Front Matter

The first step [Equation (11)] is an esterification in a solvent-free system with non-

regiospecific lipase. When immobilizedCandida antarcticalipase (NovozymeTM)

was used under optimal conditions in appropriately reduced pressure, over 90 %

yield of the targeted product was achieved from stoichiometric ratios of the sub-

strates. The reaction mixture from step (11) was then subjected to the second

step [Equation (12)] without any purification after separation of the immobilized

enzyme. The second step [12] is again an interesterification in a solvent-free system

with 1,3–regiospecific lipase (Rhizomucor miehei, LipozymeTM). When excess mo-

lar ratio of C 8 EE over tri-EPA (100/1) was used, ca. 100 % of the yield of the targeted

product was obtained (Figure 6). The unreacted C 8 EE and the byproduct (free

caprylic acid, EPAEE and free EPA) can be removed fractionally by short-pass dis-

tillation from the acylglycerol fraction which contains more than 90 % of

C 8 (EPA)C 8. It is to be noted that although molar incorporation of EPA into the gly-

cerol backbone is only one from three moles in a single cycle of the reaction, the two

remaining moles can be reused in the first step [Equation (11)] of the next cycle of the

reaction so that all the EPAEE is eventually converted to the desired sTAG. The

advantages of this process are that: (i) no organic solvent is used; (ii) isolation

and purification of the intermediates is not necessary; and (iii) the liberated EPAEE

(and small amounts of free EPA) and remaining caprylic acid ethyl ester are reusable.

Figure 7A and B depict the typical time courses of reactions shown in Equations (11)

and (12), respectively (Rosu et al., 2000).

9.4 Examples of syntheses of pure sTAG containing PUFA 165

Front Matter

With stoichiometric ratios of the two substrates in a solvent-free system and water

removal by vacuum, 98 % of 1,3-dicaprylin was obtained by the esterification

[Equation (9)] under optimal conditions. 1,3-Dicaprylin was then subjected to the

second chemical esterification [Equation (10)] by using 1,1’-dicyclohexylcarbodii-

mide (DCC) and 4-dimethylaminopyridine (DMAP) in dry chloroform. The yield

after purification by silica gel column chromatography was 42 %, and the purity

of TAG was 98 %, of which 90 % was C 8 (EPA)C 8. Thus, the chemo-enzymatic pro-

cess was unsatisfactory. 1,3-DAG can be also produced by chemical esterification of

1,3-dihydroxyacetone with DC in the presence of DMAP followed by reduction by

sodium borohydride (Awe et al., 1989; Baba et al., 1994). The use of expensive,

xenobiotic catalysts and low yields are disadvantages of chemical and chemo-enzy-

matic methods. Therefore, a higher yield of esterification to meet the industrial de-

mand may not be expected unless a more efficient catalyst is found.

More recently, the author’s group has developed a novel two-step enzymatic pro-

cess that seems more promising as shown in Equations (11) and (12) (Rosu et al.,

2000):