Master Degree / Yüksek Lisans Tezleri
Permanent URI for this collectionhttps://hdl.handle.net/11147/3008
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Master Thesis Molecular Cloning, Overexpression and Characterization of Thermostable Esterase and Lipase From Thermophilic Bacillus Sp.(Izmir Institute of Technology, 2009) Tekedar, Hasan Cihad; Şanlı Mohamed, Gülşah; Şanlı Mohamed, Gülşah; 04.01. Department of Chemistry; 04. Faculty of Science; 01. Izmir Institute of TechnologyThe organisms that reside in hot places called thermophiles become very useful tool for biotechnology. The natural consequence of adapting to hot environments for thermophiles is encoding thermostable enzymes which make them a target for scientists.We have aimed to use microorganisms that were previously isolated and characterized as a Bacillus sp. from Balçova Geotermal region in İzmir for their lipase and esterase activity. In order to measure esterase and lipase activity, the strains were incubated in the media that contain the detergent tween 20 and media containing rhodamin-B, respectively. Three strains out of almost 110 bacterial strains have displayed high lipase and esterase activity at the same time. Three different esterase (Est1, Est2, Est3) and two different lipase (Lip1, Lip2) from different environmental samples were cloned directly by PCR amplification using consensus degenerate primers from genomic DNA. The deduced amino acid sequence of the three types of esterase gene exhibited similar amino acid sequence identity with few amino acid differences. However sequenced lipase genes were complicated to explain so that characterization studies have been made for only esterases.For over expression in Escherichia coli, the esterase genes and lipase genes were sub-cloned in pET28a vector with a strong T7 promoter. A one step purification of the recombinant esterases and lipases was achieved using His-Select HF nickel affinity gel.Enzyme assays using variety of p-nitrophenyl (p-NP) esters with different acyl chain lengths (C2-C16) as the substrate have confirmed the esterase activity.All three esterase showed a very high specific activity toward all tested p-NP esters. Optimum pH and temperature, stability in terms of pH and temperature, the effect of several metal ions, inhibitors and detergents on activity were determined for purified Est1, Est2, Est3 separately and compared to each other.Master Thesis L[+]-Lactic Acid Purification From Fermentation Broth Using Ion Exchange Resins(Izmir Institute of Technology, 2002) Polat, Zelal; Harsa, Hayriye Şebnem; Harsa, Hayriye Şebnem; 03.08. Department of Food Engineering; 03. Faculty of Engineering; 01. Izmir Institute of TechnologyLactic acid exists in two optically active form, D(-) and L(+)-lactic acid. It has been used in food, leather, textile, pharmaceutical and cosmetic industries. Moreover, L(+)-lactic acid constitutes the raw material for the production of poly-L-lactic acid which is used in biomedical applications.The aim of this study was to recover and purify the microbially produced L(+)-lactic acid from the fermentation media efficiently and economically. Among the various downstream operations, ion exchange chromatography was used since it is highly selective and yields a low cost product recovery within a short period of time. The additional goals were to investigate the end product purity, to obtain new data on the adsorption/desorption behaviours of lactic acid and to investigate the applicability of the system for industrial usage. In this project, Lactobacillus casei NRRL B-441 was used for the production of L(+)-lactic acid from whey by a 12 hours fermentation process at pH 5.5 and 37 oC. The product concentration was 50 g/l with 100% L(+)-lactic acid content. Then, a suitable resin with high sorption capacity and rapid equilibrium behavior was selected. The selected resin was Dowex marathon WBA, a weakly basic anion exchanger in OH form. It reached the equilibrium state in 15 minutes. The batch sorption experiments were done at pH 7.0 and 30 oC and sampling was continued for 20 hours. Furthermore, the effect of temperature and pH was investigated and their influence was found to be unimportant. All the adsorption/desorption experiments were applied both to model lactic acid and to biomass free fermentation broth. The ion exchange equilibria of lactic acid and L(+)-lactic acid in fermentation broth on Dowex marathon WBA were explained by the Langmuir isotherm. The maximum exchange capacity (qm) for model lactic acid was 0.25 g La/g wet resin, while L(+)-lactic acid in fermentation broth has a qm value of 0.04 g La/g wet resin. The equilibrium loading and exchange efficiency of L(+)-lactic acid in fermentation broth were reduced as a result of competition by other ionic species. The competing ions inhibit the binding of L(+)-lactic acid to the free sites of ion exchanger. Moreover, column operations were applied to recover sorbed lactic acid from the ion exchanger. 