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@PHDTHESIS{Nth:834681,
author = {Nöth, Maximilian Wolfgang Stefan},
othercontributors = {Schwaneberg, Ulrich and Pich, Andrij},
title = {{E}ngineering of biocatalytic microgels and bifunctional
peptides for biohybrid systems},
school = {RWTH Aachen University},
type = {Dissertation},
address = {Aachen},
publisher = {RWTH Aachen University},
reportid = {RWTH-2021-10064},
pages = {1 Online-Ressource : Illustrationen, Diagramme},
year = {2021},
note = {Veröffentlicht auf dem Publikationsserver der RWTH Aachen
University 2022; Dissertation, RWTH Aachen University, 2021},
abstract = {Biohybrid materials and systems have great potential in the
fields of biocatalysis and materials science, as the
combination of biological and synthetic building blocks
enables the design of novel biohybrid catalyst and material
concepts that convey new functionalities. New strategies for
integrating biocatalytic functionalities into materials and
for material and surface functionalization are needed, to
develop new biohybrid materials and systems. In this thesis,
two novel biohybrid systems for biocatalysis and universal
material and surface functionalization were developed: in
the first chapter, P450 μ-Gelzymes were established by
immobilizing cytochrome P450 BM3 monooxygenase in
stimuli-responsive "smart" microgels, while in the second
chapter, a universal surface functionalization toolbox based
on bifunctional peptides was developed for the
functionalization of synthetic polymers, metals, and
silicon-based materials.Enzymes are biocatalysts evolved by
nature to perform (bio)chemical reactions at environmentally
benign reaction conditions with impressive chemo-, regio-,
and stereoselectivities. P450 monooxygenases are versatile
biocatalysts with high synthetic application potential, but
their application is yet challenged, amongst others, by
their low operational stability (e.g., low organic solvent
tolerance, enzyme inactivation after a certain reaction
time). Enzyme immobilization is one the most successful
strategies to improve enzyme stability and enables the
release, re-immobilization, and recycling of enzymes. P450
monooxygenases are challenging to immobilize, and in the
case of P450 BM3 from Bacillus megaterium, the activity of
the immobilized enzyme is often deteriorated or entirely
lost. Microgels have attracted attention as an innovative
class of "smart" and stimuli-responsive carriers for enzyme
immobilization due to their chemical and mechanical
stability, tuneable architecture, biocompatibility,
porosity, high water content, and the ability to achieve
high enzyme loadings and provide a protective environment
for the immobilized enzymes. Therefore, new strategies for
integrating enzymes in microgels and new microgel systems
need to be developed to harness the potential of
stimuli-responsive microgels as a carrier platform for
"sensitive" enzymes that are challenging to immobilize. This
work reports the first pH-independent immobilization of P450
BM3 in novel poly(N-vinylcaprolactam) microgels with
1-vinyl-3-methylimidazolium as comonomer without loss of
catalytic activity (biohybrid P450 μ-Gelzymes) and the
first systematic study of P450 μ-Gelzyme performance.
Poly(N-vinylcaprolactam) microgels were synthesized with a
pH-independent, positive charge by modifying
1-vinylimidazole moieties through a quaternization reaction
(1-vinyl-3-methylimidazolium). The pH-independent
immobilization allowed to operate biohybrid P450 μ-Gelzyme
catalysts at the pH activity optimum (pH 8). In addition,
P450 μ-Gelzymes enabled ionic-strength triggered release
and re-immobilization of P450 BM3 as well as biocatalyst
recycling for repeated use and provided initial protective
effects against organic cosolvents.The biological
transformation of materials science is paving the way to
novel biohybrid material concepts by combining and
integrating biological and synthetic building blocks in
materials (e.g., biofunctionalization of synthetic polymers,
metals, and silicon-based materials). Universally applicable
and specific material and surface functionalization
methodologies are prerequisites for the biological
transformation of materials science but pose a key challenge
due to the vastly different properties and chemistries of
materials and surfaces. Innovative material and surface
functionalization technologies have to be developed to
address this challenge. Biological surface functionalization
with material binding or anchor peptides is a simple
strategy to endow materials with biological and synthetic
functionalities and an energy-efficient and environmentally
benign alternative to chemical and physical surface
functionalization strategies. Following this notion, a novel
toolbox concept for universal material and surface
functionalization based on bifunctional peptides was
developed. Bifunctional peptides were synthesized by
specific modification of the universal anchor peptide LCI
from Bacillus subtilis with different functional amine
moieties (e.g., reactive groups for click chemistry,
fluorescent dyes, antibiotics, synthetic polymers) through
sortase-mediated ligation employing sortase A from
Staphylococcus aureus. Sortase-mediated ligation further
enabled the purification of bifunctional peptides by a
negative purification strategy using Strep‐tag II affinity
chromatography (purities > $90\%).$ In general, the
bifunctional peptide toolbox enabled surface
functionalization either as a two or one-step strategy. In
the case of the one-step strategy, the desired functionality
was directly introduced to LCI, while in the case of the
two-step strategy, LCI was modified with a reactive group
that enabled further functionalization (e.g., employing
click chemistry). For the two-step strategy, synthetic
polymers (polypropylene, polyethylene terephthalate), metals
(stainless steel, gold), and silicon were functionalized
with reactive groups for copper-free azide-alkyne click
chemistry. The one-step strategy was demonstrated by direct
functionalization of polypropylene with a fluorescent dye
and biotin. These results represent the first systematic
combination of universal anchor peptides, like LCI, and
sortase-mediated ligation in a toolbox concept for universal
surface functionalization, including the first
peptide-mediated functionalization of polypropylene and
polyethylene terephthalate with reactive groups for
copper-free azide-alkyne click chemistry.},
cin = {162610 / 160000},
ddc = {570},
cid = {$I:(DE-82)162610_20140620$ / $I:(DE-82)160000_20140620$},
pnm = {SFB 985 A01 - Mikrogel-gesteuerte chemoenzymatische
Kaskaden unter Verwendung ganzer Zellen (A01) (221465724) /
DFG project 191948804 - SFB 985: Funktionelle Mikrogele und
Mikrogelsysteme (191948804)},
pid = {G:(GEPRIS)221465724 / G:(GEPRIS)191948804},
typ = {PUB:(DE-HGF)11},
doi = {10.18154/RWTH-2021-10064},
url = {https://publications.rwth-aachen.de/record/834681},
}
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