New approaches for flavoenzyme applications: Cofactor-mediated immobilization & in vitro production of human metabolites

Krzek, M., 2017, [Groningen]: University of Groningen. 151 p.

Research output: Thesis › Thesis fully internal (DIV)

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  • Title and contents

    Final publisher's version, 218 KB, PDF document

  • Chapter 1

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  • Chapter 2

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  • Chapter 3

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  • Chapter 4

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  • Chapter 5

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  • Chapter 6

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  • Acknowledgements

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  • Complete thesis

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  • Propositions

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  • Marzena Krzek
This thesis presents new approaches towards applications of flavin-containing enzymes as biocatalysts.
The first part describes a novel method of cofactor-mediated enzyme immobilization. By coupling the flavin cofactor to carrier material, a target prototype flavoenzyme could be immobilized. This resulted in a fully active and highly stable enzyme formulation. By varying the type of chemical linkers between the carrier material and the flavin cofactor, this new method allows to tune the surface loading and distance from the solid phase to the enzyme (monolayer). To provide a better access to the required chemically modified flavin cofactors, a new chemical synthesis protocol for the preparation of alkylated flavin cofactors was also developed.
To demonstrate the value of flavin-containing enzymes as biocatalysts, work is presented on the exploration of a specific group of flavoenzymes: the flavin-containing monooxygenases (FMOs). First, a physiologically crucial human FMO, flavin-containing monooxygenase 3 (FMO3), is extensively discussed concerning the current knowledge on its sequence and (predicted) structural properties. Biochemical studies on this human enzyme are hampered by its reluctance towards heterologous expression of soluble protein. Results on the production of an active, self-sufficient hFMO3 in bacterial cells are presented. Furthermore, it is demonstrated that sequence-related microbial enzymes can be used as human FMO mimics for the synthesis of drug metabolites. Such enzymes are shown to be capable of performing enantio- and regioselective oxygenations of (human) drug molecules. This study shows that the currently available collection of thermostable microbial flavoprotein monooxygenases provides an attractive alternative to produce and study drug metabolites
Translated title of the contributionNieuwe benaderingen voor flavoenzymes toepassingen: Flavinecofactor-gemediëerde immobilisatie en in vitro productie van geneesmiddelmetabolieten
Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
Award date15-Sep-2017
Place of Publication[Groningen]
Print ISBNs9789403400969
Publication statusPublished - 2017

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