Food Lipids: Chemistry, Nutrition, and Biotechnology

without shaking for 30 minutes, whereupon the reaction was stopped as described
above. Up to 90% of the substrate was hydrolyzed under these conditions by C.
rugosa lipase when the incubation time was extended to 48 hours with periodic
additions of buffer (0.2 mL) during the incubation.
D. Microemulsion: Reverse Micelles
1. Hydrolysis
The desired amount of concentrated buffered lipase solution is poured into 5 mL of
50 mM AOT [Aerosol–OT, bis (2-ethylhexyl) sodium sulfosuccinate]–isooctane solution
containing 10% v/v of the substrate. The amount of enzyme solution depends
on the R value (e.g., 10.5). The reaction is initiated by vortexing until clear, and the
mixture is incubated at 30�C for 15 minutes; then 0.4 mL is added to 4.6 mL benzene
and 1.0 mL cupric acetate–pyridine solution, and the reaction is stopped by vortexing
(41a). Fatty acids liberated by the hydrolytic reaction are determined according to
Lowry and Tinsley (42). Other examples of studies involving hydrolysis by lipases
in organic solvent–reverse micelle systems are cited in Table 2 (41a, 41b,43–78).
2. Transesterification
Using a lipase from Rhizopus delemar, Osterberg et al. (79) used the following
system for transesterifing triacylglycerols with stearic acid: isooctane (91.65 wt %)
was mixed with AOT at 100–200 mM, aqueous 0.066 M phosphate buffer (pH 6)
(1.0%), and substrate (5.0%). The enzyme in the buffer was used at 1.5 U/mg substrate.
The reaction was carried out at 35�C under nitrogen with magnetic stirring,
and was stopped by raising the temperature to 100�C and holding at that temperature
for 10 minutes. Other examples of studies involving transesterification and synthetic
reactions in organic solvent–reverse micelle systems are cited in Table 2.
V. PROPERTIES AND REACTIONS
A. Structure
Over the past several years, the crystal structures of several mammalian and microbial
lipases have been determined. Generally, lipases are �/� proteins with a central
core of a mixed � sheet containing the catalytic triad composed of Ser ���His ���
Asp, and a surface loop restricting access of the substrate to the active site.
1. Animal Lipases
Human pancreatic lipase is folded into two domains, a larger N-terminal domain
comprising residues 1–335 and a smaller C-terminal domain (residues 336–449).
the core of the N domain is formed by a nine-stranded, �-pleated sheet in which
most of the strands run parallel to one another. Seven �-helical segments of varying
length occur in the strand connections, and six of them pack against the two faces
of the core sheet. The C domain is formed by two layers of antiparallel sheets, the
strands of which are connected by loops of varying length. The N domain contains
the active site, a glycosylation site, a Ca 2� -binding site, and possibly a heparinbinding
site. The active site is buried beneath a short amphipathic �-helical surface
loop, termed the ‘‘flap’’ or ‘‘lid’’ (80,31). Colipase (see below), binds exclusively to
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