Novel approaches for the redesign of flavoprotein oxidases

PhD ceremony: Mr. R.T. Winter, 16.15 uur, Aula Academiegebouw, Broerstraat 5, Groningen

Dissertation: Novel approaches for the redesign of flavoprotein oxidases

Promotor(s): prof. M.W. Fraaije

Faculty: Mathematics and Natural Sciences

Enzymes are a powerful class of protein based catalysts. They are able to perform almost any type of chemical reaction under mild conditions. The potential of enzymes is increasingly appreciated by industry in their search for greener alternatives to current processes.A drawback of many enzymes, however, is their inability to be directly applied in industrial processes as they have evolved to fulfill a distinct role in a defined niche, hampering broad applicability. The challenge is to redesign or “engineer” enzymes to convert pre-defined substrates under pre-defined conditions. This PhD thesis tackles this challenge for one particular class of enzymes: flavoprotein oxidases.

Flavoprotein oxidases use molecular oxygen directly to catalyze the oxidation of substrates, performing this reaction under mild conditions. As traditional oxidation chemistry is notoriously dirty, their mild and specific nature makes oxidases interesting for industrial processes. Flavoprotein oxidases catalyze the oxidation of a diverse group of substrates, including sugars, alcohols and steroids. In this thesis we present a variety of different approaches taken to engineer oxidases.We study the way oxidases channel oxygen to the site of the reaction. We develop a system for expressing oxidases on the surface of bacteria. We develop an oxidase sensor, by fusing a short peptide capable of detecting the byproduct of oxidase conversions to an oxidase. We describe a thermostable oxidase originating from a thermal-hot springs dwelling organism and try to engineer its thermostability into a less heat-tolerant oxidase. Finally, we attempt to discover new activities by creating and screening a very large collection of oxidase variants.