surfactant concentration have to be considered as independent variables, since the same
Wo value can be obtained with different combinations of the two factors. Nevertheless,
the restrictions in water content below certain levels dominate the interactions protecting
the enzyme against denaturation. The lack of mobility may in this way be favorable.
The surfactant concentration also acts upon substrate distribution and catalysis itself.
A model developed alongside the experimental work considers that a part of the overall
substrate remains in close contact with the surfactant in the micellar sub-phase due to an
adsorption effect. The increase in surfactant concentration will decrease the substrate
accessed by the enzyme, explaining the reduction of kcat by 2 to 4-fold in reversed
micellar systems. Some changes in substrate specificity corroborate the partitioning
explanation as the more hydrophilic substrates become preferred in reversed micellar
catalysis. Brown et al. (1993) detected a decrease on Rhizopus arrhizus activity with an
increase in AOT concentration that reflected the values of kcat. Other authors explain the
same phenomenon assuming a non-competitive inhibition by AOT (Marangoni, 1993;
Tsai et al., 1995).
Water activityAnother way to express the hydration level of reversed micelles is the use of the
thermodynamic activity concept. Although the multiphase systems can be analysed in
terms of concentrations, the use of water activity (aw) offers advantages (Hailing, 1994).
In general, the reversed micellar systems are prepared with small amounts of water
(although sufficient to attain the aw value of 1) as a low water content does not warrant a
low aw. However, at very small Wo values aw tends to decrease sharply.
Among the components of the reversed micellar system that can compete for the water
are the organic solvent, protein, surfactant, substrates/products, buffer salts and co-
surfactants such as alcohols (when present).
Competition between these components and the biocatalyst for available water may
create “apparent” effects. For instance, high concentrations of a medium chain alcohol,
used as substrate, may reduce the aw, creating an apparent inhibitory effect if this water is
necessary as a substrate of the reaction or if it integrates the “hydration shell” of the
enzyme. The same may occur when raising the surfactant concentration.
Only very few papers (Hoppert et al., 1994; Jorba et al., 1992; Peng and Luisi, 1990;
Stamatis et al., 1995) report the control of aw in reversed micellar systems and usually the
methods used are the pre-equilibrating ones. This is probably a consequence of the
constraints of the aw determination, which exist in all systems but increase dramatically in
reversed micelles due to the system components. One of the general problems in
determining the aw that applies to all systems is the handling of samples, due to the
volatility and adsorption to materials which is even more complicated at higher
temperatures.
Diagrams demonstrating the correspondence of Wo and aw have been published by
Luisi et al. (1988) (from the work of Higuchi and Misra, 1962) and more recently by
Crooks et al. (1995). At W 0 5 the aw is 0.8 and increases to 0.9 at W 0 10. Hailing and co-
workers (unpublished data) also measured the aw in reversed micelles containing enzyme
and detected a sharp increase from Wo 0 to 10 and then a smooth evolution until aw 1.
Multiphase bioreactor design 198