Dokument: Glycosynthases - Tuning glycosidase activity towards glycoside diversification and synthesis
| Titel: | Glycosynthases - Tuning glycosidase activity towards glycoside diversification and synthesis | |||||||
| URL für Lesezeichen: | https://docserv.uni-duesseldorf.de/servlets/DocumentServlet?id=51808 | |||||||
| URN (NBN): | urn:nbn:de:hbz:061-20191217-104535-3 | |||||||
| Kollektion: | Dissertationen | |||||||
| Sprache: | Englisch | |||||||
| Dokumententyp: | Wissenschaftliche Abschlussarbeiten » Dissertation | |||||||
| Medientyp: | Text | |||||||
| Autor: | Dr. Hayes, Marc Richard [Autor] | |||||||
| Dateien: |
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| Beitragende: | Prof. Dr. Pietruszka, Jörg [Gutachter] Prof. Urlacher, Vlada [Gutachter] | |||||||
| Stichwörter: | Glycosynthase, Glycoside, Glycodiversification, Biocatalysis, Site-directed mutagenesis | |||||||
| Dewey Dezimal-Klassifikation: | 500 Naturwissenschaften und Mathematik » 540 Chemie | |||||||
| Beschreibung: | With the aim of producing and applying new glycosynthases towards the synthesis of glycosides, the results obtained during the work of this thesis highlighted various aspects not only of the glycosynthase catalysed reaction but also of the process of transforming a glycosidase into a glycosynthase.
The influence of temperature on glycosynthetic reactions was examined in detail by applying mutated glycosidases with hyperthermophilic, mesophilic, and psychrophilic properties. An elevated temperature proved as unsuitable for glycosynthetic reactions utilising glycosyl fluoride donors, due to the high thermal degradation of the glycosyl fluoride donor molecule. This unsuitability of elevated temperatures turned the focus towards low and moderate temperatures for the glycosynthetic reaction in order to minimalise glycosyl fluoride hydrolysis. The mesophilic and psychrophilic β glucosidase mutants Abg E358S and BglU E377A were generated and successfully applied in the diversification of flavonoid glycosides. A glycosynthase activity assay was developed in a microplate format with a short assay time and high maximal detection limit. Application of the assay was demonstrated by the determination of the conversion of the glycosyl fluoride donor, kinetic measurements, substrate scope, and optimal acceptor : donor ratio of the generated glycosynthases. The coniferin specific β glucosidase Cbg1 of R. radiobacter was identified as a glycosynthase candidate, which could potentially glycosylate phenolic compounds. Structural analysis identified the catalytic residues and nine mutant variants were created by mutagenesis and tested for hydrolytic and glycosynthase activity utilising the glycosyl fluoride or the chemical recovery method. Additionally, transglycosylation experiments were attempted applying phenolic compounds. The conversion of the α L rhamnosidase RhaB of Bacillus sp. GL1 into a rhamnosynthase was attempted, by applying the strategy of an in situ production and transfer of the β glycosyl azide donor. The mutant variants were tested for hydrolytic activity, recovered activity by chemical recovery, and glycosynthetic activity. A colourimetric detection assay for azide ions by acidic ferric solution was adapted for a microplate format and applied for the identification of glycosynthetic activity of RhaB mutant variants. In cooperation with the group of Prof. Fujiyama (Osaka University) the synthetic potential of the glycosynthase variant Endo CC N180H of C. cinerea for the diversification of flavonoids was evaluated. The transfer of a sialo biantennary glycan catalysed by Endo CC N180H was successfully demonstrated for different substrates. | |||||||
| Lizenz: |  Urheberrechtsschutz | |||||||
| Fachbereich / Einrichtung: | Mathematisch- Naturwissenschaftliche Fakultät » WE Chemie » Bioorganische Chemie | |||||||
| Dokument erstellt am: | 17.12.2019 | |||||||
| Dateien geändert am: | 17.12.2019 | |||||||
| Promotionsantrag am: | 10.07.2019 | |||||||
| Datum der Promotion: | 24.09.2019 |
