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@PHDTHESIS{Anand:816648,
author = {Anand, Deepak},
othercontributors = {Schwaneberg, Ulrich and Böker, Alexander},
title = {{C}hiral separation of arginine based on tailor-made
{F}hu{A} β-barrel protein},
school = {RWTH Aachen University},
type = {Dissertation},
address = {Aachen},
publisher = {RWTH Aachen University},
reportid = {RWTH-2021-03346},
pages = {1 Online-Ressource : Illustrationen, Diagramme},
year = {2020},
note = {Veröffentlicht auf dem Publikationsserver der RWTH Aachen
University 2021; Dissertation, RWTH Aachen University, 2020},
abstract = {Chirality of chemical compound is ubiquitous in nature
performing central function in metabolism of many nutrients
and pharmaceuticals. Even-though enantiomers of a compound
have similar chemical and physical properties it can have
completely different biological activity. Thus chiral
molecules are of large economic value in chemical,
pharmaceutical, and food industries. Many applications in
these industries require the isolation and use of single
chiral isomers (enantiomers) of chiral compounds. As a
result, there is an ever increasing necessity of optically
pure compounds but it is a challenging task to obtain it.
Since the first optical resolution of tartaric acid,
performed by Louis Pasteur in early 1848, various techniques
have been developed for chiral resolutions of enantiomeric
compounds. Methods such as chromatographic or enzymatic
techniques are commonly used. However, they are limited by
difficulties for large-scale productions. Crystallization
can be used in large-scales but often several rounds of
crystallization and recrystallization are required to obtain
enantiopure compounds due to entrapments of the unwanted
enantiomer during crystal growth, which often leads to
significant reduction in yields (up to 50 $\%).$ Each and
every one of these techniques has their own advantages and
disadvantages but the membrane based approach is of
particular interest because of its use in continuous
operation and its ease of scale-up. But the problem with the
membrane based technology developed till now is that of
non-uniform pore size and limited number of pores. Inspired
by nature, membrane-based approaches for chiral separation
using a barrel protein can be a suitable alternative. Chiral
protein-polymer membranes would be an attractive,
cost-effective, and scalable method. However, the main
challenges lie in the design of “filter regions” and the
generation of “screening systems” to identify chiral
channel proteins. In the present thesis, ferric hydroxamate
uptake component A (FhuA) was engineered to generate chiral
channel for separation of arginine enantiomers which can be
used as a scaffold for the generation of protein-polymer
membrane. FhuA is a large monomeric transmembrane protein of
Escherichia coli which folds into a barrel consisting of 22
antiparallel β-strands and a barrel-plugging
“corkdomain”. FhuA was chosen as a functional nanopore
because of its high tolerant towards organic solvents,
thermal resistant, and has a high robustness towards
reengineering. Structurally, FhuA contains a water channel
wherein two flexible loops in the cork domain(loop1; residue
35-40 and loop2; residues 135-145) were shortened to
generate two selectivity filter regions (filter1 and
filter2). Additionally, cork domain was stabilized inside
the barrel by substituting three amino acids (Q62D, R81W,
and N117L) resulting in generation of FhuAΔLvariant having
higher interaction between barrel and cork domain. In order
to generate a chiral selective variant, a directed evolution
protocol was developed to generate a chiral FhuAΔLvariant.
A novel whole cell calorimetric screening system based on
amino acid utilizing enzyme (argininedeiminase) was
developed in order to identify the enantioselective variant
from mutant libraries of FhuAΔL. Screening of mutant
libraries led to the identification of FhuAF4 variant (amino
acid substitutions: G134S, G146T) showing approximately two
times higher transport of L-arginine compared to parent
FhuAΔL with E-value=1.92; ee $\%=23.91$ at 52.39 $\%$
conversion. Steered molecular dynamics (SMD) simulations
were carried out for molecular understanding of observed
altered enantiopreference of FhuAF4 variant towards
L-arginine compared to the FhuAΔL variant. In FhuAΔL
variant, less interactions of both D- and L-arginine with
the filter region 2 were observed and both enantiomers pass
freely. Interestingly, in FhuAF4 variant, transport of
D-arginine is hindered and steered transport is slowed down,
indicated by a longer residence time close to the
selectivity filter region 2. The obtained results provide
first proof of principle of engineering of a membrane
protein towards chiral separation of amino acids and provide
insight into the mechanism of chiral separation within the
FhuA channel. It is likely that with the identified filter
region and OmniChange libraries further improvements are
achievable for other amino acids and a broader range of
enantiomers. The chiral FhuA channel proteins would be an
excellent scaffold for generation of chiral membrane based
on protein polymer conjugates with a high potential for
novel and scalable downstream processes in pharmaceutical
and food industries.},
cin = {162610 / 160000},
ddc = {570},
cid = {$I:(DE-82)162610_20140620$ / $I:(DE-82)160000_20140620$},
typ = {PUB:(DE-HGF)11},
doi = {10.18154/RWTH-2021-03346},
url = {https://publications.rwth-aachen.de/record/816648},
}
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