Master Degree / Yüksek Lisans Tezleri
Permanent URI for this collectionhttps://hdl.handle.net/11147/3008
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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, AlperProteases 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 Investigation of Gas Phase Fragmentation Mechanism of Doubly Charged a Ions by Mass Spectrometry(Izmir Institute of Technology, 2019) Kızılkoca, Doğacan; Yalçın, Talat; Arslanoğlu, AlperThis dissertation presents studies of gas-phase fragmentation mechanism of doubly-charged a7 ions from basic amino acid containing model peptides under low-energy collision-induced dissociation (CID). The study includes three sets of C-terminal amidated model peptides which are alanine series containing basic amino acids (His – Arg – Lys). Position of His, Arg and Lys residue is varied from N-to-C terminal. Both positional effect and peptide sequence effect were examined for the fragmentation reactions of doubly-protonated a7 ions for these heptapeptides. The CID-MS4 mass spectra of doubly-protonated a7 ions have internal amino acid losses which provide an evidence for macrocyclization reaction. The proposed reaction mechanism involves production of doubly-charge a ions and charge-separation reaction of doubly-protonated a ions in the gas-phase which generates a protonated direct and non-direct a ion. All model peptides were also studied to understand behavior of doubly-protonated a ions better. Direct and non-direct sequence fragmentations which are singly or doubly protonated were observed for all studies. The reactions mechanisms were adjusted according to the results. In conclusions, the results presented in this dissertation can be used to elucidate the correct and reliable peptide sequences, and this improve protein identification strategies which is required for high-throughput proteomic studies.Master Thesis Cloning, Heterologous Expression and Purification of Various Wax Ester Synthases in Escherichia Coli(Izmir Institute of Technology, 2017) Ovacık, Kamil; Arslanoğlu, AlperBiodiesel, 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 Investigation of the Effects of Hiv-1 and Siv Agm Tat Proteins on Slpi Gene Expression in Chlorocebus Sabaeus Kidney (vero) Cell Line(Izmir Institute of Technology, 2017) Özkan, Aysu; Arslanoğlu, AlperEven though Human Immunodeficiency Virus (HIV) can infect Old World Monkey (i.e African Green Monkey) cells, replication of the virus is hampered with different kinds of restriction mechanisms before the integration of viral genome to host genome. After viral integration to host genome, HIV-1 TAT protein is expressed, which plays an important role for the generation of other proteins for viral production. Previous studies which were performed in our laboratory indicate that Secretory Leukocytes Protease Inhibitor (SLPI) overexpressed in existence of HIV-1 TAT protein. This result was confirmed with 2D-PAGE, qRT-PCR and Western Blot Analysis. The aim of this study is to understand how Vero Stable Cell Lines which produces HIV-1 TAT protein can affect expression levels of SLPI. At mRNA level of SLPI expression was confirmed with qRTPCR. In order to measure SLPI expression at the protein level in the presence of HIV-1 TAT,Western Blot and Sandwich Elisa methods were used. Luciferase Assay process was performed in order to measure the effects of SLPI on HIV-LTR. Consequently, expression of SLPI in Vero Stable Cell Line increased in the existence of HIV-1 TAT protein. Additionally, SLPI inhibits HIV-LTR function through suppressing NF- κB promoter which exists in HIV-LTR.Master Thesis Investigation of the Effects of Siv Agm Tat Protein on Slpi Gene Expression in Primate Cells(Izmir Institute of Technology, 2017) Demirci, Dilara; Arslanoğlu, AlperAfrika Yeşil maymunu (AYM) gibi Eski Dünya maymunları HIV-1’e karşı dirençlidirler. Türlerine spesifik simian bağışıklık yetmezlik virüsü (SIV) ile enfekte olabilmelerine rağmen AIDS benzeri bir sendrom geliştirmezler. Bazı restriksiyon faktörlerinin, HIV-1 replikasyonunu engelleyerek bu organizmaları enfeksiyona karşı koruduğu bilinmektedir. Eski Dünya maymunları hücreleri çok yüksek viral yük kullanıldığında, başarılı bir şekilde enfekte edilebilmelerine rağmen, bu hücrelerde virüs sayısının zamanla saptanamayacak seviyelere düşmesi, bu hücrelere dirençlilik sağlayan henüz tanımlanmamış