Dokument: Heterologous expression and artificial maturation of the [FeFe]-hydrogenase from Desulfovibrio desulfuricans -Characterization and comparison with the native enzyme-
| Titel: | Heterologous expression and artificial maturation of the [FeFe]-hydrogenase from Desulfovibrio desulfuricans -Characterization and comparison with the native enzyme- | |||||||
| URL für Lesezeichen: | https://docserv.uni-duesseldorf.de/servlets/DocumentServlet?id=35161 | |||||||
| URN (NBN): | urn:nbn:de:hbz:061-20150827-111038-7 | |||||||
| Kollektion: | Dissertationen | |||||||
| Sprache: | Englisch | |||||||
| Dokumententyp: | Wissenschaftliche Abschlussarbeiten » Dissertation | |||||||
| Medientyp: | Text | |||||||
| Autor: | Wrede, Kathrin [Autor] | |||||||
| Dateien: |
| |||||||
| Beitragende: | Prof. Dr. Lubitz, Wolfgang [Gutachter] Prof. Dr. Jaeger, Karl-Erich [Gutachter] | |||||||
| Stichwörter: | Hydrogenase, Desulfovibrio desulfuricans, heterologous expression, EPR | |||||||
| Dewey Dezimal-Klassifikation: | 500 Naturwissenschaften und Mathematik » 540 Chemie | |||||||
| Beschreibung: | A better understanding of the hydrogen reaction cycle in hydrogenases would help to design new bio-based catalysts for hydrogen production. Therefore, the [FeFe]-hydrogenase from Desulfovibrio desulfuricans was studied in this thesis.
An important issue in studying proteins is the availability of high amounts of pure protein. Chapter 3 shows the improvement of the cell growth and purification methods for the native enzyme from Desulfovibrio desulfuricans. Additionally, and for the first time, the development and establishment for an overexpression system for the [FeFe]-hydrogenase from D. desulfuricans is reported. For that the sequences encoding for the protein were codon-optimized. Afterwards a plasmid was constructed that contained these codon optimized sequences for both subunits of the hydrogenase, one of which additionally contained the sequence for an affinity tag for easy purification. From this, high amounts of pure, unmaturated enzyme could be prepared. Spectroscopic studies of the heterologously expressed, unmaturated enzyme by EPR spectroscopy are reported in Chapter 4, as well as its crystallization. It is shown that artificial maturation of this enzyme with a synthetic [2Fe]H-mimic is possible and leads to fully active protein. The optimization for hydrogen production and consumption assays is also reported. Afterwards the maturated enzyme was studied by EPR and FTIR spectroscopy and compared with the native enzyme. To get better insight into the involvement of the accessory iron-sulfur clusters in the function of the [FeFe]-hydrogenase, site-directed mutagenesis experiments to change redox potentials of these clusters are reported in Chapter 5. Maturation and activity measurements are shown here to see the effect of the mutations on the protein behaviour. The unmaturated enzymes were studied by EPR spectroscopy and compared to the heterologously expressed wild type protein. The native [FeFe]-hydrogenase from D. desulfuricans shows an inactivation at high potentials (HPI) in protein film electrochemistry (PFE) experiments, which is not fully understood. Therefore, Chapter 6 reports experiments designed to provide further understanding of this phenomenon. It is reported that chloride seems to be essential for HPI and that HPI is also observed in the absence of hydrogen. Furthermore, the unmaturated and maturated [FeFe]-hydrogenase were measured for the first time with this technique to compare it with the native enzyme and to determine the redox potentials of the different clusters. | |||||||
| Lizenz: |  Urheberrechtsschutz | |||||||
| Fachbereich / Einrichtung: | Mathematisch- Naturwissenschaftliche Fakultät | |||||||
| Dokument erstellt am: | 27.08.2015 | |||||||
| Dateien geändert am: | 27.08.2015 | |||||||
| Promotionsantrag am: | 23.05.2015 | |||||||
| Datum der Promotion: | 13.07.2015 |
