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%0 Thesis
%A Sommerfeld, Isabel Katja
%T Microgels as protein and enzyme carriers: a pathway to biocatalytic glycan synthesis
%I RWTH Aachen University
%V Dissertation
%C Aachen
%M RWTH-2024-08211
%P 1 Online-Ressource : Illustrationen
%D 2024
%Z Veröffentlicht auf dem Publikationsserver der RWTH Aachen University
%Z Dissertation, RWTH Aachen University, 2024
%X The combination of functionalized microgels with biomacromolecules presents versatile opportunities in biomedical and biotechnological fields, offering enhanced biocatalytic applications. This thesis therefore focuses on the synthesis of different microgels for the loading of proteins and enzymes. First, stimuli-responsive microgels containing ionizable functional groups were synthesized utilizing precipitation polymerization. These microgels were characterized using various techniques such as Infrared Spectroscopy, Dynamic and Electrophoretic Light Scattering, NMR relaxometry, and Electron Microscopy. The responsiveness of the microgels to various stimuli was elucidated. Most importantly, these biocompatible microgels demonstrated their utility for ionic binding of positively charged cytochrome c, thereby showcasing their potential for protein immobilization. Moreover, immobilizing His6-tagged hyaluronan synthase onto these microgels via metal affinity binding, particularly using nickel ions, enabled repetitive enzymatic production of hyaluronic acid with unmatched yields, as monitored through capillary electrophoresis. Additionally, other glycosyltransferases were immobilized onto poly(ethylene glycol)-based microgels synthesized using droplet-based microfluidics. Enzyme immobilization through on-chip encapsulation was compared to post-attachment of enzymes to pre-synthesized microgels. For enzyme attachment, non-selective thiol Michael addition and selective SpyTag-SpyCatcher interaction were utilized, complemented by immobilization through mainly non-covalent interactions. Analysis of microgels involved optical microscopy for size determination, infrared spectroscopy for determination of chemical composition, and permeability assays for porosity assessment. Additionally, enzyme immobilization was confirmed using Bradford and fluorescamine assays while High-Performance Liquid Chromatography (HPLC) revealed the enzymatic activities for each glycosyltransferase. Utilizing the most suitable glycosyltransferase-microgels, cascade reactions were demonstrated, showcasing potential pathways toward a larger library of glycan products and a more efficient glycan synthesis. These findings underscore the synergistic potential of combining microgels with biomacromolecules in biomedical and biotechnological applications, particularly in the enzymatic production of complex glycans.
%F PUB:(DE-HGF)11
%9 Dissertation / PhD Thesis
%R 10.18154/RWTH-2024-08211
%U https://publications.rwth-aachen.de/record/992494