butanol (Hayes and Marchio, 1998). The exclusion of water is likely to be a consequence
of the increase in interfacial curvature. Moreover, W 0 values lower than 10 improved
protein expulsion whereas above 10, the W 0 parameter did not influence the process.
Alcohols may also improve the enzyme’s stability. According to Hayes and Gulari,
(1994), their effect on stability is due to the strengthening of intermicellar attractive
interactions (sometimes with clusters formation) and to a change in interface flexibility
that hinders the enzyme-surfactant interactions (especially important when anionic
denaturing surfactants are present).
Schübel and Ilgenfritz (1997) studied the influence of oligo and polyethylene glycols
dissolved in AOT reversed micelles verifying a strong influence on the phase boundaries
and on percolation of the system. The objective of the authors was the replacement of a
portion of water by macromolecules, and they used PEG as a model polymer. In the
stabilisation field this kind of work to surpass the unfolding process through the
modification of the water pools microenvironment is welcomed.
Sorbitol has been reported to be a stabiliser of microencapsulated enzymes such as (β-
D-fructofuranosidase (Subramani et al., 1996), whereas glycerol has little effect. Glycerol
is a water-miscible cosolvent that improves the aqueous ordering and the rigidification of
proteins which enhances its stability. Rariy et al. (1998), verified this effect with (α-
chymotrypsin and Shah et al. (1997) with α-amylase.
The addition of ethylene glycol and lauric acid on the behavior of AOT reversed
micelles was investigated by Hayes and Gulari (1995). The former compound increased
the solubilisation of water, whereas the later also improved the water solubilisation
associated with ethylene glycol.
The effect of poly(oxyethylene) (POE) was analysed separately in reversed micelles of
AOT and in reversed micelles of C 12 E 5 (Meir, 1996). In AOT reversed micelles, the
attractive interactions between polymer and surfactant lead to the adsorption of the
polymer at the interface, whereas in the case of the non-ionic surfactant the polymer
molecules are repelled from the interface and forced into the droplet interior. In AOT the
preferential location of POE in the interface may lead to the interconnection of droplets to
form clusters which is supported by conductivity measurements (Meir, 1996).
Some substrates also induce stabilisation of enzymes. This is the case with acyl
substrates (palmitic and oleic acids) which raise the stability of Candida cylindracea
lipase in reversed micelles (Ayyagari and John, 1995).
Organic solventThe choice of solvent is normally determined by its compatibility with the surfactant and
biocatalyst. Several correlations have been established between solvents and their
properties affecting the enzymes’ behavior and stability. Laane et al. (1987) related the
stability of biocatalysts with the logarithm of partition coefficient of the organic solvent
in the system octanol/water (log P), where solvents with high log P were usually less
detrimental to enzyme stability.
Gupte et al. (1995a) proposed a more detailed approach to the use of log P in reverse
micelles. The author uses the concept of log Pb, which refers to the log P of the
microenvironment of the biocatalyst. Log Pb depends on log P of surfactant, on the log P
of cosurfactant and on the molar ratio of the co-surfactant to the surfactant. This approach
Reversed micellar bioreaction systems 201