Handbook of Solvents - George Wypych - ChemTech - Ventech!

14.4.3 Choice of solvent for enzymatic reaction 875
ferent solvents, maximum reaction rate was observed at widely different water content, but
if water content was qualified in terms of a w, the optimum was observed at almost the same
a w (Figure 14.4.3.1). However, as seen from Figure 14.4.3.1, the profile does not lay on a
single curve, and the absolute optimal reaction rate varies depending on the kind of the solvent,
implying that a w is not almighty.
2) Hydrophobicity (or polarity), logP
A hydrophobicity parameter, logP, was first proposed for microbial epoxidation of propene
and 1-butene. 9,10 logP is the logarithm of P, where P is defined as the partition coefficient of
a given compound in the standard n-octanol/water two phase:
solubility of a given compound in n −octanol
phase
P =
solubility of a given compound in water phase
[14.4.3.2]
Laane et al. 10 concluded as a general rule that biocatalysis in organic solvents is low in
polar solvents having a logP < 2, is moderate in solvents having a logP between 2 and 4, and
is high in apolar solvents having a logP > 4. They also stated that this correlation between
polarity and activity paralleled the ability of organic solvents to distort the essential water
layer bound to the enzyme that stabilized the enzyme. Since logP can easily be determined
experimentally, or be estimated from hydrophobic fragmental constants, many biotechnologists
have tried since then to correlate effects of organic solvents on biocatalysts they
studied with logP approach. Their results have been successful, not completely but only partially.
A number of exceptions to the ‘logP rule’ have been in fact reported.
3) Dielectric constant (or dipole moment), ε (or D)
Interactions between an enzyme and a solvent in which the enzyme is suspended are mostly
non-covalent ones as opposed to interactions in water. These strong non-covalent interactions
are essentially of electrostatic origin, and thus according to Coulomb’s law, their
strength is imposed dependent on the dielectric constant, ε, (which is higher for water than
for almost all organic solvents). It is likely that enzymes are more rigid in anhydrous solvents
of low εthan in those of high ε. Thus, εof a solvent can be used as a criterion of rigidity
of the enzyme molecule. For the enzyme to exhibit its activity, it must be dynamically flexible
during its whole catalytic action so that its activity in a solvent of lower ε should be less
than in a solvent of higher ε. On the other hand, its selectivity or specificity becomes higher
when its flexibility decreases so that the selectivity in a solvent of lower ε should be higher
than in a solvent of higher ε.
14.4.3.4 Properties of enzymes affected by organic solvents
1) Thermal stability (half-life), t1/2 Stability of an enzyme in an organic solvent is estimated by its half-life, t1/2, when its activity
is plotted as a function of incubation time. Although, t1/2 during the enzymatic reaction is
more informative for practical purposes, t1/2 under no substrate is often reported because of
its easiness of measurement. Inactivation of an enzyme is caused mostly by change in its native
conformation, or irreversible unfolding of its native structure. Water, especially enzyme-bound
water makes a major contribution to the protein folding through van der Waals
interaction, salt-bridges, hydrogen bonds, hydrophobic interaction, etc. When the enzyme
molecule is put into organic solvent, water molecules bound to the enzyme molecule are
more or less re-equilibrated, depending on the free water content. Therefore, both the nature
of organic solvent and the free water content have profound effects on its stability.