0.7 g immobilized lipase was incubated at 40 8 C with shaking. Figure 8 shows a
typical time course of the change of fatty acid contents in the glyceride fraction.
The palmitic and stearic acid contents in glycerides rapidly decreased until 10 h,
and gradually thereafter. The increase in AA content in glycerides was completely
associated with the decrease in the palmitic and stearic acid contents. The AA con-
tents after 24-and 34-h reactions reached 59 and 63 mol%, respectively.
Glycerides obtained from a 24-h reaction were analyzed by high-performance
liquid chromatography (HPLC) (Figure 9). The components eluted between 12
and 14 min were diglycerides, as shown by thin-layer chromatography, and the com-
ponents eluted after 18 min were triglycerides. The structure of each triglyceride was
estimated from the fatty acid composition, and are shown in Figure 9. The contents of
AAA, APA, ASA, APP, and APS were calculated to be 7.3, 75.9, 3.1, 12.4, and 1.3
wt% from their peak areas, respectively. High purity of APA could be obtained by
preparative HPLC (yield of HPLC, 56 %).
Acidolysis of tripalmitin with AA produced 7 wt% of triarachidonin as a bypro-
duct. Because the positional specificity ofRhizopuslipase was very strict as de-
scribed below, it was strongly suggested that triarachidonin was not generated by
enzymatic exchange of fatty acids at the 2-position for AA. The presence of 4.6
wt% of diglycerides showed that hydrolysis occurred concomitantly with the acid-
olysis. In addition, concomitant esterification could be expected to occur during the
reaction. Therefore, triarachidonin may be generated via the following processes: a
diglyceride with AA at the 1(3)-position and palmitic acid at the 2-position is gen-
erated by hydrolysis, and the palmitic acid migrates spontaneously to the 3(1)-posi-
tion. Palmitic acid at 3(1)-position is acidolyzed with AA, and diarachidonin is gen-
erated. After AA at the 1(3)-position migrated to the 2-position, triarachidonin is
synthesized by esterification of free AA to the 1(3)-position. It was actually con-
firmed that diarachidonin was present in the reaction mixture, because the peak
142 8 Production of Functional Lipids Containing Polyunsaturated Fatty Acids with Lipase
Figure 8. Time course of acidolysis of tripalmitin with AA. A mixture of 30 g AA/tripalmitin (5 : 1,
w/w) and 2.1 g immobilizedRhizopus delemarlipase was incubated at 40 8 C with shaking., Palmitic
acid content in glycerides;, stearic acid content;&, AA content.