Master Degree / Yüksek Lisans Tezleri

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  • Master Thesis
    Cloning and Expression of the Pseudomonas Sp Ke38 Extra-Cullular Protease Gene in E.coli
    (01. Izmir Institute of Technology, 2023) Bozlak, Esma Nur; Arslanoğlu, Alper
    Proteases are enzymes that hydrolyze proteins into smaller pieces by breaking the peptide bonds. Protease enzyme is produced by all living things on Earth. Pseudomonas sp. KE-38 is a cold adapted bacterium isolated from soil at high altitude in Erciyes mountain, Kayseri. The purpose of this thesis was to clone the cold-active extracellular protease gene from Pseudomonas sp. KE-38, partial purification and characterization of the extracellular protease enzyme. The partial sequence of protease gene from Pseudomonas sp. KE-38 was analysed. The estimated size encoded by this gene after sequence analysis was 105 kDa. However, the size of this enzyme that was purified in this thesis was found to be approximately 50 kDa as evaluated of gelatine zymography analysis. Further investigation of the proteins in the partially purified enzyme sample by Liquid Chromatography Mass Spectrophotometry, revealed the presence of a metalloprotease enzyme with a predicted mass of 50 kDa. These results showed that the purified and characterised protease enzyme was not the same enzyme of which its gene was amplified gene was amplified and sequenced. Nevertheless, the partial characterization of the extracellular metalloprotease was performed, and the optimum temperature and pH was found to be 30°C and 8.0 respectively. The enzyme showed high activity in the presence of calcium, ethanol. The enzyme showed extremely high stability up to 25°C above this temperature; the stability dropped sharply which confirmed that the protease was a cold active enzyme and can have a potential to be used in cold temperature applications.
  • Master Thesis
    Preparation and Characterization of Antifouling Nanofiltration Membranes From a Responsive Pentablock Copolymer
    (Izmir Institute of Technology, 2018) Çağlar, Nağahan; Alsoy Altınkaya, Sacide
    The most substantial factor restricting the extensive application of membrane processes is the fouling problem resulting from the deposition of solutes in water on the surface or within the pores of membranes. The frequently used chemical washing procedure to eliminate the fouling issue causes environmental pollution and shortens the membrane life. In order to overcome these disadvantages, the development of membranes possessing low fouling potential is needed. In recent years, the stimuliresponsive polymers have received attention for developing membranes possessing low fouling potential. The antifouling property of these membranes is controlled through the change in their conformation and hydrophilic/hydrophobic characteristics as a response to change in external stimuli such as pH, temperature and ionic strength. The aim of this study was to design antifouling nanofiltration membranes (NF) using a pentablock copolymer which consists of temperature responsive Pluronic F127 (PEO-b-PPO-b- PEO) in the middle block and pH responsive poly(N,N-(diethylamino)ethyl methacrylate) (PDEAEM) in the end blocks. Effects of pH and temperature responsiveness on the membrane fouling were investigated. Fouling tendencies of the membranes were evaluated by using Bovine Serum Albumin (BSA), Alginate (ALG) as organic foulant and Escherichia coli (E.coli) as biological foulant. NF membranes were characterized by scanning electron microscope (SEM), contact angle and zeta potential measurements. It was demonstrated that pentablock copolymer coated membranes displayed antifouling resistance by changing filtration pH and temperature.
