Food Lipids: Chemistry, Nutrition, and Biotechnology

of the interface as a measure of substrate concentration was proven by Bezonana
and Desnuelle (49), who measured the rates of hydrolysis in coarse and fine emulsions
(Fig. 10). When the rate of the reaction for the two emulsions was plotted as
a function of substrate mass, the same maximal rates of hydrolysis were obtained;
however, there was a difference in the values for K m. In contrast, when initial velocities
were plotted as a function of interfacial area, the values of K m and V max were
constant for both fine and coarse emulsions, indicating that the concentration of
substrate at the interface (mol/m 3 ) directly determines the rate of the reaction
(11,12,46,50). Reaction rates have also been shown to be a function of the emulsion
concentration. If a stock emulsion is diluted to different concentrations, a progress
curve of rate versus concentration will be obtained, which upon plotting gives a
straight line in the Lineweaver–Burke plot. A relative K m can be obtained from this
plot, but in order to obtain the absolute value for K m, the area of the interface must
be known (11). It is very difficult to obtain an accurate assessment of the interfacial
area due to several factors. In free enzyme solutions, the size distribution of emulsion
droplets and the degree of adsorption of enzyme to the interface must be known. It
is difficult to estimate the surface area of the interface due to size heterogeneity and
the possibility of coalescence of emulsion droplets. With immobilized enzyme, the
size distribution and surface area of support particles and pores must be determined,
as well as the degree of loading of the lipase (47). Due to the difficulty in measuring
these factors accurately, only relative K m values are determined.
E. Kinetics and Mechanism of Action
Interesterification is a multisubstrate reaction, with the main substrates being glycerides,
fatty acids, and water. This reaction can be considered a special case of
chemical group transfer, involving sequential hydrolysis and esterification reactions
(51). Lipase-catalyzed interesterification follows a Ping-Pong Bi-Bi reaction for multisubstrate
reactions (50,51). The actual mechanism of acylation and deacylation of
Figure 10 Lineweaver–Burk plot of lipase activity as a function of (a) mass of substrate
at the interface and (b) area occupied by substrate at the interface. The comparison was made
with assays containing a coarse emulsion (�) and a fine emulsion (�). (Adapted from Ref.
49.)
Copyright 2002 by Marcel Dekker, Inc. All Rights Reserved.