Enzymatic H₂O₂ for biocatalysis

Angew. Chem. Int. Ed. https://doi.org/10.1002/anie.201902380 (2019)

Several biocatalytic oxidation and oxyfunctionalization reactions rely on H₂O₂ — a clean oxidant — which causes inactivation of the enzymes over time. A strategy to overcome this incompatibility is to produce H₂O₂ in situ for its use in biocatalysis.

The commonly used biocatalytic H₂O₂ generation system is based on glucose oxidase, but its practicality is limited due to the high amount of produced waste (196 g of gluconate waste per mol H₂O₂ generated) and a high viscosity of the reaction medium at large scales.

Now, Frank Hollmann and colleagues tested a recently reported formic acid oxidase from Aspergillus oryzae (AoFOx) as an alternative enzyme for in situ generation of H₂O₂ using formate as reductant. Hereby, during the reductive activation of O₂ only 44 g of volatile CO₂ waste — which does not accumulate in the reaction medium — are produced per mol H₂O₂.

First, the authors tested AoFOx in a model oxyfunctionalization reaction — the selective hydroxylation of ethyl benzene into (R)-1-phenylethanol — catalysed by a peroxygenase from Agrocybe aegerita (AaeUPO). The authors showed that this enzymatic cascade operates optimally at pH 6 and 25 °C. A relative ratio of H₂O₂-generating and H₂O₂-consuming enzymes of 1:5 resulted in good productivities and stable product formation for at least 24 hours. Increasing the availability of O₂ was a critical factor in enabling a ten-fold increased productivity at higher O₂ pressures compared to the reaction under ambient atmosphere and without stirring.

Finally, the authors demonstrated the compatibility of the presented H₂O₂ generation system with different enzymes (cytochrome c, lipase B, chloroperoxidase) in biocatalytic oxidation reactions such as epoxidation, sulfoxidation and hydroxyhalogenation. Remarkably, the turnover numbers achieved with the AoFOx system in these H₂O₂-dependent reactions proved to be orders of magnitude higher compared to the utilization of established H₂O₂ generation systems.

Overall, the AoFOx H₂O₂ generation system is simple, practical and shows excellent performance and good compatibility in coupled H₂O₂-dependent reactions. This system can be further engineered towards a lower K_m value towards formate or a broader optimal temperature range.