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 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.