Influence of water-miscible organic solvents on kinetics and enantioselectivity of the (R)-specific alcohol dehydrogenase from Lactobacillus brevis.

Using the organic solvents acetonitrile and 1,4-dioxane as water-miscible additives for the alcohol dehydrogenase (ADH)-catalyzed reduction of butan-2-one, we investigated the influence of the solvents on enzyme reaction behavior and enantioselectivity. The NADP(+)-dependent (R)-selective ADH from Lactobacillus brevis (ADH-LB) was chosen as biocatalyst. For cofactor regeneration, the substrate-coupled approach using propan-2-ol as co-substrate was applied. Acetonitrile and 1,4-dioxane were tested from mole fraction 0.015 up to 0.1. Initial rate experiments revealed a complex kinetic behavior with enzyme activation caused by the substrate butan-2-one, and increasing K(M) values with increasing solvent concentration. Furthermore, these experiments showed an enhancement of the enantioselectivity for (R)-butan-2-ol from 37% enantiomeric excess (ee) in pure phosphate buffer up to 43% ee in the presence of 0.1 mol fraction acetonitrile. Finally, the influence of the co-solvents on water activity of the reaction mixture and on enzyme stability was investigated.