Master Degree / Yüksek Lisans Tezleri
Permanent URI for this collectionhttps://hdl.handle.net/11147/3008
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Master Thesis Cloning, Expression and Characterization of Sericin Like Octamer-Repeat Protein(01. Izmir Institute of Technology, 2021) Şahin, Alper; Sürmeli Eraltuğ, Nur Başak; 01. Izmir Institute of TechnologySilk proteins are natural polymers which are synthesized by insects like silkworms and spiders. There are two proteins in the silk obtained from the cocoon of B. mori, these are silk fibroin and silk sericin. While the silk fibroin is the main part of the cocoon with a high commercial importance, sericin is the "glue" protein that holds fibroins together. Sericin has wide variety of applications from cosmetics to biomaterial research. Because of its high fibrillar structure, it can be used in wound healing or drug delivery studies. It can be obtained from the degumming process of cocoons which includes chemical treatment like alkali treatment and boiling, or biological treatment like enzymatic digestion. However, sericin obtained by these processes are not consistent in structure, and the sequence of the protein obtained depends on the extraction methodology and conditions. However, proteins obtained by recombinant production can be standardized, and repeat-chain length can be adjusted as desired. In this thesis study, a sericin like protein which includes eight repeats of the 38 amino acid sequence of the natural sericin was cloned and expressed in E. coli. The resulting protein, Ser-8mer, was analyzed in terms of fibril structure and secondary structure. It was found out that the protein has beta sheet conformation in contrast to commercial sericin with random coils. And as a result of this conformation, it forms insoluble self-assembled fibril structures which shows a promising contribution to the biomaterial research.Master Thesis Characterization of Designed Novel Cytochrome P450 for Industrial Biocatalysis(01. Izmir Institute of Technology, 2020) Sakallı, Tuğçe; Sürmeli Eraltuğ, Nur Başak; 01. Izmir Institute of TechnologyBiocatalysts are increasingly applied in chemical synthesis due to their high level of regioselectivity and enantioselectivity. P450s are important biocatalysts due to their ability to hydroxylate unactivated carbon atoms using molecular oxygen. P450s catalyze monooxygenation reactions by using nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) as electron donor and electron transfer proteins. P450s can also utilize hydrogen peroxide (H2O2) instead of NAD(P)H and redox partners through a H2O2-shunt pathway. However, P450s are inefficient in oxygenation reactions with H2O2. Thermophilic enzymes demonstrates high stability at different temperatures, pH and organic solvents, so it is expected to increase implementations of enzymes. CYP119 is an acidothermophilic P450 from Sulfolobus acidocaldarius. In our laboratories, directed evolution was used to create improved mutants of CYP119 with higher oxidation activity when using H2O2. T213R/T214I CYP119 was such a variant. The aim of the study is investigations of T213R/T214I CYP119 whether it is a stable and efficient biocatalyst for selective oxidation of hydrocarbons, which does not require expensive cofactors and electron transfer proteins or not. T213R/T214I CYP119 was expressed and isolated under optimized conditions. Peroxidase activity of T213R/T214I CYP119 was tested and compared to wild type (WT) CYP119. Characterization of T213R/T214I CYP119 shows higher peroxidation activity of enzyme for Amplex® Red, guaiacol and ABTS and epoxidation of enzyme for styrene substrates compared to CYP119. T213R/T214I CYP119 have higher affinity for progesterone and lower affinity for lauric acid. Mutations on Thr213 and Thr214 residues the active site will shed light on the design of novel CYP119 mutants in the future.Master Thesis Rational Design of Hemoproteins for Peroxidation Reactions(01. Izmir Institute of Technology, 2020) Güralp, Gülce; Güralp, Gülce; Sürmeli Eraltuğ, Nur Başak; 01. Izmir Institute of TechnologyBiocatalysts are important for the synthesis of fine chemicals and steroidal drugs in the biopharmaceutical industry. Cytochromes P450 (P450) monooxygenases are significant biocatalysts due to their high selectivity for oxidation reactions. CYP119 is the first characterized thermoacidophilic P450. CYP119 was isolated from Sulfolobus acidocaldirius. CYP119 enzyme shows high