h1

TY  - THES
AU  - Mertens, Alan
TI  - Untersuchung verschiedener Ein- und Dreikomponenten-P450-Systeme für die Anwendung in der Biokatalyse
PB  - RWTH Aachen
VL  - Dissertation
CY  - Aachen
M1  - RWTH-2016-00818
SP  - 1 Online-Ressource (VII, 262 Seite) : Diagramme
PY  - 2015
N1  - Veröffentlicht auf dem Publikationsserver der RWTH Aachen University 2016
N1  - Dissertation, RWTH Aachen, 2015
AB  - Cytochrome P450 monooxygenases are enzymes of high interest which are applied frequently in biocatalysis because they are able to catalyze hydroxylation reactions and many other reactions. As these enzymes need electrons for catalysis, cytochrome P450 monooxygenases can be grouped into different classes according to the system used for electron transfer. P450 enzymes which belong to three-component systems require redox proteins like ferredoxins and ferredoxin reductases while one-component P450 monooxygenases are redox-self-sufficient and therefore do not depend on additional redox proteins.The first project of this thesis dealt with the investigation of putative electron transfer proteins from the thermophilic bacterium <i>Thermobifida fusca</i> in order to identify the native redox partners of CYP154H1, a thermostable three-component P450 monooxygenase. Furthermore, thermostable redox partners for their application in biocatalysis with other three-component P450 monooxygenases should be identified. In the first step, selected redox proteins from <i>T. fusca</i> (five ferredoxins, one flavodoxin and four ferredoxin reductases) were recombinantly expressed in <i>E. coli</i>. After activity determination, a number of redox protein combinations were applied in biocatalysis experiments with several cytochrome P450 monooxygenases (CYP154H1, CYP154C5 from <i>Nocardia farcinica</i> and CYP106A2 from <i>Bacillus megaterium</i>). The data analysis revealed that none of the tested combinations resulted in a conversion which differed significantly from the conversion in the respective negative controls. Under the premise that the natural redox partners for CYP154H1 would have been active and correctly folded, these findings indicate that no natural redox partners were among the tested electron transfer proteins. In addition, the different redox proteins were obviously not capable to transfer electrons to CYP154C5 and CYP106A2 under the applied conditions.The second project of this thesis comprised the investigation of the self-sufficient cytochrome P450 monooxygenases CYP102K1 from the gram-negative bacterium <i>Azorhizobium caulinodans</i> and CYP102<sub>Nmu</sub> from the gram-positive bacterium <i>Nakamurella multipartia</i>. The heme domain of these enzymes and the heme domain of CYP102A1 (a well-characterized self-sufficient P450) share a sequence identity of less than 40 
LB  - PUB:(DE-HGF)11
UR  - https://publications.rwth-aachen.de/record/567406
ER  -