tina sui
(Tina Sui)
#1
1991). The organic and aqueous excess phases contained only the hydrolysis reaction
products, monoglyceride and fatty acid soaps, respectively (Sonesson and Holmberg,
1991). The rate of reaction was slightly slower than hydrolysis in the AOT system
(Sonesson and Holmberg, 1991). The drawbacks of this approach were the high ionic
strength of the aqueous phases (1.5 M) and the low substrate loading (Sonesson and
Holmberg, 1991). When the substrate (trimyristin) concentration in isooctane was
greater than 0.07 (w/w), the microemulsion system transformed into a Winsor II
system, i.e., consisting of a w/o-ME phase in equilibrium with an excess aqueous
phase (Sonesson and Holmberg, 1991).
With regard to solvent types, hydrocarbon solvents that can penetrate the surfac-
tant layer have been demonstrated to yield the highest rate of enzymatic reaction.
Typically, isooctane, and C 6 –C 8 alkanes perform optimally (Han and Rhee, 1985b;
Hayes and Gulari, 1990). Esterification has also been catalyzed by lipase encapsu-
lated in AOT/near-critical liquid propane w/o-MEs (Murakata et al., 1996).
3.2.5 Stability and conformation of encapsulated lipases
As mentioned above, lipases encapsulated in AOT w/o-MEs can rapidly inactivate,
with strong activity loss occurring almost immediately (Han and Rhee, 1985b; Hayes
and Gulari). However, the activity loss is greatly reduced whenwois small (Fletcher
and Robinson, 1985; Han and Rhee, 1985b; Hayes and Gulari, 1990; Shiomori et al.,
1996) and when fatty acyl substrate is present (Hayes and Gulari, 1990; Rao et al.,
1991; Patel et al., 1995). The improvement of stability with lowwoalso holds true for
CTAB (Valis et al., 1992). Perhaps the lower interfacial curvature at smallworeduces
enzyme–surfactant interaction. It is believed that fatty acyl groups lessen inactiva-
tion due to the conformational changes in lipase upon formation of an acyl–enzyme
intermediate. In addition, it has been demonstrated that short-chain alcohols (1-bu-
tanol, glycerol) improve the activity and stability of encapsulated lipases (Hayes and
Gulari, 1990; 1994). Hayes and Gulari speculate that the improvement occurs be-
cause of the reduction of interfacial tension and disruption of surfactant packing.
Both events would reduce enzyme adsorption at the interface (Hayes and Gulari,
1994). In agreement, the presence of long-chain co-surfactant, known to promote
ordered surfactant packing, reduced enzyme stability (Hayes and Gulari, 1994).
Hayes and Gulari designed a series of experiments that allowed determination of
lipase stability in the presence of both fatty acid and glycerol. Their results demonsts-
rated that the encapsulated lipase possessed remarkable stability (Figure 4). When
tetradecane, which promotes similar structural changes as 1-butanol, was employed
as bulk solvent, lipase possessed a half-life of ca. 3 weeks (Figure 4). [In agreement,
Freedman and co-workers also reported enhanced stability for w/o-ME-encapsulated
enzymes in long-chain oils (Skrika-Alexopoulos et al., 1987).] Furthermore, it ap-
pears that solubilization of lipase by a liquid–liquid extraction process (Section 3.3)
yields a more stable lipase than that encapsulated by the injection of aqueous lipase
buffer in organic media (Prazeres et al., 1992).
The loss of activity retention in AOT systems coincides with changes in the sec-
ondary structure of encapsulated lipases, as observed using circular dichroism and
56 3 Lipid Modification in Water-in-Oil Microemulsions
