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@PHDTHESIS{Hofzumahaus:228826,
author = {Hofzumahaus, Sebastian},
othercontributors = {Schallmey, Anett},
title = {{V}erbesserung der {S}ubstrataufnahme in
{G}anzzellbiotransformationen mit {E}scherichia coli},
address = {Aachen},
publisher = {Publikationsserver der RWTH Aachen University},
reportid = {RWTH-CONV-143921},
pages = {IX, 165 S. : Ill., graph. Darst.},
year = {2013},
note = {Prüfungsjahr: 2013. - Publikationsjahr: 2014; Aachen,
Techn. Hochsch., Diss., 2013},
abstract = {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.},
keywords = {Cytochrom P-450 (SWD) / Biokatalyse (SWD) / Steroide (SWD)
/ Carrier-Proteine (SWD)},
cin = {160000 / 162610},
ddc = {570},
cid = {$I:(DE-82)160000_20140620$ / $I:(DE-82)162610_20140620$},
typ = {PUB:(DE-HGF)11},
urn = {urn:nbn:de:hbz:82-opus-48998},
url = {https://publications.rwth-aachen.de/record/228826},
}
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