Substrate Walking for Selective Ene Reduction

ABSTRACT Ene‐reductases (EREDs) efficiently reduce activated C═C bonds, but their activity toward tetra‐substituted alkenes has been largely unexplored. Here, we report the first systematic study demonstrating that several EREDs catalyze the stereoselective reduction of tri‐ and tetra‐substituted cyclohexenones while investigating the influence of different cofactors. To enable a scalable application, the best enzyme (YqjM) was co‐immobilized with glucose dehydrogenase on Ni–NTA cellulose beads, achieving >99% immobilization efficiency and high operational stability. The immobilized system was successfully scaled to 200 mL, reaching full substrate conversion without enzyme degradation over 9 days.