Master Degree / Yüksek Lisans Tezleri

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Author: search.filters.author.Bedir, ErdalSubject: search.filters.subject.Cytochrome P450 proteins

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  • 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 Technology
    Directed 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.