Directed evolution of a cytochrome P450 enzyme to increase peroxidation activity
Başlar, Muhammet Semih, author.

Directed evolution of a cytochrome P450 enzyme to increase peroxidation activity

Başlar, Muhammet Semih, author.

Yazar Ek Girişi
Başlar, Muhammet Semih, author.

Fiziksel Tanımlama
x, 61 leaves: charts;+ 1 computer laser optical disc.

Directed 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.

Konu Başlığı
Cytochrome P-450.
Enzymes -- Synthesis.
Enzymes -- Biotechnology.

Yazar Ek Girişi
Eraltuğ Sürmeli, Nur Başak,

Tüzel Kişi Ek Girişi
İzmir Institute of Technology. Bioengineering.

Tek Biçim Eser Adı
Thesis (Master)--İzmir Institute of Technology: Bioengineering.
İzmir Institute of Technology: Bioengineering--Thesis (Master).

Elektronik Erişim
Access to Electronic Versiyon.

LibraryMateryal TürüDemirbaş NumarasıYer NumarasıDurumu/İade Tarihi
IYTE LibraryTezT002115TP248.E5 B31 2019Tez Koleksiyonu