Food Lipids: Chemistry, Nutrition, and Biotechnology

imization of interesterification, acyl migration is usually minimized simply by reducing
reaction times. Lipases can be made more active and soluble in organic
solvent systems by attachment of an amphiphilic group such as polyethylene glycol
(PEG). PEG reacts with the N-terminal or lysine amino groups, rendering the lipase
more soluble in organic solvents (69). The activity of lipases in organic solvent
depends on the solubility of the solvent in water. Lipases are only active in waterimmiscible
solvents, since water-miscible organic solvents extract the water of hydration
layer from the vicinity of the enzyme, thereby inactivating them (44). Since
the success of an interesterification reaction depends on the concentration of water
in the system, the hydration state of the lipase plays a key role because a minimal
amount of water is needed to maintain the enzyme in its active form. The use of
hydrophobic solvents limits the flexibility of the enzyme, preventing it from assuming
its most active conformation. Therefore, if organic solvents are used, the enzyme
must be in its active conformation prior to addition of the organic solvent. This can
be accomplished by exposing the enzyme to an inhibitor or substrate, then drying it
in its active conformation (12,70). The advantage of using organic solvents in lipasecatalyzed
interesterification reactions is that the water content can be carefully controlled.
A water content higher than 1% can produce high degrees of hydrolysis,
whereas water levels lower than 0.01% can prevent full hydration of the lipase and
reduce the initial rate of hydrolysis (1). Therefore, water levels between these two
extremes are necessary to maximize the effectiveness of enzymatic interesterification
in organic solvents. In microaqueous organic solvent systems, the effect of pH on
lipase activity is complex because water levels are so low. It has been proven that
enzymes in organic solvent systems have a memory of the pH of the last aqueous
environment in which they were. Elliott and Parkin (65) found that porcine pancreatic
lipase has an optimum activity in hexane after being exposed to pH values between
6.5 and 7.0. At pH 8.5, the decrease in activity was attributed to a change in the
ionization state of the histidine in the active site.
A common form of organic solvent system used in lipase-catalyzed interesterification
is that of reverse micelles. Reverse micelles, or micoremulsions, are defined
as nanometer-sized water droplets dispersed in organic media with surfactants stabilizing
the interface (29,71). A common surfactant used is an anionic double-tailed
surfactant called sodium-bis(2-ethylhexyl)sulfosuccinate (AOT). Reverse micelles
are used in interesterification reactions because they increase the interfacial area and
improve the interaction between lipase substrate (29). As well, the use of microemulsions
makes it possible to use polar and nonpolar reagents in the same reaction
mixture (72). Reverse micelles can be formed by gently agitating a mixture of AOT,
lipid substrate, organic solvent, and lipase dissolved in buffer until the solution becomes
clear. The lipase is trapped in an aqueous medium in the core of the micelle,
avoiding direct contact with the organic medium (61). Lecithin has been used to
promote the formation of reverse micelles and to protect the lipase from nonpolar
solvents (73,74). At ionic strengths higher than 1 M, activity is decreased due to
decreased solubility and activity of the lipase. The water content required for microemulsion
systems is dependent on the desired reaction, although some level of
water is necessary to hydrate the enzyme. For example, Holmberg et al. (75) found
that 0.5% water was the optimum for production of monoacylglycerols from palm
oil in a microemulsion. The composition of the substrate can also affect the rate of
interesterification in reverse micelles. Substrates with more amphiphilic properties
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