restriksiyon faktörlerinin varlığını göstermektedir. Eski Dünya maymunları viral proteinlerin üretimine karşılık olarak bazı genlerinin ekspresyonlarını değiştirerek bir direnç mekanizması oluşturmuş olabilirler. HIV-1 Tat, virüsün hücre içine girişinden sonra üretilen ilk viral protein olduğundan ve diğer HIV-1 proteinlerinin sentezlenmesi için varlığı gerekli olduğundan, bu maymunların geliştirdiği direnç mekanizmasında önemli bir rol oynaması mümkündür. Laboratuarımızda yapılan ön çalışmalarda, antiproteaz, antimikrobiyel ve antiviral aktivitesi olan Salgısal Lökosit Proteaz İnhibitor (SLPI) proteini olası bir restriksiyon faktörü olarak belirlenmiştir ve AYM hücrelerinde HIV tat varlığında bu proteinin aşırı ifadelendiği gözlemlenmiştir HIV ve SIV tat genlerinin işlevleri benzer olduğu halde, aminoasit dizilimleri çok farklıdır (≈%40 benzerlik). Bu nedenle HIV-1 Tat ve SIV-Tat proteinlerinin SLPI geni ifadelenmesine etkileri farklı olabilir. Bu çalışma AYM hücrelerinde SIV AYM Tat proteininin SLPI üretimine etkisini incelemeyi amaçlamaktadır. Bu amaçla, SIV tat genini kalıcı olarak ifadeleyen AYM hücrelerinde SLPI ifadelenmesi Q-PCR ve Western blotlama yöntemleriyle incelenmiştir. SIV Tat varlığında SLPI ifadelenmesi belirgin olmayan bir şekilde azalırken, HIV Tat varlığında anlamlı bir artış gözlemlenmistir.Master Thesis Real-Time Pcr as a Molecular Tool for the Enumeration of Probiotics in Commercial Products(Izmir Institute of Technology, 2016) Öz, Ödül; Baysal, Ayşe Handan; Arslanoğlu, AlperQuantitative Real-Time PCR (qPCR) assays targeting the 16S rDNA was developed as a genus and species specific detection tool for Bifidobacterium and Lactobacillus, and Bifidobacterium animalis subsp. lactis BB-12 and Lactobacillus acidophilus LA-5, respectively. Standard curves were established to quantify these probiotic bacteria. The linear regression of standard curves indicated high correlations between the log numbers of pure probiotic culture cells and the Ct values. The assay had a high efficiency and the limit of detection was estimated to be 1.54 ng DNA (corresponding to 104 cells). Results show that qPCR method may be very useful as a rapid, sensitive and specific tool for detecting and quantifying B. animalis subsp. lactis BB-12 and L. acidophilus LA-5 in probiotic supplements. FTIR spectroscopy was used for the first time to determine the ratios of different microorganisms in commercial probiotic supplements. FTIR analysis was also performed for the pure probiotic cultures of B. animalis subsp. lactis BB-12 and L. acidophilus LA-5. Results obtained in this study showed that FTIR spectroscopy is potentially a rapid method for determining probiotic cell components and their ratios in the supplements and verification their detection and identification.Master Thesis Development of Antibacterial Polymer Based Nanocomposite Materials(Izmir Institute of Technology, 2015) Abatay, Ezgi; Arslanoğlu, Alper; Tanoğlu, MetinHuman beings are often infected by microorganisms such as bacterium, mold, yeast, virus, etc. in the living environment. It became a requirement and a necessity to create sterile fields in areas. Composite stones are one of the main materials that can be used for the contact surfaces in indoor and outdoor places due to their being of highly resistant to abrasives, chemicals and impacts. Research has been intensive in antibacterial material containing various inorganic substances. The aim of this thesis is investigating the antibacterial effect of inorganic substances such as silver, zinc oxide, calcium oxide, titanium oxide and magnesium oxide on stone products. This study also deals with the silver doped zinc oxide powder and their antibacterial efficacies. Stone product is formed of mainly two type compound which are quartz aggregates as reinforced and filler and thermoset polyester resin as matrix. The manufacturing process begins with selection of raw quartz materials. They are crushed and blended in the ratio of 90 % quartz aggregates to 10% polyester matrix and other additives such as antibacterial agent, pigment. These united constituents are used for production of composite stones by applying those combined vacuum, vibration and pressing processes which are named as vibropress, simultaneously. Following it, they are subjected to surface preparation and polishing processes. In this