Highly productive biocatalytic reductions were established using an isolated alcohol dehydrogenase (ADH) under water-deficient conditions. First, a solvent-free system was evaluated for the reduction of 2-butanone catalyzed by ADH evo-1.1.200 promoted by the “smart cosubstrate” 1,4-butanediol. ADH evo-1.1.200 excelled by its activity and stability under high reagent concentrations and hence was the enzyme of choice. However, conversion of 2-butanone was limited to <1 % in 10 days under the solvent-free conditions. Therefore, water-immiscible organic solvents were evaluated whereby the highest conversions were achieved in MTBE and toluene. MTBE was chosen as its different boiling point compared to other reaction components (e.g., 2-butanone, 2-butanol, diol cosubstrate, and lactone coproduct) would simplify the downstream processing. Further on, by tuning substrate loading, the productivity of the ADH evo-1.1.200 was successfully increased to a turnover number (TON) of 64 000.
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