Peroxygenases require a controlled supply of H2O2 to operate efficiently. Here, we propose a photocatalytic system for the reductive activation of ambient O2 to produce H2O2 which uses the energy provided by visible light more efficiently based on the combination of wavelength-complementary photosensitizers. This approach was coupled to an enzymatic system to make formate available as a sacrificial electron donor. The scope and current limitations of this approach are reported and discussed.

An increasing number of biocatalytic oxidation reactions rely on H 2 O 2 as a clean oxidant. The poor robustness of most enzymes towards H 2 O 2 , however, necessitates more efficient systems for in situ H 2 O 2 generation. In analogy to the well‐known formate dehydrogenase to promote NADH‐dependent reactions, we here propose employing formate oxidase (FOx) to promote H 2 O 2 ‐dependent enzymatic oxidation reactions. Even under non‐optimised conditions, high turnover numbers for coupled FOx/peroxygenase catalysis were achieved.

The pilot-scale production of the peroxygenase from Agrocybe aegerita (r Aae UPO) is demonstrated. In a fed-batch fermentation of the recombinant Pichia pastoris, the enzyme was secreted into the culture medium to a final concentration of 0.29 g L –1 corresponding to 735 g of the peroxygenase in 2500 L of the fermentation broth after 6 days. Due to nonoptimized downstream processing, only 170 g of the enzyme has been isolated. The preparative usefulness of the so-obtained enzyme preparation has been demonstrated at a semipreparative scale (100 mL) as an example of the stereoselective hydroxylation of ethyl benzene. Using an adjusted H 2 O 2 feed rate, linear product formation was observed for 7 days, producing more than 5 g L –1 ( R )-1-phenyl ethanol. The biocatalyst performed more than 340.000 catalytic turnovers (942 g of the product per gram of r Aae UPO).

Natural deep eutectic solvents (NADES) are proposed as alternative solvents for peroxygenase‐catalysed oxyfunctionalization reactions. Choline chloride‐based NADES are of particular interest as they can serve as solvent, enzyme‐stabiliser and sacrificial electron donor for the in situ H2O2 generation. This report provides the first proof‐of‐concept and basic characterisation of this new reaction system. Highly promising turnover numbers for the biocatalysts of up to 200,000 have been achieved.

The use of natural deep eutectic solvents (NADES) as multifunctional solvents for limonene bioprocessing was reported. NADES were used for the extraction of limonene from orange peel wastes, as solvent for the chemoenzymatic epoxidation of limonene, and as sacrificial electron donor for the in situ generation of H2O2 to promote the epoxidation reaction. The proof‐of‐concept for this multifunctional use was provided, and the scope and current limitations of the concept were outlined.