Food Lipids: Chemistry, Nutrition, and Biotechnology

is a thin layer located directly next to the surface of the support. In the case of
hydrophobic supports and hydrophobic substrates, such as triacylglycerols, the concentration
of substrates in the Nernst layer is more concentrated than in the bulk
solution since the hydrophobic substrate tends to partition toward the hydrophobic
support material. Another layer surrounding the support particles is a diffusion or
boundary layer. A concentration gradient is established between the diffusion layer
and the bulk phase as substrate is converted to product by the lipase. The product
concentration in the diffusion layer is higher than in the bulk phase as it must diffuse
from the surface of the support into the bulk phase. Consequently, due to the higher
product concentration in the diffusion layer, the substrate concentration is lower than
in the bulk phase, producing concentration gradient with more substrate diffusing
toward the support and immobilized lipase. Differences in substrate concentration
between the Nernst layer and/or the boundary layer and the bulk phase can affect
the determination of K m since substrate concentration will be measured in the bulk
layer, which may not be the concentration of substrate closer to the lipase. With a
lower substrate concentration at the support in comparison with the bulk phase, the
apparent K m will appear higher and the activity will appear lower than its actual
values. The opposite will occur with a higher substrate concentration at the interface.
A third factor that can affect the activity of immobilized lipase is electrostatic
effects. If the support and substrate possess the same charge then they will experience
repulsion, whereas if they have opposite charge they will be attracted. This factor
can have an effect on the apparent K m. As well, electrostatic effects can have an
impact on other components in the reaction. For example, if the support was anionic,
the local concentration of hydrogen ions would be higher in the vicinity of the
immobilized lipase, which would cause a decrease in the pH around the enzyme.
Combining the electrostatic effects and the effect of the Nernst layer, the value
of the apparent K m can be modified as follows (88):
� �
x RT
K� m = K m � Vmax
D RT � xzFV
where K� m is the apparent Km of the lipase, x is the thickness of Nernst layer, R is
the universal gas constant, T is the absolute temperature, z is the valence of the
substrate, F is Faraday’s constant, V is the magnitude of the electric field around the
enzyme support, and D is the diffusion coefficient of the substrate.
If the thickness of the Nernst layer decreases, then the ratio x/D would decrease
and K�m would decrease, approaching Km. B. Stability of Immobilized Enzymes
The stability of immobilized enzymes depends on the method of immobilization and
the susceptibility of the enzyme to inactivation. Inactivation can be caused by contaminants
and changes in temperature, pH, and ionic strength. High shear, microbial
contamination, fouling, and breakage of support particles have also been found to
inactivate immobilized enzymes. Depending on the strength of the immobilization
method, the enzyme can also be desorbed from the support. The stability of immobilized
enzymes is evaluated by determining the half-life of the enzyme under the
reaction conditions. In diffusion-limited systems, there is a linear decay in enzyme
activity in time, as enzymes on the surface of the support are inactivated and the
Copyright 2002 by Marcel Dekker, Inc. All Rights Reserved.