study, mechanical, thermal, and morphological properties of the particles, polyester matrix and stone product were investigated. Antibacterial efficacies of these were investigated based on colony-count method against gram negative (E.coli) and gram positive (Bacillus subtilis) bacteria. Silver-containing stone samples showed best antibacterial property about ninety-nine percent reduction.Master Thesis Isolation and Identification of a Lipase Producing Psychrotrophic Bacteria From Soil: Cloning and Partial Characterization of Its Lipase(Izmir Institute of Technology, 2009) Adan, Aysun; Arslanoğlu, AlperLipases are serine hydrolases and catalyze both the hydrolysis and synthesis of long-chain triacylglycerols. Lipases have great importance since their wide usage in industry. Lipases are produced by microorganisms (bacteria and fungi), plants and animals. However, microbial lipases, especially from bacteria, more useful than their plant and animal derivatives because of several important properties. The primary goals of this thesis were to isolate and identify a lipase producing bacterium from soil sample from Erciyes mountain in Kayseri by 16S rRNA sequence analysis, find the sequence of lipase gene by degenerate PCR and inverse PCR and analyze of lipase gene for some important features like active site residues. The other purposes of this study were determination of lipase production conditions like optimum production time, partial purification and characterization of native lipase enzyme. Purification was performed by ammonium sulfate precipitation and gel filtration. Spectrophotometric lipase assay was used for enzyme characterization. As conclusion, a lipase producer bacterium was isolated from soil using rhodamine B-olive oil plate assay and identified as a strain of Pseudomonas fluorescens based on 16S rRNA sequence homology. Its partial lipase gene was obtained and it was suggested that the lipase belong to group 3 Pseudomonas lipases according to gene and amino acid homology search. Moreover, native lipase partially purified and characterized. The molecular mass of purified lipase was estimated to be approximately 43 kDa by SDS-PAGE. The optimum temperature and pH for the lipase were found to 45°C and pH 8, respectively.Master Thesis Phylogenetic Analysis of Bacterial Communities in Kefir by Metagenomics(Izmir Institute of Technology, 2008) Ünsal, Burcu; Arslanoğlu, AlperKefir is a traditional fermented milk beverage which is produced by adding kefir grains into milk and is allowed for fermentation. Grains contain vital complex flora of microorganisms (bacteria and yeast) that live in harmony. Since health and food safety of fermented milk products is important, population structure of food-type microbes involve in fermentation should be known very well. Rapid determination of kefir bacterial composition may accelerate the determination of food quality and also may facilitate specification of bioactive products that obtain from kefir. The goal of this thesis was to analysis the genomic structure of bacterial communities of the fermented kefir drink and grains by both culture-dependent and culture-independent methods (metagenomic approach). Total Genomic DNA was purified from each analysis methods and the partial small subunits of 16S rDNA were amplified by a pair of universal bacterial primers. 16S rDNAs fragments were cloned and then sequenced. The vast quantities of data were screened in NCBI database by BLASTN program according to similarity scores with related sequences. 7 different bacteria were identified to species level composed of Lactococcus lactis subsp. lactis , Lactobacillus kefiranofaciens, Lactobacillus helveticus, Acetobacter lovaniensis, Acetobacter syzygii, Leuconostoc mesenteroides, Enterococcus faecium and 1 bacteria to genus level named Lactobacillus kefiri or parabucheri. The results of this study showed that the combination of both methods is more efficient to identify high percentage of species than using only one of them. Finally, phylogenetic relationships among identified species inferred from partial 16S rRNAs gene sequencing were determined by Neighbor-joining algorithm.Master Thesis Cloning and Expression of the Pseudomonas Ke38 Extra-Cellular Lipase Gene in E. Coli(Izmir Institute of Technology, 2013) Karakaş, Fulya; Arslanoğlu, AlperLipases 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.