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Daniel Eggerichs, Carolin Mügge, Julia Mayweg, Ulf-Peter Apfel, Dirk Tischler

• Styrene and indole monooxygenases (SMO and IMO) are two-component flavoprotein monooxygenases composed of a nicotinamide adenine dinucleotide (NADH)-dependent flavin adenine dinucleotide (FAD)-reductase (StyB or IndB) and a monooxygenase (StyA or IndA). The latter uses reduced FAD to activate oxygen and to oxygenate the substrate while releasing water. We circumvented the need for the reductase by direct FAD reduction in solution using the NAD(P)H-mimic 1-benzyl-1,4-dihydronicotinamide (BNAH) to fuel monooxygenases without NADH requirement. Herein, we report on the hitherto unknown solvent tolerance for the indole monooxygenase from \(\textit {Gemmobacter nectariphilus}\) DSM15620 (\(\it Gn\)IndA) and the styrene monooxygenase from \(\textit {Gordonia rubripertincta}\) CWB2 (\(\it Gr\)StyA). These enzymes were shown to convert bulky and rather hydrophobic styrene derivatives in the presence of organic cosolvents. Subsequently, BNAH-driven biotransformation was furthermore optimized with regard to the applied cosolvent and its concentration as well as FAD and BNAH concentration. We herein demonstrate that \(\it Gn\)IndA and \(\it Gr\)StyA enable selective epoxidations of allylic double bonds (up to 217 mU \(mg^{−1}\)) in the presence of organic solvents such as tetrahydrofuran, acetonitrile, or several alcohols. Notably, \(\it Gn\)IndA was found to resist methanol concentrations up to 25 vol.%. Furthermore, a diverse substrate preference was determined for both enzymes, making their distinct use very interesting. In general, our results seem representative for many IMOs as was corroborated by in silico mutagenetic studies.