Glycosynthases - tuning glycosidase activity towards glycoside diversification and synthes
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....
| Main Author: | |
|---|---|
| Format: | Doctoral or Postdoctoral Thesis |
| Language: | English |
| Published: |
Forschungszentrum Jülich GmbH Zentralbibliothek, Verlag
2019
|
| Subjects: | |
| Online Access: | https://juser.fz-juelich.de/record/867847 https://juser.fz-juelich.de/search?p=id:%22FZJ-2019-06449%22 |
| Summary: | 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 reaction utilising glycosyl fluoride donors. The application of a hyperthermophilic enzyme was attempted with mutants of the putative β-galactosidase BglC, which was provided by the group of Prof. Elling (RWTH Aachen). A general unsuitability of high temperatures for glycosynthetic reactions was determined as thermal degradation of the glycosyl fluoride donor dominated the reaction. The 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 β-glucosidase mutant Abg-E358S acted, not only as an example of a mesophilic glycosynthase, but also as a positive control as this variant was literature known to possess glycosynthetic activity. The psychrophilic glucosidase BglU of M. antarcticus was characterised and the catalytic residue mutated with the aim of producing a cold-adapted β-glycosynthase. Mutation of the nucleophilic residue and β-glucosidase activity screening via esculin-agar resulted in the discovery of the glycosynthetic active variant BglU-E377A. Similar to the wt, the glycosynthase BglU-E377A exhibited a lower synthetic activity than the mesophilic Abg-E354S and despite the lower activity, a repeated glycosylation of the product similar to the reaction catalysed by Abg-E358S was observed. |
|---|