8%), blaSHV (4.4%) blaFOX (68.9%), blaACC (45.6%), blaCIT (41.1%), blaMOX (10%), blaDHA (2.2%), blaEBC (2.2%), and plasmid mediated quinolone resistance genes: qepA (25.6%), aac(6’)-Ib-cr (16.7%), oqxB (64.4%), oqxA (16.7%), qnrB (2.2%), qnrA (14.4%) and qnrS (45.6%) were detected in the E. coli isolates, while, blaTEM (78.1%), blaOXA-1-LIKE (75%), blaSHV (68.8%) blaFOX (75%), blaACC (34.4%), blaCIT (6.3%), qepA (18.8%), aac(6’)-Ib-cr (21.9%), oqxB (100%), oqxA (71.9%), qnrB (9.4%), and qnrS (28.1%) were detected in the K. pneumoniae isolates. This study concluded that multidrug-resistant E. coli and K. pneumoniae, harbouring clinically significant resistance genes are present in livestock which could be transferred to humans through direct contact or the food chain. Plasmid-mediated quinolone resistance and beta-lactamases genes, often reported in clinical settings, were detected in some of the isolates in this study.
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    Production, purification and characterization of keratinase from keratinase producing bacteria isolated from selected chicken Feature dumpsites in Ile Ife Nigeria
    (Department of Microbiology, Faculty of science, Obafemi Awolowo Universty, Ile Ife, 2021) Oluwatobi Sanson Ayegun
    This research study focused on the isolation and identification of keratinase-producing bacteria from soil samples of selected chicken feather dump sites. It also explored the optimum conditions required for the production of keratinase by the isolated keratinase producing bacterium, thereby leading to the partial purification and characterization of the keratinase produced using its physicochemical and kinetic parameters. Soil samples were obtained from selected feather dumpsites in Ile-Ife from where keratinase producing bacteria isolated and purified using nutrient agar. The isolates were screened on skimmed milk agar and the isolates with high yield were subjected to submerged fermentation in order to select the best bacterial isolate for keratinase production. After 72 hours of incubation at a temperature of 37 oC, the mineral salt media were centrifuged at 4000 rpm for 20 minutes in a cold condition and the supernatants obtained were used as crude enzymes which were later quantified using keratinase assay and protein concentration. The optimum conditions of various parameters were investigated for the optimal production of keratinase by the isolate. Afterwards, all the optimum parameters were combined and used for the bulk production of crude enzyme. The crude enzyme produced was partially purified using 90 % Ammonium sulphate saturation and ion exchange chromatography and the partially purified enzyme was later characterized. A total of 110 bacteria were isolated and screened for keratinase production, 98 of the bacterial isolates tested positive to the screening process using Skimmed Milk agar. The best keratinase producer using the results of the keratinase assay and protein concentration from the submerged fermentation process was molecularly identified as Bacillus subtilis 2C-9B. The optimal temperature required for the production of keratinase by Bacillus subtilis 2C-9B was discovered to be 35 oC and at a pH 9.0. The carbon and nitrogen source that gave an optimal production of keratinase was discovered to be feather meal and yeast extract respectively. The bulk enzyme produced was partially purified using 90 % ammonium sulphate saturation and the result gave a 0.68-fold purification and this was further purified by ion exchange chromatography yielding an active protein peak at 4.9-fold purification. The purification process was followed by the characterization of the partially purified keratinase. An optimum pH in the alkaline region of pH 11.0 and optimum temperature of 45ºC was recorded. Metal ions like Ba2+, Hg2+ and Mn2+ inhibited the enzyme activity whereas Cu2+ and Pb2+ exhibited a slight inhibition. Phenylmethylsulphonyl fluoride (PMSF) completely inhibited the activity of the partially purified keratinase. However, EDTA did not inhibit the enzyme which implies that the partially purified enzyme belongs to the serine protease family and not the metalloprotease family. The study concluded that the keratinase produced by the isolated Bacillus subtilis 2C-9B strain was an alkalophilic enzyme which implies that it is suitable for industrial applications in leather processing and detergent industry.