Master Degree / Yüksek Lisans Tezleri
Permanent URI for this collectionhttps://hdl.handle.net/11147/3008
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Master Thesis Chemical Characterization of Caldanaerobacter Subterraneus Subsp. Tengcongensis Heme-Nitric Oxide/Oxygen Binding Protein(01. Izmir Institute of Technology, 2020) Sürmeli, Nur Başak; Sürmeli, Nur Başak; Sürmeli, Nur Başak; 03.01. Department of Bioengineering; 03. Faculty of Engineering; 01. Izmir Institute of TechnologyHemoproteins, which contain the heme prosthetic group , take part in different biological processes in many stages of life. Their ability to catalyze important biosynthesis reactions makes them good candidates for understanding and elucidating complex mechanisms for biocatalysis. In this study, the catalytic properties of thermophilic Thermoanaerobacter tencogensis nitric oxide/oxygen binding protein, a heme protein reshaped by rational design, were investigated and chemical characterization was carried out. The peroxidase activity of the enzyme was investigated by the oxidation reactions of guaiacol, amplex red and 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid (ABTS). Kinetic parameters of the reactions were determined. These obtained results demonstrated that, in the presence of H2O2, wild type and Y140H TtH-NOX proteins are able to catalyze oxidation reactions of guaiacol, Amplex red and ABTS. Comparison of the kinetic parameters showed that Y140H mutant catalyzed the guaiacol and ABTS oxidation 3-fold and 15 -fold faster than wild type enzyme, respectively. The stability of TtH-NOX proteins were investigated in the presence of organic solvents. Results were demonstrated that WT TtH-NOX was more stable than Y140H mutant in the presence of organic solvents In addition to these, for the first time, thermophilic TtH-NOX proteins were immobilized with a novel enzyme immobilization method and organic-inorganic hybrid nanostrucrures were obtained. Copper ion incorporated TtH-NOX-based hybrid nanoflowers were synthesized at different pH values. SEM and EDX analysis of TtH-NOX-based hybrid nanoflowers proved that free TtH-NOXs were immobilized successfully.Master Thesis Optimization of Expression and Isolation of a Thermophilic P450 Enzyme(Izmir Institute of Technology, 2018) Aslantaş, Yaprak; Sürmeli, Nur Başak; Şanlı Mohamed, Gülşah; Sürmeli, Nur Başak; Şanlı Mohamed, Gülşah; 03.01. Department of Bioengineering; 04.01. Department of Chemistry; 03. Faculty of Engineering; 04. Faculty of Science; 01. Izmir Institute of TechnologyCytochrome P450 enzymes (CYP or P450) are monooxygenases that catalyze the oxidation of hydrocarbons with high efficiency and selectivity, and many other reactions like hydroxylation, epoxidation, reduction, demethylation. CYP119, is a thermophilic P450 from Sulfolobus acidocaldarius. Thanks to thermophilic properties, CYP119 has potential to be widely used as a biocatalyst in production of fine chemicals and pharmaceuticals. However, production and purification of CYP119s is quite difficult and time consuming. Here, through recombinant protein production techniques, the optimum production and purification of heat-tolerant CYP119 has been successfully carried out. N-terminal and C-terminal histidine tags were cloned to CYP119. Protein expression was induced in Escherichia coli BL21 (DE3) cells with isopropyl β-D-1-thiogalactopyranoside (IPTG). δ-aminolevulinic acid (ALA) was also used to increase the heme biosynthesis. Different IPTG and ALA concentrations, expression temperature and duration were used to optimize production. CYP119 was isolated and purified with Ni-NTA affinity column. The thermostability of purified N (N-His-CYP119) and C (C-His-CYP119) terminal His-tagged were compared with wild type CYP119 (Wt-CYP119). Oxidation reaction of CYP119 and variants carried out and compared at 25 °C and 65 °C. Also, epoxidation of styrene was performed with N-His-CYP119 in different temperatures. The effects of histidine tags on stability and activity of the CYP119s were observed. Here, conditions for the production of CYP119 were optimized and the histidine tags were found to cause changes in stability and function of proteins. This project will lead to increase in the production of the important enzyme CYP119, which will increase its utilization in the industry.Master Thesis Generation of Mutant Libraries for Directed Evolution of a Thermophilic P450 Enzyme(Izmir Institute of Technology, 2018) Haklı, Emre; Sürmeli, Nur Başak; Bedir, Erdal; Sürmeli, Nur Başak; Bedir, Erdal; 03.01. Department of Bioengineering; 03. Faculty of Engineering; 01. Izmir Institute of TechnologyDirected evolution, inspires from natural selection, is a frequently utilized approach in protein engineering for designing enzymes. It allows iterative evolution of existing proteins towards the ones with desired characteristics by the application of random mutagenesis in the laboratory. However, library construction constitutes the most fundamental part of directed evolution. Application of different construction methods affects both the number and diversity of variants created and the screening/selection techniques used. Early procedures including error-prone PCR, mutator strains, chemical mutagens and gene shuffling have been successful in whole gene mutagenesis yet have been required more screening/selection effort by leading larger libraries. On the other hand, recent approaches such as use of degenerate primers and site saturation mutagenesis have decreased the screening/selection effort by allowing random mutagenesis of amino acids located at specific positions in the polypeptide chain. Especially, active site residues of biocatalysts were chosen as targets and the catalytic efficiencies were enhanced. CYP119, a member of cytochrome P450 protein family, from Sulfolobus Acidocaldarius is a thermostable enzyme capable of catalyzing peroxidation, monooxygenation and oxidoreduction reactions. Here, a library of mutants consist of CYP119 variants was created via application of combinatorial active site saturation test (CAST) in amino acid positions 213 – 214 and an effective fluorescence-based method was developed to screen the library for increased peroxidase activity while utilizing hydrogen peroxide as oxidant. After screening of mutant library, a variant with Thr213Arg – Thr214Ile substitutions showed 1.32-fold increased peroxidase activity in the catalysis of Amplex Red compared to wild type CYP119.