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.
