Toward Kilogram-Scale Peroxygenase-Catalyzed Oxyfunctionalization of Cyclohexane

Abstract

Mol-scale oxyfunctionalization of cyclohexane to cyclohexanol/cyclohexanone (KA-oil) using an unspecific peroxygenase is reported. Using AaeUPO from Agrocybe aegerita and simple H₂O₂ as an oxidant, cyclohexanol concentrations of more than 300 mM (>60% yield) at attractive productivities (157 mM h^-1, approx. 15 g L^-1 h^-1) were achieved. Current limitations of the proposed biooxidation system have been identified paving the way for future improvements and implementation.

Scheme 1. Oxidation of Cyclohexane

Outlined are the established aerobic oxidation procedures (upper) and the proposed biocatalytic alternative (lower).

Figure 1

35 L-reactor used for the reaction on 10 L scale. (a) Process flow diagram (T1: temperature sensor, pH 01: pH sensor and display, P1: pump, HT in/out: hot water in/out, R-01: reactor). (b) Photograph of the reactor setup. (c) Photograph of the pump setup.

Figure 2

rAaeUPO-catalyzed oxidation of cyclohexane on a 10.9 L scale. Two individual batches are shown (black, red). (black circle, red circle): total product formed; (black triangle, red triangle): H₂O₂ concentration. Reaction conditions: 500 mM substrate (starting) concentration, 50 vol % ACN, 100 mM potassium phosphate buffer, pH 6.0, 20 μM AaeUPO (concentrated supernatant), H₂O₂ dosing rate: 120 mL h^–1 from 12.75 M stock, (pump flow rate was 2 mL per min). The reaction was stirred at 225 rpm at 25 °C. The product concentration was calculated from calibration curves. GC analysis on achiral column (CP-Wax 52GB).