Food Lipids: Chemistry, Nutrition, and Biotechnology

of buffer A containing 1 M ammonium sulfate and then eluted with 1 L
of a linear gradient from 1 to 0 M ammonium sulfate in buffer A at a flow
rate of 60 mL/h; 4 mL fractions are collected.
4. Ultrogel-HA hydroxyapatite column chromatography. The pooled active
fractions from the Toyopearl column are further purified by passage
through an Ultrogel-HA column (3.0 cm � 9 cm), preequilibrated with 10
mM phosphate buffer (pH 7.0). After being washed with 10 mM phosphate
buffer, the column is eluted with a 1 L linear gradient from 10 to 500 mM
phosphate buffer at a flow rate of 50 mL/h, and 5 mL fractions are collected.
The active fractions are pooled and stored at �20�C.
These procedures are carried out at 4�C. The purity of lipase active fractions
is determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis.
IV. ASSAY OF LIPASES
A. Review of Emulsion Systems
Lipases are assayed in emulsions where high substrate surface areas can be achieved.
Since, in addition to high interfacial area, the chemical and physical environments
at the substrate–water interface are important to the adsorption and activity of lipases,
it is useful to understand the nature of the different types of emulsion used
for lipase assay or in various applications. Emulsions are mixtures of two immiscible
liquids (e.g., oil and water); one of the components is dispersed as very small droplets,
or particles, and the mixture is stabilized by a surface-active agent, or surfactant.
Emulsions are classified as macro- or microemulsions where the dispersed particles
are either greater or smaller than one micrometer in diameter, respectively. Macroemulsions
are turbid, milky in color, and thermodynamically unstable (i.e., they will
ultimately separate into the two liquid phases). On the other hand, microemulsions
are homogeneous and stable.
Depending on the conditions of formation, and particularly the nature of the
surfactant, macroemulsions may be normal phase (oil-in-water) or reversed phase
(water-in-oil), invert). In the former, the oil is emulsified into the aqueous phase,
and the surfactant forms a monolayer film around the dispersed oil droplets, whereby
the hydrophobic moiety of the surfactant extends into the oil and the polar moiety
is at the droplet surface (Fig. 1). In reversed phase emulsions, the aqueous phase
is dispersed in the oil with the orientation of the surfactant molecules reversed
(Fig. 1).
Amphiphilic (surfactant) molecules undergo self-organization into spheroidal
particles when dissolved in certain organic solvents such as isooctane with the polar
head groups oriented inward and hydrophobic tails outward. These particles are referred
to as reverse micelles and are less than 1 �m in diameter (Fig. 1). Water can
be ‘‘solubilized’’ in the organic solvent by becoming entrapped in the particles at up
to several dozens of molecules per molecule of surfactant. Typically, reverse micelles
are formed when the molar ratio of water to surfactant is less than 15, which is
expressed as water activity, w0 (i.e., molarity of water to molarity of surfactant) or
R. Enzymes such as lipases can be entrapped within the aqueous phase particles of
the invert emulsions or reverse micelles of microemulsions, where they retain their
activity. In the latter case, the enzymes are isolated from the organic solvent. Luisi
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