Food Lipids: Chemistry, Nutrition, and Biotechnology

of the enzyme. Animal and plant lipases are usually less thermostable than extracellular
microbial lipases (99). In a solvent-free system, the temperature must be
high enough to keep the substrate in the liquid state (84,102). Temperatures do not
need to be as high in systems containing organic solvents since they easily solubilize
hydrophobic substrates. However, for food industry applications, where organic solvents
are avoided, the reaction temperatures are usually higher. Sometimes the temperature
has to be increased to as high as 60�C to liquify the substrate. Such high
temperatures can seriously reduce the half-life the lipase, although immobilization
has been found to improve the stability of lipases under high temperature conditions.
Immobilization fixes the enzyme in one conformation, which reduces the susceptibility
of the enzyme to denaturation by heat. The optimal temperature for most
immobilized lipases falls within the range of 30–62�C, whereas it tends to be slightly
lower for free lipases (50). Immobilized lipases are more stable to thermal deactivation
because immobilization restricts movement and can reduce the degree of
unfolding and denaturation. Hansen and Eigtved (103) found that even at a temperature
of 60�C, immobilized lipase from Mucor miehei has a half-life of 1600 hours.
C. Water Content and Water Activity
The activity of lipases at different water contents or water activity is dependent on
the source of the enzyme. Lipases from molds seem to be more tolerant to low water
activity than bacterial lipases. The optimal water content for interesterification by
different lipases ranges from 0.04% to 11% (w/v), although most reactions require
water contents of less than 1% for effective interesterification (15,50,104). The water
content in a reaction system is the determining factor as to whether the reaction
equilibrium will be toward hydrolysis or ester synthesis. Ester synthesis depends on
low water activity. Too low a water activity prevents all reactions from occurring
because lipases need a certain amount of water to remain hydrated, which is essential
for enzymatic activity (34,105). As stated previously, lipases tend to retain the
greatest degree of original activity when immobilized on hydrophobic supports.
When the immobilized lipase is contacted with an oil-in-water emulsion, the oil phase
tends to associate with and permeate the hydrophobic support, so that there is no
aqueous shell surrounding the enzyme and support. It can be assumed that there is
an ordered hydrophobic network of lipid molecules surrounding the support. Any
water that reaches the enzyme for participation in hydrolysis and interesterification
reactions must diffuse there from the bulk emulsion phase. Therefore, to avoid diffusional
limitations, the oil phase must be well saturated with water (50). Too much
water can inhibit interesterification, probably due to decreased access of hydrophobic
substrates to the immobilized enzyme. Abraham et al. (106) found that in a solventfree
system, interesterification dominated hydrolysis up to a water-to-lipase ratio of
0.9, after which hydrolysis became the predominant reaction. During interesterification,
the reaction equilibrium can be forced away from ester synthesis due to
accumulation of water, 1 mol of which is produced for every mole of ester synthesized
during the reaction. The equilibrium can be pushed back toward ester synthesis
by continuous removal of water produced during the reaction. Water activity can be
kept constant by having a reaction vessel with a saturated salt solution in contact
with the reaction mixture via the gas phase in order to continuously remove the
water produced in the course of interesterification. Another method of water activity
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