Luisi, 1989; 1990; O’Connor and Cleverly, 1994), NIR (Hanna et al., 1996), and FT-
(^1) H-NMR (Rees and Robinson, 1995) have been developed. Furthermore, Walde has
developed an assay to measure the formation of free fatty acids (FFA) during lipo-
lysis by encapsulating the fluorescent dye phenol red in the w/o-MEs (Walde, 1990).
The dye’s absorbance spectrum undergoes a 100 nm blue shift upon binding with
FFA. Alternatively, the release of FFA was determinedin situthrough a lipase-lip-
oxygenase coupled reaction scheme, yielding hydroperoxides, which were detected
spectrophotometrically (250 nm) (Hochkoeppler and Palmieri, 1990). Microemul-
sion-encapsulated lipases have been used to screen for lipase type in their activity
toward unusual free fatty acids (O ̈sterberg et al., 1989; Derksen and Cuperus, 1992;
Hayes and Kleiman, 1993a; 1993b).
3.2.2 Substrate partitioning controls the kinetics
There is sufficient circumstantial evidence that indicates that lipases catalyze reac-
tions at the interface. In other words, the partitioning of substrates and products
between the interface, the bulk solvent, and the dispersed aqueous phase plays a
strong role in controlling the observed kinetics. For instance, w/o-ME-encapsulated
lipase hydrolyzed the wax ester 1-butyl laurate to a small extent, but esterified 1-
butanol and lauric acid strongly in the forward direction (Figure 2) (Hayes, 1991).
These results reflect the greater degree of interfacial acticity of lauric acid and 1-
butanol relative to butyl laurate, demonstrated by dynamic light scattering data re-
sults (w/o-ME size and attractive interaction parameters) (Hayes, 1991). Note the
agreement of equilibrium concentrations from hydrolysis and esterification reac-
3.2 Lipases encapsulated in water-in-oil microemulsions 49
Figure 2. Synthesis (smid symbols) and hydrolysis (open symbols) of 1-butyl laurate yield the same
equilibrium position, independent of lipase type. 100 mM AOT and 970 mM water in isooctane (i.e.,w 0 =
9.7), 23 8 C; all substrates present at 100 mM.Candida rugosa(,) andRhizopus delemar(&,&) lipase at
micro-aqueous concentration of 2.0 and 1.0 mg mL, respectively. One lipase Unit is defined as the amount
to produce 1 mM of ester per hour atw 0 = 8.0. (Reproduced with permission from Hayes, 1991).