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A strategy for the enantioselective synthesis of chiral short chain alcohols using biocatalysis is developed employing substrate and enzyme dependent in situ cofactor regeneration. Enzyme characterization shows strong dependence of activity on diverse reaction parameters. The obtained results are transferred to one-phase batch synthesis of®- and (S)-2-butanol where conversion and enantioselectivity are strongly dependend on reaction conditions. Due to limitations like low substrate solubility the synthesis is transferred to two-phase reaction systems where MTBE is the solvent of choice. Conversion and selectivity are positively influenced. The same is found for two-phase reactions in a continuous reaction set-up. With the optimum reaction conditions obtained from the batch experiments conversion and selectivity are improved. ADH and cofactor show exceptionally high stability and high TTN. Together with further development of a work-up strategy the continuous two-phase reaction set-up will be a strong tool to produce enantiopure short chain alcohols on preparative relevant scale.
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