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Identifier 000433969
Title Expression of cytochromes P450 and investigation of their selectivity towards bioactive compounds
Alternative Title Έκφραση κυτοχρωμάτων P450 και μελέτη της εκλεκτικότητας αυτών προς βιοδραστικές ενώσεις
Author Σακολέβα, Θάλεια Γ.
Thesis advisor Παυλίδης, Ιωάννης
Reviewer Χανιωτάκης, Δημήτριος
Bornscheuer, Uwe T.
Abstract Cytochromes P450 (CYPs) or P450 monooxygenases belong to the superfamily of heme b containing monooxygenases and they are involved in the oxidation of a variety of organic compounds, such as xenobiotics, antibiotics, steroids, fatty acids, and others. In a typical reaction catalyzed by P450s, an alcohol via hydroxylation and water are formed under the consumption of reduced nicotinamide adenine dinucleotide phosphate (NADPH). NADPH acts as an electron donor via redox partner protein systems for the hydroxylation of the target substrate, but the complexity of these systems limits the wider application of the CYPs. The purpose of the present thesis is the creation of a collection of redox biocatalysts which will be able to synthesize bioactive compounds. The project is separated into two parts. In the first part, after previous researchers’ bioinformatics analysis on sequencing, a list of twenty different P450 candidates was screened firstly for the co-expression with different redox partner systems, and second, for the highly selective hydroxylation of a steroid of interest. The reaction was performed in whole-cell systems, using E. coli strains as expression host. The studies aimed for the successful recombinant expression with proper protein folding, and substrate conversion using the proper redox partner. The expression level was tested using SDS-PAGE, the proper folding was indicated with CO titration and the product formation was studied using chromatographic methods. The results indicated that although several of the studied CYPs were properly folded, only OleP was able to hydroxylate the steroid of interest. Using OleP as the most promising P450 monooxygenase candidate, reactions were performed, using other steroid substrates, such as cholic acid and deoxycholic acid. From the whole-cell biocatalysis, but also using purified proteins, it could be shown that OleP can hydroxylate deoxycholic acid, but not cholic acid, using either PdR/PdX system or BMR reductase. Using bioinformatic analysis, substrate-selectivity of CYPs was investigated. The analysis could not highlight the underlying reason of the selectivity of OleP towards deoxycholic acid, while it was inactive against cholic acid. Nevertheless, it could predict the right binding conformation of deoxycholic acid, while – via this analysis – several positions were selected for mutagenesis experiments, which could potentially lead to different regioselectivity. In the second part, the development and optimization of expression protocols to maximize the production of fused CYPs with a molecular weight of ~120 kDa in a properly folded form was investigated. The high molecular weight can be challenging for the protein expression and the protein stability in bacterial systems. The successful expression and purification of these CYPs in proper form was followed from photometric analysis of reactions with limonene as substrate. Literature data was confirmed for (R)-limonene, as NADPH was consumed during these reactions, however, despite the reports of activity with (S)-limonene as well, no activity could be monitored. CYPs identified in this thesis and were expressed in proper folded conformation can be further studied in the future concerning the substrate scope they accept, in order to get access to further products of potential biotechnological interest.
Language English
Subject Hydroxylation
Redox partner
Μονοοξυγενάσες P450
Οξειδοαναγωγικά συστήματα
Issue date 2020-11-27
Collection   Faculty/Department--Faculty of Sciences and Engineering--Department of Chemistry--Post-graduate theses
  Type of Work--Post-graduate theses
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