2.0 M HCl was found to be a suitable eluting agent to recover the bound L(+)-lactic acid with a flowrate of 1 ml/min at ambient temperature. About 95 % of bound L(+)-lactic acid was recovered from Dowex marathon WBA.Master Thesis The Effect of Surface Modification of Biomaterials on the Cellular Interactions(Izmir Institute of Technology, 2008) Özgür, Melek; Çiftçioğlu, Muhsin; Çiftçioğlu, Muhsin; 03.02. Department of Chemical Engineering; 03. Faculty of Engineering; 01. Izmir Institute of TechnologyThe preparation and characterization of chitosan-hydroxyapatite composite scaffolds and protein adsorption chracteristics of these scaffolds have been investigated in this study. The effects of different chitosan/hydroxyapatite contents of the low density composites on the protein adsorption behaviour were experimentally examined.Bradford method at 595 nm and 280 nm UV protein absorption methods were used for the determination of adsorbed amount of bovine serum albumin (BSA) and human serum protein (HSP). In this study low molecular weight chitosan and hydroxyapatite have been used for the preparation of the scaffold composites by freeze drying and SEM was used for microstructural analysis. The thermal behaviour of the composites was investigated by DSC and TGA. Composite scaffolds were prepared by using different amounts of chitosan and hydroxyapatite (HA) and six different scaffolds were prepared and coded as C100H0, C80H20, C70H30, C50H50, C30H70, and C20H80. The porous low density scaffolds had 93.5-96.3% porosity with a slight increase in density with increasing HA content. The interconnected pore network was formed from 50-250 .m relatively uniform size pores with thin pore walls. The HA particles were fully embedded in the polymer matrix in the pore walls. The TGA curves have shown that the freeze dried phase seperation induced biopolymer sturucture degrates at lower temperatures faster than the original raw polymer. The adsorptions of BSA and HSP onto composites have been studied as a function of time, protein concentration and pH. Adsorption experiments were also conducted with commercial HA powder. The adsorption kinetics experiments have indicated that protein adsorption was almost completely achieved in the first 2-3 hours with relatively high uptake values of up to 45-60 mg/g and 40-60 mg/g for 595 nm Bradford and 280 nm methods. The adsorption behaviour did not fit to the commonly known Langmuir and Freundlich isotherms. This was attributed to the swelling/degradation tendency of the freeze-dried chitosan containing scaffolds. The HSP uptake of 30 and 50 wt% HA containing composites were in the 50-60 mg/g range which was higher than other composites and the raw unprocessed chitosan.Master Thesis Determination of Genetic Diversity and Antioxidant Content of the National Melon (cucumis Melo) Collection(Izmir Institute of Technology, 2008) Şığva, Hasan Özgür; Frary, Anne; Frary, Anne; 04.03. Department of Molecular Biology and Genetics; 04. Faculty of Science; 01. Izmir Institute of TechnologyIn this study, characterization with 19 morphological criteria and two types of PCRbased molecular marker systems, Simple Sequence Repeats (SSRs, microsatellites) and Amplified Fragment Length Polymorphisims (AFLPs), were used to characterize genetic variability among 238 national melon (Cucumis melo L.) accessions and 12 different members of the Cucurbitaceae family.According to morphological analysis, all accessions were vigourous. The accessions showed a great variety of fruit shape, 53 (38.4%) accessions were globular (round), 51 (37.0%) were eliptical, 15 (10.9%) were ovate, 7 (5.1%) oblate, 2 (1.4%) elongate and one accession was flattened. Another agronomically important criterion was predominant fruit skin colour. A total of 55 (39.9%) accessions had light yellow predominant skin colour, 34 (24.6%) were pale green, 29 (21.0%) were orange, 9 (6.5%) were cream, 6 (4.4%) were green, 4 (2.9%) were blackish-green and 1 (0.7%) was dark green.In accordance with genetic characterization, a total of 345 polymorphic AFLP fragments (products of 10 AFLP primer combinations) and 93 SSR fragments (products of 12 SSR markers) were detected and used to calculate genetic distance using DICE matrix and UPGMA (Unweighted Pair Group Method) arithmetical averages. The average polymorphic AFLP fragments per combination was 34.5 and SSR fragments per marker was 7.75. The phylogenetic tree showed that groups were clearly separated by both marker systems. This study allowed the identification of the relationship between national melon accessions based on genetic similarity or differences.Forty three melon accessions were also analyzed for total water soluble antioxidant and total phenolic compound activities.Master Thesis Enztmatic Co2 Sequestration by Carbonic Anhydrase(Izmir Institute of Technology, 2008) Kanbar, Bora; Özdemir, Ekrem; Özdemir, Ekrem; 03.02. Department of Chemical Engineering; 03. Faculty of Engineering; 01. Izmir Institute of TechnologyCarbonic Anhydrase (CA) was immobilized within Polyurethane (PU) Foam and characterized for CO2 sequestration purposes. The catalytic activities for the free and immobilized CA were estimated