- Unspecific peroxygenases (UPO; EC 1.11.2.1) represent a group of secreted heme-thiolate proteins that are capable of catalyzing the mono-oxygenation of diverse organic compounds, using only H2O2 as a co-substrate. Here we show that the four peroxygenases AaeUPO, MroUPO, rCciUPO and rNOVO catalyze the stepwise hydroxylation of cyclohexane to cyclohexanol and cyclohexanone. The catalytic efficiencies (kcat/Km) for the initial hydroxylation were in the same order of magnitude for all four peroxygenases (∼104 M−1 s−1), whereas they differed in the second step. The conversion of cyclohexanol by AaeUPO and rCciUPO was 1–2 orders of magnitude less efficient (∼102 M−1 s−1) than by MroUPO and rNOVO (∼104 M−1 s−1). The highest conversion rate in terms of H2O2 utilization was accomplished by MroUPO under repeated addition of the peroxide (87% in relation to the total products formed). Using the latter UPO, we successfully established a micro-mixing reaction device (SIMM-V2) for the oxidation of cyclohexane. As cyclohexanone is a chemical of highUnspecific peroxygenases (UPO; EC 1.11.2.1) represent a group of secreted heme-thiolate proteins that are capable of catalyzing the mono-oxygenation of diverse organic compounds, using only H2O2 as a co-substrate. Here we show that the four peroxygenases AaeUPO, MroUPO, rCciUPO and rNOVO catalyze the stepwise hydroxylation of cyclohexane to cyclohexanol and cyclohexanone. The catalytic efficiencies (kcat/Km) for the initial hydroxylation were in the same order of magnitude for all four peroxygenases (∼104 M−1 s−1), whereas they differed in the second step. The conversion of cyclohexanol by AaeUPO and rCciUPO was 1–2 orders of magnitude less efficient (∼102 M−1 s−1) than by MroUPO and rNOVO (∼104 M−1 s−1). The highest conversion rate in terms of H2O2 utilization was accomplished by MroUPO under repeated addition of the peroxide (87% in relation to the total products formed). Using the latter UPO, we successfully established a micro-mixing reaction device (SIMM-V2) for the oxidation of cyclohexane. As cyclohexanone is a chemical of high relevance, for example, as starting material for polymer syntheses or as organic solvent, new enzymatic production pathways for this compound are of interest to complement existing chemical and biotechnological approaches. Stable and versatile peroxygenases, as those presented here, may form a promising biocatalytic platform for the development of such enzyme-based processes.…