Baeyer-Villiger Monooxygenases from a Dietzia sp. - Enzyme Discovery, Characterization and Engineering

University dissertation from Division of Biotechnology, Lund University

Abstract: Popular Abstract in Undetermined Most of the things that we use daily are the product of chemical reactions. Plastics derive from the polymerization of smaller molecules, drugs are synthesised rigorously by pharmaceutical industries, and products for the house and personal care contain a number of different chemicals and fragrances. All these compounds are produced by different branches of the chemical industry that rearranges raw material into more complex molecules. Industrial chemical reactions can be subdivided into two categories: classical synthetic reactions and “green” biocatalysed reactions. Classic synthetic approaches often need high temperature and pressure to perform the reaction. Organic reactants are often toxic and generation and maintenance of high temperature and pressure consumes a great amount of energy. In the perspective of a world in which resources are limited and pollution is a problem for mankind, it is important to improve chemical reaction processes with the aim to reduce their energy requirements and to use non-toxic reactants. For this reason biocatalysed reactions are gaining more and more importance. Biocatalysis as the name implies use biological catalysts, often refered as enzymes, to transform the raw material into product. Enzymes are produced by microrganisms including bacteria, yeast and fungi. Unlike chemical catalysts, enzymes are biodegradable, require mild conditions (low temperature and atmospheric pressure) to catalyse reactions, they are safe and specific. This thesis work aims at developing enzymes that can be used in the future to catalyse reactions. Baeyer-Villiger monooxygenases are enzymes which insert one oxygen atom from atmospheric air into a substrate and release water as a by-product. The classical chemical process to catalyse the same reaction requires strong oxidants, that are toxic and carry a high risk of explosion and corrosion, to catalyse the same reaction. These enzymes can be potentially used to catalyse reactions for the production of drugs, perfumes and other chemicals for various aplications such as in the production of biodegradable polymers, nylon, plastics etc. This thesis focuses on Baeyer-Villiger monooxygenases (BVMOs) and in particular it deals with the discovery of new enzymes. Since existing BVMOs are rather unstable, meaning they lose the ability to catalyse the reaction in a short time, their application in industrial processes is hindered. Therefore there is a need for new and better industrial enzymes. In this thesis work, four new enzymes are discovered from a bacterium called Dietzia sp. D5. One of these new BVMOs, is relatively stable compared to other enzymes of its kind and catalyses a number of reactions, including the synthesis of a precursor for the anti-inflammatory drug family of profen (the drug marketed as Ipren in Sweden). This enzyme was also manipulated in order to produce more caprolactone, a precursor for the sysnthesis of different polymers. Mutation of the enzyme improved the production of caprolactone by 12-fold. The work behind this thesis is just part of the global effort in moving towards a more sustainable chemical industry. It contributes by identifying a new source of oxygenase and novel BVMOs which can be potentially applied in the future to industrial processes.