Identification and characterization of flavoprotein monooxygenases for biocatalysis

Biocatalysis is maturing to be a serious alternative to classical chemical transformations and this hopefully can contribute to a greener industry and new products at the same time. This thesis aimed to be a contribution to the field of biocatalysis by evaluating diverse enzymatic features of several flavoproteins, with an emphasis on Baeyer-Villiger Monooxygenases (BVMOs). Chapter 1 provides an overview on the catalytic properties of BVMOs. In chapter 2, a protocol is described for the generation of a small library of fusion proteins with varying linkers. This was used to evaluate the effect of the length of a glycine-rich linker in a monooxygenase-dehydrogenase fusion. Chapter 3 describes a comprehensive study on the production of reduced oxygen species by flavoprotein oxidases and monooxygenases. Insight in which hydrogen peroxide and superoxide are formed and what conditions can influence their formation. The second part of this thesis was focused in the discovery of novel flavoprotein monooxygenases by genome mining. In chapter 4 were studied two bacterial monooxygenases from the biosynthetic routes of lobatamide A and oocydin. In chapter 5, the flavin-containing monooxygenases HdFMO (from Hypsibius dujardini) and CbFMO (from Chloroflexi bacterium) were identified and characterized. Finally, in chapter 6, we discovered two BVMOs from the genome of the actinobacterium Streptomyces leeuwenhoekii C34 from Atacama Desert. Specifically, these biocatalysts exhibited a broad acceptance for cyclic ketones and high regio- and enantioselectivity. Overall, this thesis delivered new enzymes for the toolbox collection of known flavoproteins monooxygenases and insights into their molecular functioning as oxidative biocatalysts.