Identification of novel unspecific peroxygenase chimeras and unusual YfeX axial heme ligand by a versatile high-throughput GC-MS approach

Abstract

Catalyst discovery and development requires the screening of large reaction sets necessitating analytic methods with the potential for high-throughput screening. These techniques often suffer from substrate dependency or the requirement of expert knowledge. Chromatographic techniques (GC/LC) can overcome these limitations but are generally hampered by long analysis time or the need for special equipment. The herein developed multiple injections in a single experimental run (MISER) GC-MS technique allows a substrate independent 96-well microtiter plate analysis within 60 min. This method can be applied to any laboratory equipped with a standard GC-MS. With this concept novel, unspecific peroxygenase (UPO) chimeras, could be identified, consisting of subdomains from three different fungal UPO genes. The GC-technique was additionally applied to evaluate an YfeX library in an E. coli whole-cell system for the carbene-transfer reaction on indole, which revealed the thus far unknown axial heme ligand tryptophan.