Enhancing the Peroxygenase Activity of a Cofactor‐Independent Peroxyzyme by Directed Evolution Enabling Gram‐Scale Epoxide Synthesis

Peroxygenases selectively incorporate oxygen into organic molecules making use of the environmentally friendly oxidant H2O2 with water being the sole by-product. These biocatalysts can provide ‘green’ routes for the synthesis of enantioenriched epoxides, which are fundamental intermediates in the production of pharmaceuticals. The peroxyzyme 4‑oxalocrotonate tautomerase (4‑OT), catalysing the epoxidation of a variety of α,β-unsaturated aldehydes with H2O2, is outstanding because of its independence from any cost-intensive cofactor. However, its low-level peroxygenase activity and the decrease in the enantiomeric excess of the corresponding α,β-epoxy-aldehydes under preparative-scale conditions is limiting the potential of 4‑OT. Herein we report the directed evolution of a tandem-fused 4‑OT variant, which showed an ~150-fold enhanced peroxygenase activity compared to 4-OT wild type, enabling the synthesis of α,β-epoxy-aldehydes in milligram- and gram-scale with high enantiopurity (up to 98% ee) and excellent conversions. This engineered cofactor-independent peroxyzyme can provide new opportunities for the eco-friendly and practical synthesis of enantioenriched epoxides at large scale.