Monohydroxylated metabolites of vitamin D₃ (cholecalciferol) and vitamin D₂ (ergocalciferol), generically known as 25-hydroxycalciferol, are better for several diseases, and other applications, than vitamin D (calciferol). This work describes a novel biotechnological approach for the preparation of 25-hydroxycalciferols, starting from readily available cholecalciferol and ergocalciferol. This approach enables the regioselective (100 %) hydroxylation of these compounds (at the C-25 position) under mild and environmentally friendly conditions by using a peroxidase from the fungus Coprinopsis cinerea (gene model CC1G_08427T0 from the sequenced genome), which catalyzes monooxygenation with H₂O₂ as the only co-substrate (peroxygenase). Hydroxylation of cholecalciferol and ergocalciferol is a true peroxygenation, as demonstrated by incorporation of ¹⁸O from H₂¹⁸O₂ into the products. The peroxygenase has additional advantages related to its recombinant nature, enabling enzyme engineering and low-cost overexpression in an industrial host. Therefore, the peroxygenase is a promising biocatalyst for the production of vitamin D active metabolites.

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