by using p-Nitrophenyl Acetate (p-NPA) as the substrate. The activities were estimated in tris buffer containing 10% Acetonitrile.Because, the p-NPA is only soluble up to 4mM in aqueous phase. Lineweaver-Burk relationship was employed to estimate the Michaelis-Menten kinetic parameters for the free and immobilized CA. The kcat, KM, and kcat/KM values for free enzyme were found to be 1.21s-1, 12.2mM, and 148.1M-1s-1, respectively. The KM value for immobilized BCA was estimated to be 9.59mM in tris buffer (50mM, pH.7.5), in the presence of 10% acetonitrile at the same conditions. The immobilized CA was stable and did not loose any activity over seven consecutive washings and activity tests. In addition, while the free CA lost its activity in 45 days stored at 4oC in fridge, the immobilized CA was stable and did not loose any activity over 45 day period. The optimum temperature for the immobilized BCA was found to be between 35oC and 45oC. No activity was observed for the immobilized CA at 60oC. Thermal deactivation energies for the free and immobilized CA were found to be 29kcal/mol and 86kcal/mol, respectively. The immobilized CA was employed in CaCO3 precipitation. It was found that the CaCO3 particles were less than 100nm and more evenly dispersed. It was concluded that the immobilized CA could be used in CO2 sequestration.Master Thesis Characterization of Antioxdant Activity and Protein Functionality in Some Legume Cultivars Grown in Turkey(Izmir Institute of Technology, 2008) Aydemir, Levent Yurdaer; Yemenicioğlu, Ahmet; Yemenicioğlu, Ahmet; 03.08. Department of Food Engineering; 03. Faculty of Engineering; 01. Izmir Institute of TechnologyTurkish chickpeas (4 cultivars) and lentils (6 cultivars) show similar total phenolic contents and free radical scavenging capacities in aqueous extracts ranging between 2869 and 4312 mg gallic acid equivalents/kg legume and 24.42 and 38.20 mmol Trolox equivalents/kg legume, respectively. However, the free radical scavenging capacity of lentil and chickpea protein extracts, range between 110 and 185 mmol Trolox/kg protein and 58 and 144 mmol Trolox/kg protein, respectively, clearly showed the higher free radical scavenging capacity of lentil proteins than chickpea proteins.Protein extracts of chickpeas and lentils showed considerable emulsifying and foaming capacities in almost at the same range, but emulsions and foams formed by chickpea proteins are more stable than those of lentil proteins. The lentil protein extracts are highly soluble and showed poor water absorption and gelling characteristics. In contrast, chickpea protein extracts showed moderate water absorption and gelling capacity.Chickpea protein extracts are also good oil absorbers with almost 1.5 to 2 fold better oil adsorption capacity than lentil protein extracts. Thus, chickpea proteins are suggested as soy and whey protein alternatives for functional proteins used in the food, drug and cosmetics industries. Considering functional properties of proteins for different cultivars, the outstanding Turkish chickpea cultivars are Gökçe and Cevdetbey, while the outstanding Turkish lentil cultivar is Alidayı. Variations in the functional properties of protein suggest the diversity of genes in chickpeas and lentils responsible for these properties. Thus, this study showed the possibility of improving functional properties of chickpeas and lentils by breeding programs.Master Thesis Physical and Antimicrobial Properties of Biofilms Containing Natural Antimicrobial Agents(Izmir Institute of Technology, 2008) Türe, Hasan; Özen, Fatma Banu; Özen, Fatma Banu; 03.08. Department of Food Engineering; 03. Faculty of Engineering; 01. Izmir Institute of TechnologyThe goal of this study is to evaluate physical and antimicrobial properties of biopolymers prepared from gluten (WG) and methyl cellulose (MC) with incorporation of activated lactoferrin (ALF), rosemary extract (RE) and natamycin (NA). The effectiveness of agents and antimicrobial films were investigated by agar disc diffusion method. Mechanical and water vapor permeability (WVP) of antimicrobial films were also examined. Film structure was investigated by scanning electron microscopy (SEM) and Fourier-transform infrared spectroscopy (FTIR). Application of films was studied on Kashar cheese inoculated with target molds during 30 days storage at 10oC.ALF and RE did not show inhibitory activity against tested microorganisms.Minimum inhibitory concentration (MIC) of NA was found 750 ppm against Aspergillus niger and Penicillium roquefortii. Both films containing NA showed antifungal activity concentrations at and above 2 and 1 mg NA/10 g film solution (fs) against A. niger and P. roquefortii, respectively. RE in combination with NA reduced MIC of NA against A. niger to 1.5 mg NA/10g fs in both films. FTIR spectroscopy did not indicate any interaction between NA and both films. SEM observations showed that NA crystallizes at high concentrations in biopolymers. There was no significant change in WVP of films containing NA. Incorporation of NA to WG film did not cause major