Optimization of expression and isolation of a thermophilic P450 enzyme
Aslantaş, Yaprak, author.

Optimization of expression and isolation of a thermophilic P450 enzyme

Aslantaş, Yaprak, author.

Yazar Ek Girişi
Aslantaş, Yaprak, author.

Fiziksel Tanımlama
xii, 56 leaves: illustrarions, charts;+ 1 computer laser optical disc.

Cytochrome 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.

Konu Başlığı
Enzymes -- Biotechnology.
Thermophilic fungi

Yazar Ek Girişi
Sürmeli, Nur Başak,

Tüzel Kişi Ek Girişi
İzmir Institute of Technology. Biotechnology and Bioengineering.

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

Elektronik Erişim
Access to Electronic Versiyon.

LibraryMateryal TürüDemirbaş NumarasıYer NumarasıDurumu/İade Tarihi
IYTE LibraryTezT001892TP248.65.E59 A83 2018Tez Koleksiyonu