Robust monooxygenase biocatalysts: discovery and engineering by computational design

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This PhD thesis describes research in the field of biocatalysis. We use enzymes, i.e. proteins that facilitate chemical transformations in living organisms, and explore their potential for application in the chemical and pharmaceutical industry. With a focus on enzymes that use oxygen as a green and abundantly available oxidant, we aim to replace the currently applied chemical transformations in industry, which use hazardous and wasteful catalysts. In a future, bio-based economy, biocatalysis can also boost the productivity and cost-effectiveness of the production of complex molecules such as drugs, because it allows novel and highly selective synthesis routes.
I describe here the identification and characterization of several monooxygenases that display properties which are interesting for application: unlike many other enzymes, they show good stability when exposed to conditions typical for industrial processes, such as high temperature and the presence of high concentrations of chemicals. Our research also encompasses the unravelling of the molecular origin of such properties and how natural enzymes can be tuned to deliberately introduce them. In this context I also describe here a computational protocol that allows to identify soft spots in unstable proteins and predicts substitutions that can stabilize them. An experimental guide on how to create these variants is also given. This was then applied to create a thermostable variant of an enzyme that can produce a precursor of Nylon-6. A related protein was also subjected to the Nobel Prize-awarded method of directed evolution, which allowed to change the outcome of the catalyzed reaction. A study of the same enzyme then also allowed us to address one of the greatest puzzles in enzymology: why are some enzymes very specific for one compound, while other enzymes can act on many? We found that here, the individual amino acids in the protein’s center do not bind molecules specifically.
Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
  • University of Groningen
  • Fraaije, Marco, Supervisor
  • Janssen, Dick, Co-supervisor
Award date21-Jun-2019
Place of Publication[Groningen]
Print ISBNs978-94-028-1563-4
Electronic ISBNs978-94-034-1795-0
Publication statusPublished - 2019


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