Master Degree / Yüksek Lisans Tezleri
Permanent URI for this collectionhttps://hdl.handle.net/11147/3008
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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 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.