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%0 Thesis %A Hofzumahaus, Sebastian %T Verbesserung der Substrataufnahme in Ganzzellbiotransformationen mit Escherichia coli %C Aachen %I Publikationsserver der RWTH Aachen University %M RWTH-CONV-143921 %P IX, 165 S. : Ill., graph. Darst. %D 2013 %Z Prüfungsjahr: 2013. - Publikationsjahr: 2014 %Z Aachen, Techn. Hochsch., Diss., 2013 %X Biocatalysis is a method to produce fine and bulk chemicals environmentally friendly by either using isolated enzymes or by using whole cells. In whole cell biocatalysis substrates are converted by (recombinant) microorganisms, expressing the enzymes intracellularly. A good conversion requires therefore the efficient uptake of the substrate molecules into the cells. However, especially big hydrophobic substrates often do not fulfill this requirement. Because of the composition of the cell wall of gram-negative bacteria hydrophobic molecules can enter the cells only with very slow diffusion rates. Hence, organic solvents, detergents, surfactants and cyclodextrins are applied to improve the solubility of hydrophobic substrates and to increase the permeability of the cell walls. Unfortunately, these additives are either very expensive or can lead to the destabilization of the cell wall. To avoid these problems, the improvement of the substrate conversion was tested in this work by applying elicitins in whole cell biocatalysis reactions. As model reaction served the hydroxylation of six steroids with resting E. coli cells, coexpressing the P450 monooxygenase from CYP154C5 Nocardia farcinica and the electron transfer components putidaredoxin (Pdx) and Putidaredoxinreduktase (PdR) from Pseudomonas putida. Elicitins are small 10 kDa sterol carrier proteins, which are secreted by the Oomycetes Phytophthora and Pythium into their surroundings. Since elicitins could be heterologously expressed solubly only in Pichia pastoris so far, initially the expression of beta-cinnamomin from Phytophthora cinnamomi had to be established in E. coli. First, the cytoplasmic expression of the elicitin was tested, which, however, resulted in the formation of insoluble inclusion bodies because of the three disulfide bonds of beta-cinnamomin. To express the elicitin solubly, it was therefore expressed into the periplasm of E. coli with the Sec-dependent secretion signal sequence MalEss of the maltose binding protein E. By cloning the gene of beta-cinnamomin in frame with a C-terminal His-tag, the heterologously expressed elicitin could be purified by immobilized metal ion affinity chromatography in a single chromatographic step with high purity. Following, the expression of another elicitin, beta-cryptogein from Phytophthora cryptogea, could be carried out successfully under the same conditions. In the second part of this work it could be shown, that the substrate conversion of whole cell biotransformations of the six steroids pregnenolone, testosterone, progesterone, dehydro¬epiandrosterone, androstendione and nandro¬lone, could be significantly improved by adding purified elicitin in the whole cell biocatalysis reactions and by using E. coli cells, coexpressing beta-cinnamomin with CYP154C5, Pdx and PdR, in the whole cell biotransformations. %K Cytochrom P-450 (SWD) %K Biokatalyse (SWD) %K Steroide (SWD) %K Carrier-Proteine (SWD) %F PUB:(DE-HGF)11 %9 Dissertation / PhD Thesis %U https://publications.rwth-aachen.de/record/228826