Food Lipids: Chemistry, Nutrition, and Biotechnology

the glyceride in the active site is shown in Figure 11. During acylation, a covalent
acyl–enzyme complex is formed by nucleophilic attack of the active site serine on
the carbonyl carbon of the substrate. The serine is made a stronger nucleophile by
the presence of histidine and aspartic acid residues. The histidine imidazole ring
becomes protonated and positively charged, stabilized by the negative charge of the
active site aspartic acid or glutamic acid residues. A tetrahedral intermediate is subsequently
formed, stabilized by two hydrogen bonds formed with oxyanion-stabilizing
residues (12). A break in the carbon–oxygen bond of the ester causes release of
the alcohol. During the reaction, the acylglycerol is associated with the catalytic triad
through covalent bonds. Histidine hydrogen bonds with both serine and the oxygen
of the leaving alcohol. Nucleophilic attack by water or an alcohol causes the addition
of a hydroxyl group to the carbonyl carbon, producing a tetrahedral intermediate,
which will rearrange, releasing the altered acylglycerol and regenerating the active
site serine (42,52).
The first stage of interesterification involves hydrolysis of triacylglycerols with
consumption of water to produce diacylglycerols, monoacylglycerols, and free fatty
acids. Accumulation of hydrolysis products will continue during interesterification
until an equilibrium is established (51). Since lipases are involved in multisubstrate,
multiproduct reactions, more complex kinetic mechanisms are required. Interesterification
involves acylation and deacylation reactions, either of which can be the ratelimiting
step (50,53). The basic mechanism for a Ping-Pong Bi-Bi reaction using
multiple substrates is shown in Figure 12.
Under steady-state conditions,
v [AX][BX]
=
Vmax Km BX[AX] � Km AX[B]
� [A][B]
where AX is the first substrate and BX is the second substrate (41). It is difficult to
study the kinetics of Ping-Pong Bi-Bi mechanisms due to the presence of two substrates.
In order to study the kinetics, one substrate concentration is usually held
constant while the other one is altered. In the case of lipase-catalyzed interesterification
under aqueous conditions, there is the additional difficulty that the lipid substrate
is also the reaction medium, which is in excess compared with other components.
Even with measurable amounts of lipid substrate, it is difficult to develop rate
equations since all species involved have to considered (50).
F. Specificity
The main advantage of lipases that differentiates enzymatic interesterification from
chemical interesterification is their specificity. The fatty acid specificity of lipases
has been exploited to produce structured lipids for medical foods and to enrich lipids
with specific fatty acids to improve the nutritional properties of fats and oils. There
are three main types of lipase specificity: positional, substrate, and stereo. Positional
and fatty acid specificity are usually determined by partial hydrolysis of synthetic
triacylglycerols and separation by thin-layer chromatography with subsequent extraction
and analysis of the products. Other methods include conversion of the fatty
acids produced during hydrolysis to methyl esters for gas chromatographic analysis
(54).
Copyright 2002 by Marcel Dekker, Inc. All Rights Reserved.