  • Master Thesis
    Development of Gold Nanoparticle-Based Plasmonic Assay Platform for Esherichia Coli Detection
    (Izmir Institute of Technology, 2017) Erdoğan, Duygu; Yıldız, Ümit Hakan; Arslan Yıldız, Ahu
    The traditional methods for pathogen detection have long detection time and insufficient sensitivity. Optical methods can overcome these drawbacks. There are solution based nanoparticle growth in the literature to enhance a surface sensitivity for biosensing applications. In this project, surface refractive index (RI) sensitivity was enhanced on solid support via gold growth to develop a label free, simple and costeffective methodology for bacteria screening. The gold nanoparticles (GNPs) were grown on solid support by using 20 μl of HAuCl4 / 80 μl of NH2OH at varied incubation times. Firstly, about 20 nm GNPs were synthesized and immobilized on polystyrene surfaces. Then, these GNPs were utilized as seed particles, and grown on solid support. During GNPs growth, a red shift in the plasmonic wavelength was observed. Morphological characterization showed that almost uniform gold growth could be achieved. The plasmonic platform sensitivity was validated by varied concentrations of sucrose, ethanol and BSA solutions, showing that the plasmonic platform gave a response to any small RI change. Next, two different E.coli bacterial strains’ adsorption was tested. Adsorption screenings for about 107 E.coli DH5-alpha cells/ml and 107 E.coli BL21(DE3) cells/ml in Phosphate Buffer Saline were made on growth gold surfaces. Further, E.coli BL21(DE3) containing milk and apple juice were also adsorbed on these gold surfaces with a 30 min incubation time. The results showed that these gold surfaces exhibit higher binding kinetics for bacteria. Therefore, the proposed LSPR-based label free methodology can be an alternative to the bacteria screening in water or food samples.
  • Master Thesis
    Cloning, Heterologous Expression and Purification of Various Wax Ester Synthases in Escherichia Coli
    (Izmir Institute of Technology, 2017) Ovacık, Kamil; Arslanoğlu, Alper
    Biodiesel, known all around theWorld, is a diesel fuel containing fatty acid methyl esters (FAMEs) and fatty acid ethyl esters (FAEEs) with different molecular weights. The recent studies which are about the development of FAEE focused on production of FAEEs in vivo syntheses. This synthesis is catalyzed by wax ester synthases (WS). Bifunctional wax ester synthase/acyl-coenzyme-A (acyl-CoA): diacylglycerol acyltransferase (WS/DGAT) synthesizes wax ester by processing a certain range of fatty alcohols and fatty acyl-CoAs. It is considered as the final enzyme in biosynthetic process of wax ester production. Aim of the research is cloning, heterologous expression, purification and crystallization trial of was ester synthases from M. aquaeolei VT8 (MaWES) and R. opacus PD630 (RoWES). MaWES was cloned into pET expression vector and heterologous expression of MaWES was carried out in E.coli BL21 (DE3) strain. Three chromatography systems were used for purification of MaWES. After Immobilized Metal Affinity Chromatography (IMAC), buffer exchange and gel filtration chromatography, enzyme was purified with approximately 100 mg yield. This project can pave the way for structural studies WS/DGAT enzymes mentioned above. In summary, the findings of this study will circuitously help for solving the relationship between function and structure of these enzymes. It may lead to increased generation of FAEE based biodiesel.
  • Master Thesis
    Cloning and Expression of the Pseudomonas Ke38 Extra-Cellular Lipase Gene in E. Coli
    (Izmir Institute of Technology, 2013) Karakaş, Fulya; Arslanoğlu, Alper
    Lipases are serine hydrolases that catalyze both the hydrolysis and synthesis of insoluble or poorly soluble long-chain triacylglycerols with an acyl chain length ≥ 10 carbon atoms based on the presence or absence of water. Lipases are produced and secreted by all kingdoms of life that are eukaryotes including plants, animals, fungi and prokaryotes including bacteria and archaea. However, microbial lipases, especially from bacteria, more useful than their plant and animal derivatives because of several important properties. Because of their acitivities in both aqueous and nonaqueous environments, lipases have specific applications in industry and medicine. The primary goals of this thesis were to clone and express the extra-cellular lipase gene from Pseudomonas sp. KE38, isolated from soil samples of Erciyes mountain in Kayseri, in E. coli and partial purification of the gene product. To achieve this aim, genome walking technique was used to obtain lipase gene from Pseudomonas sp. KE38, that gene was then cloned into pET28a expression vector and expressed in E. coli. The lipase expression of E. coli BL21 and its activity was screened with olive oil-Rhodamin B plate assay. After expression recombinant lipase was partially purified via inclusion body isolation. Moreover the optimum lipase production time of E. coli BL21 cells were determined and analyzed with SDS-PAGE. According to SDS-PAGE analysis the recombinant lipase was approximately 64 kDa and lipase production reached to the highest level after two hours of IPTG induction. As conclusion, recombinant lipase from Pseudomonas sp. KE38 was cloned into E. coli, expressed and partially purified.