stability at low pH and high temperature. CYP119 can utilize the peroxidase shunt pathway in the catalytic cycle of P450. These abilities make CYP119 attractive biocatalyst for production of fine chemicals and drugs. In this study, Leu69Gly mutant CYP119 enzyme was cloned by site-directed mutagenesis. L69G and WT CYP119 was expressed successfully in Escherichia coli BL21 (DE3) cells with isopropyl β-D-1- thiogalactopyranoside (IPTG). This study shows that L69G mutation is important for binding to progesterone. This was predicted by in silico mutagenesis in a previous computational study. Isolation and purification of the WT and L69G CYP119 were carried out. Activity assays and substrate binding studies of the enzymes were performed and compared each other. L69G mutation did not cause significant effect on Amplex Red® oxidation and styrene epoxidation activities. L69G CYP119 (KS: 34.55 ± 7.4 μM) showed higher affinity for progesterone compared to WT CYP119 (KS: 69.8 ± 48.9 mM). A new product, thought to be hydroxylated progesterone, was formed as result of hydroxylation of progesterone by L69G CYP119 using peroxidase shunt pathway.Master Thesis Optimizing the Transfection Conditions for the Generation of Stable Transgenic Cell Lines(Izmir Institute of Technology, 2020) Özçivici, Engin; Özçivici, Engin; Sürmeli Eraltuğ, Nur Başak; 03.01. Department of Bioengineering; 03. Faculty of Engineering; 01. Izmir Institute of TechnologyTransgenic cell lines that produce biopharmaceutical proteins are widely utilized in the biotech industry and the demand is not predicted to decline in the near future. For obtaining an industrially usable cell line, process components like expression vector, host cell line and transfection method need to be carefully selected. Due to all practical reasons, the industry prefers to use the most conventional Chinese hamster ovary (CHO) cell line. The generation of recombinant cell lines is known to be time-consuming, labor-intensive and expensive. Therefore, the several steps of this process are under constant development. One of the first work packages is transfection, where genes encoding for the therapeutic protein are taken into mammalian host cells. In this study, we aimed to generate a more cost-effective transfection procedure using the electroporation based technology of nucleofection. This method is favored by the researchers for its high and reproducible transfection efficiency, but also known for the high cost and lack of public information on its components and related consumables. As a result of this study, a novel nucleofection solution was developed for the transfection of CHO-DG44 cells, showing comparable if not better performance over the commercial Lonza's solution in terms of transient and stable expression of recombinant proteins. The transfection was further improved by selecting a more effective nucleofection program and by linearizing the plasmid prior to transfection. These enhancements, optimized on the basis of the biotherapeutic protein production, are potentially advantageous for any research requiring a large number of efficient transfection experiments.Master Thesis Directed Evolution of a Cytochrome P450 Enzyme To Increase Peroxidation Activity(Izmir Institute of Technology, 2019) Başlar, Muhammet Semih; Sürmeli Eraltuğ, Nur Başak; 01. Izmir Institute of TechnologyDirected evolution, mimicking the natural selection, is a powerful tool to create novel enzymes. Evolution of natural enzymes to achieve desired properties are performed in iterative rounds of random mutagenesis followed by a screening/selection method. Enzyme activity can be enhanced with substituting the active site amino acids in the enzyme. CYP119, a member of cytochrome P450 protein family, is a thermophilic enzyme extracted from Sulfolobus acidocaldarius that exhibits monooxygenase, peroxidase and oxidoreductase activity. These properties give CYP119 a potential to be used in production of fine chemicals and pharmaceuticals. Herein, two different mutant libraries of CYP119, containing substituted amino acids at Thr213-Thr214 and Val151- Phe153 positions, constructed via combinatorial active site saturation test (CAST), and screened for improved peroxidation activity. Additionally, fluorescence based Amplex Red peroxidation activity assay using hydrogen peroxide as cofactor of CYP119 was optimized. Screening of mutant libraries resulted four improved CYP119 mutant enzymes from Thr213-Thr214 mutant