Recently, a model has been proposed to explain the distribution of hexanol in reversed
micelles of AOT/isooctane (Carvalho et al., 2000a). The distribution concentration of
alcohols is of great importance in the micellar systems due to their properties as co-
surfactants. Besides these properties, alcohols also act as substrates and stabilising agents,
which give them further interest to model.
Cutinase is a suitable enzyme for examination of this issue, since it establishes a
bridge between esterases and Lipases (Carvalho et al., 1999c; Martinez et al., 1992). Its
activity and stability strongly depends on the presence of hexanol which plays a multiple
role as substrate, as a co-surfactant and greatly improves cutinase stability (Carvalho et
al, 1997, 1999a; Sebastião et al., 1993).
The non-homogeneity of microemulsions reflects on the analysis of kinetics
performed, since the quantities of substrate accessed by the biocatalyst were not in
straightforward relation to the substrate amount added to the reaction medium.
Experimentally it was observed that the addition of hexanol up to 400–450 mM led to
reaction velocities that do not follow the Michaelis-Menten kinetics. The deviation was
overcome by calculating an effectiveness factor (η), which was related to overall
substrate concentration. Moreover, with low hexanol concentrations an analogous effect
of substrate “deficiency” was observed while performing the thermostability assays, in
spite of the fact that there was enough time for diffusion to occur, as hexanol stays in the
system some hours or days before the activity evaluation. From the aforementioned
results, it can be inferred that the lowest hexanol concentrations are comparatively less
effective than would be expected. This suggests the substrate does not distribute equally
in all the micellar components.
The experimental results discussed above were the start-point in developing a model
to explain both kinetics and stability. This model encompasses three main processes:
adsorption, partition and diffusion, and their relative importance is analysed.
In general, alcohols may distribute among the three main components of the micellar
system, which are: the organic bulk phase (Sorg), the aqueous internal solution (Sw) and
the interface with the surfactant molecules (Saot,f and Saot,ads).
The overall substrate concentration (ST) is given by
(1)The substrate concentrations are indicated as S* as they refer to the overall concentration
in the reversed micellar solution.
represents the overall substrate concentration related to the AOT,
either adsorbed (Saot,ads) and consequently unavailable to participate in the reaction or in
the free form (Saot,f).
Henceforth,
(2)
The effective substrate concentration (S*ef), which is available to reach the enzyme
microenvironment, is given by:
(3)
Multiphase bioreactor design 204