Directed evolution of dynamical elements in enzymes using microfluidic chips

Enzymes provide delicate synchronisation of numerous processes using dynamic elements like tunnels and gates. Tunnels and gates mediate exchange of ligands between the solvent and enzyme’s active site or between two buried active sites. Mobile gates allow access of the preferred substrates and co-factors, while preventing the entry of non-cognate ligands, inhibitors or solvent. Targeting these natural hotspots can provide impressive refinement of valuable enzyme properties and represents a promising strategy in protein engineering. However, targeting dynamical elements such as gates, consisting of several inseparable elements, requires simultaneous mutagenesis in a larger number of positions leading to creation of libraries as large as of >10E6 mutants. In this project, microfluidic ultrahigh-throughput screening method will be developed and applied for screening of these libraries. This newly developed method will enable a real-time sorting and multiple adaptions of proteins in a very unique way.