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

Abstract: With the emergence of Green Chemistry, biocatalysis is becoming an important approach in many laboratory and industrial processes. Enzymes catalyse chemical reactions with high regio- and stereoselectivity at mild conditions and, most importantly, are able to form products that are not possible to obtain by conventional synthetic chemistry. Monooxygenases are a fascinating group of enzymes which oxidise substrates using atmospheric oxygen and release water as a by-product. These enzymes have great potential applications and indeed there are already some industrial showcases. Monooxygenases are of different types and one of the important groups is known as Baeyer-Villiger monooxygenases (BVMOs). BVMOs oxidise ketones to esters and heteroatoms to the corresponding oxide, which are intersting reactions for pharmaceutical and fine chemical industry. To date, several BVMOs have been discovered and characterised; however, there are many limitations, such as poor stability which hindered the wide application of these enzymes. Thus, the search for better BVMOs has continued. This thesis reports the discovery of four BVMOs from the genome sequence of Dietzia sp. D5, a microorganism rich in oxygenases. The genes were cloned and expressed, and two of them were characterised. One of the enzymes, named BVMO3 readily oxidises linear aliphatic ketones. Characterization of the other BVMO, called BVMO4, revealed that it is the second most stable native BVMO ever reported and it oxidises a wide range of substrates. The oxidation of sulfides and aldehydes has been investigated further. Aldehydes were oxidised by this BVMO with a rare regioselectivity, producing carboxylic acid rather than formate ester which is the commonly observed product. Site saturation mutagenesis of selected amino acid residues in the proximity of the active site increased the cyclohexanone oxidation efficiency of BVMO4 by twelve-fold.