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.
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