Control of subtilisin substrate specificity by solvent engineering in organic solvents and supercritical fluoroform

We have investigated the effect of physical properties of 30 nonaqueous solvents on the specificity of Subtilisin Carlsberg for nucleophiles in the transesterification of N-acetyl-L-phenylalanine ethyl ester by methanol, 1-propanol, and 1-butanol at fixed thermodynamic water activity. in organic solvents, enzyme activity and nucleophile specificity are solvent-dependent, while in supercritical fluoroform, the activity and specificity are pressure-dependent. Losses in catalytic efficiency and substrate specificity are observed when subtilisin is exposed to hydrophilic organic solvents such as dioxane, tetrahydrofuran, and acetonitrile as compared to hydrophobic solvents (hexane and heptane). Log P is an important descriptor for correlating both the rate and the specificity of deacylation with solvent properties. A linear model of log initial rate against both log P and nonpolar unsaturated area provides the best two-variable fit to the data for solvents of high log P. A nonlinear model of specificity against log P provides the best fit for the complete data set. Correcting the activity for partitioning of nucleophilic substrates shows a similar trend for the intrinsic activity dependence of nucleophiles as a function of log P. In propane, under subcritical conditions, there is no significant effect of pressure on either the activity or the nucleophilic specificity of subtilisin In fluoroform, however, where the physical properties of the solvent are pressure-dependent, the specificity of the enzyme is solvent density-dependent.