library. Val151-Phe153 mutant library did not yield any improved peroxidation activity mutants which indicated amino acid substitutions at 151- 153 positions do not have any effect on peroxidation activity of CYP119. Furthermore, effect of substituted amino acids at predetermined positions were analyzed. Substrate, Amplex Red, makes single or double hydrogen bond when molecular docking was performed on improved mutant enzymes also distance of nitrogen atom in Amplex Red to heme iron is closer than wild type CYP119 in improved mutant enzymes. Thus, increasing the peroxidation activity of mutant CYP119 enzymes.Master Thesis Cloning of Sericin-Like Proteins for Recombinant Production in Bacteria(Izmir Institute of Technology, 2019) Bostan, Fatmanur; Bulmuş Zareie, Volga; Sürmeli Eraltuğ, Nur Başak; Bulmuş, Volga; 03.01. Department of Bioengineering; 03. Faculty of Engineering; 01. Izmir Institute of TechnologySilk consists of two main proteins called fibroin and sericin. While fibroin is used in textile production and various biomaterial applications, sericin is considered as waste material in the textile industry. Sericin is a multi-component protein with an indefinite structure and it has been shown to be biocompatible and has biological activity. Because of the positive effects on keratinocytes and fibroblasts have led to the development of sericin-based biomaterials for the repair of skin tissue. Sericin from silkworm cocoons can be obtained by chemical treatment, enzymatic treatment and boiling in water. Although sericin can be separated from fibroin by chemical, enzymatic and boiling in water treatment methods, all these treatment methods are not enough to obtain recovery of high-quality sericin. Moreover, in these treatment methods, the exposure of sericin protein to high temperature causes even sericin protein obtained by the same method to indicate different characteristics. The fact that the obtained sericin demonstrate such major changes in the structure according to treatment methods bring inconsistencies in the quality of sericin produced as a biomaterial. The aim of the study is to produce native sequence of sericin that forms a tetramer contain each containing 38 amino acids with recombinant production in E.coli and to characterize structural properties Thus, obtaining sericin protein from the bacteria with recombinant methods will solve these problems in question The results indicate that for the first time, the conformational properties of recombinant sericin were obtained similar to the native sericin structure.Master Thesis Bioinformatics Based Approach To Design a Thermophilic P450 Fot Industrial Biocatalysis(Izmir Institute of Technology, 2019) Kestevur Doğru, Ekin; Sürmeli Eraltuğ, Nur Başak; 01. Izmir Institute of TechnologyEnzyme catalyzed biosynthesis of steroidal drugs is an important process for pharmaceutical manufacturing. Cytochrome P450 (P450) monooxygenases are important for hydroxylation of steroid structures because they can catalyze the oxidation of inactive carbon bonds with high selectivity and efficiency. CYP119 is an acidothermophilic P450 from Sulfolobus acidocaldarius, which has the potential to be used as biocatalyst for industrial production since it shows activity at high temperature and low pH conditions. In this work we aim to use CYP119 for selective hydroxylation of progesterone, which is not the original substrate of CYP119, for production of precursor molecules of important hormones like cortisone and aldosterone. Crystal structure of CYP119 (PDB ID: 1F4T) was used for selecting residues that were mutated according to structural alignment with other CYPs that can catalyze progesterone hydroxylation naturally. Progesterone-docking performed with CYP119 to identify residues that create clashes with substrate. Finally selected 12 residues (Leu69, Val151, Phe153, Leu155, Leu205, Ile208, Ala209, Thr213, Thr214, Val254, Thr257, Leu354) were mutated with PyRosetta program to Gly, Glu, Phe, Met, Ala, His, Arg and Ile. Progesterone-docking performed with using DockMCM Protocol of PyRosetta. We used two different starting coordinates of progesterone for docking and results were eliminated according to their energy scores. Best mutants were used for creating double/triple mutants and second round of docking and elimination process were performed with using double/triple mutant enzymes. Final number of 11 mutants with best scores were selected and their possible products were identified.