The use of oxidoreductases in organic-aqueous biphasic systems is advantageous (effective solvation of reactants, minimization of substrate/product-induced inhibition, improved volumetric productivity, and straightforward downstream processing). This paper explores the effects of organic solvents on horse liver alcohol dehydrogenase (HLADH) by combining experimental and computational studies. Various organic solvents displaying a broad range of hydrophobicity and functionalities are used, namely, ethyl acetate, 2-methyltetrahydrofuran, methyl tert-butyl ether, cyclopentyl methyl ether, toluene, cyclohexane, heptane, and dodecane. The catalytic performance of model enzyme horse liver alcohol dehydrogenase concerning its activity, stability, and selectivity is experimentally evaluated. The results are interpreted with molecular dynamics simulations by assessing the (i) protein location in biphasic media, (ii) organic solvent distribution, and (iii) enzyme conformation. Herein, the stability states the robustness of the enzyme while storing it in biphasic media without catalysis taking place. Overall, different toxicities of the solvent to the enzyme can be pinpointed: "molecular toxicity", related to the solvent functional groups, and "interfacial toxicity", related to the position of the enzyme at the interface. Likewise, some solvents are more prone to be located close to the active site of the enzyme, triggering other effects on the enzymatic performance. Thus, methyl tert-butyl ether resulted as an optimal option for the enzyme, whereas other solvents like toluene and 2-methyltetrahydrofuran were detrimental. The combined forces of experiments and simulations have been shown to be useful tools to study the effects of reaction media, thus guiding solvent selection.
