Figure 7.1 Effect of adding increasing
amounts of water to form AOT
reversed micelles. Water molecules
may be classified in three states:
immobilised ( ), hydration ( ), or
free ( ) (adapted from Goto et al.,
1995).
Moreover, the microviscosity of the micellar water is higher than that of the bulk
solutions (Andrade and Costa, 1996; Hasegawa and Kitahara, 1994), especially at low W 0
values. In these conditions the activity is usually improved, possibly due to the
elimination of spontaneous fluctuations of protein structure, which disturb the catalytic
conformation in aqueous solution (Martinek et al., 1989).
Another decisive factor in choosing the Wo value is the type of catalysed reaction.
Hydrolytic or synthetic reactions have different water requirements and this also accounts
for the overall reversed micellar catalytic system performance. By medium engineering it
is possible to design systems with optimal enzyme activity, without detrimental effects on
stability.
The thermal unfolding of small globular proteins was measured by Luisi et al. (1990)
using differential scanning calorimetry (DSC), in the AOT/isooctane reversed micellar
system. The authors verified that the thermal stability of the proteins hosted in reversed
micelles depends on the water content, decreasing when the Wo value increases. The
lowest Wo value used was 11, due to the limits of technique application, but it is possible
that the use of lower values enhances the stability even more. Barbaric and Luisi (1981),
studied enzymes in lower water content micelles (0.6–1% water) and verified a stability
greater than in aqueous solutions.
Reversed micelles are capable of rearranging to accommodate a protein with
dimensions higher than the water pool. Experimental evidence has shown that in some
conditions 3 water-filled, but protein-free droplets, may be necessary to accommodate
one molecule of protein (Gupte et al., 1995a).
Since the use of very low W 0 values is possible and this minimises the water
molecules, which promote unfolding, the W 0 is an important variable to take into account
in stabilisation strategies.
Furthermore, substrate solubility in the aqueous sub-phase may also influence the
choice of hydration level. The substrate concentration in the enzyme microenvironment is
greatly influenced by this factor and for hydrophilic substrates it may be convenient to
use higher W 0 values. Additionally, there might be other variables such as the catalyst
stability, which may influence the choice of W 0.
It should be emphasised that the W 0 concept is usually associated only with the
hydration level, although it is dependent on another variable—the concentration of
surfactant. To establish their dependence it is very important to define the phase diagrams
of each system by relating the phases with the concentrations of solvent, water and
surfactant.
When the surfactant interacts with the enzyme accelerating the denaturing process,
which often succeeds with anionic surfactants, e.g. SDS, AOT, the water content and
Reversed micellar bioreaction systems 197