changes in its mechanical properties. Reduction in tensile strength of MC films was observed at high NA concentration. Application studies demonstrated the efficacy of WG and MC films including NA against A. niger and P.roquefortii inoculated on Kashar cheese.Master Thesis Characterization of Peppers for Antioxidant Content and Virus Resistance(Izmir Institute of Technology, 2008) Keçeli, Mehmet Ali; Doğanlar, Sami; Doğanlar, Sami; 04.03. Department of Molecular Biology and Genetics; 04. Faculty of Science; 01. Izmir Institute of TechnologyIn the first part of this study, total water soluble antioxidant activity, phenolic compounds and vitamin C content of 29 Turkish and 14 non-Turkish pepper cultivars were determined. Significant trait diversity was found in the cultivars with the most variation (7.4-fold) seen for total antioxidant capacity which ranged from 2565 to 18957 .mol Trolox/kg. Vitamin C content for the peppers ranged from 522 to 1631 mg/kg, a 3.1-fold difference, while total phenolic content for the pepper cultivars ranged from 607 to 2724 mg/kg, a 4.5-fold difference. A strong correlation between total antioxidant capacity and phenolic content (r . 0.71) was also seen. Significant variation for antioxidant content in Turkish germplasm indicates that this material can be used for improvement and genetic mapping of nutritional content in pepper. In the second part of this study, amplified fragment length polymorphism (AFLP), cleaved amplified polymorphism (CAPs) and simple sequence repeat (SSR) markers were used to map gene(s) for cucumber mosaic virus resistance. The mapping was conducted using a F2 population derived from a cross between Capsicum annuum cv. Jupiter (susceptible) X C. frutescens (resistant). The F2 population, consisting of 76 individuals, was screened with 13 AFLP primer pairs, 60 CAPs markers and 30 SSR markers to detect markers possibly linked to CMV resistance gene(s). As a result, 7 QTL were found which are related with resistance to CMV in pepper. This will provide pepper breeders an opportunity to use these markers for marker-assisted selection for CMV resistance in pepper.Master Thesis Identification of Micrornas Involved in Camptothecin-Induced Apoptosis in Jurkat T Cell Line(Izmir Institute of Technology, 2008) Erdoğan, İpek; Erdoğan, İpek; Akgül, Bünyamin; Akgül, Bünyamin; 04.03. Department of Molecular Biology and Genetics; 04. Faculty of Science; 01. Izmir Institute of TechnologyMicroRNAs which are non-coding RNAs 19-25 nt in length regulate gene expression at post-transcriptional and translational stages. Although it is known that they play a role in critical processes such as development and differentiation of T cells a major component of the immune system, the function of miRNAs in T cell apoptosis is unknown. This study has aimed to identify miRNAs. involvement in camptothecininduced T cell apoptosis in the Jurkat T cell leukemia cell line model. Following the enrichment of the apoptotic population by magnetic seperation, the negative and apoptotic fractions were profilled and compared according to the expression levels of microRNAs. Out of the 866 miRNAs in the miRBASE, 37 and 58 of them were downand up-regulated in the apoptotic fraction, respectively. 7 miRNAs were members of the clusters that have predicted targets as anti-apoptotic genes and tumour suppressor proteins. 66 miRNAs have no known function. Candidate miRNAs, selected based on their higher differential expression levels with predicted apoptotic/antiapoptotic targets, will be verified by qPCR. These candidate then will be further characterized by overxpression and knock-down studies.Master Thesis Identification of Genes That Play Roles in Boron Metabolism/(Izmir Institute of Technology, 2008) Kaya, Alaattin; Koç, Ahmet; Koç, Ahmet; 04.03. Department of Molecular Biology and Genetics; 04. Faculty of Science; 01. Izmir Institute of TechnologyBoron, is an essential microelement that plays a role in plant and animal development. However, an excess amount of boron is toxic for both types of organisms.The yeast Saccharomyces cerevisiae is an ideal model organism for describing the mechanism and regulation of metal ion transport in eukaryotes. We screened a yeast genomic DNA library to find genes that confer boron resistance to wild-type cells.Thirty transformants were isolated that were able to grow in the presence of a toxic amount of boron and all of them contained the multidrug efflux transporter gene ATR1 (YML116w) in the expression cassette. Our subsequent analysis revealed that ATR1 deletion mutants (.atr1) were sensitive to boron treatment and hyper-accumulated boron inside cells, whereas wild-type cells overexpressing the ATR1 gene were resistant to boron and hypoaccumulated boron in cells. We also analysed the global gene expression pattern in response to boron treatment and found that expression of ATR1 is upregulated along with many other transporter and amino acid biosynthesis genes. Our data suggest that the ATR1 gene functions as a boron efflux pump and is reqired for boron tolerance in yeast cells.