Lipase-catalyzed fractionation of conjugated linoleic acid isomers

The abilities of lipases produced by the fungus Geotrichum candidum to sele ctively fractionate mixtures of conjugated linoleic acid (CLA) isomers duri ng esterification of mixed CLA free fatty acids and during hydrolysis of mi xed CLA methyl esters were examined. The enzymes were highly selective for cis-9,trans-11-18:2. A commercial CLA methyl ester preparation, containing at last 12 species representing four positional CLA isomers, was incubated in aqueous solution with either a commercial G. candidum lipase preparation (Amano GC-4) or lipase produced from a cloned high-selectivity G. candidum lipase B gene. In both instances selective hydrolysis of the cis-9,trans-1 1-18:2 methyl ester occurred, with negligible hydrolysis of other CLA isome rs. The content of cis-9, trans-11-18:2 in the resulting free fatty acid fr action was between 94 (lipase B reaction) and 77% (CC-4 reaction). The comm ercial CLA mixture contained only trace amounts of trans-9,cis-11-18:2, and there was no evidence that this isomer was hydrolyzed by the enzyme. Analo gous results were obtained with these enzymes in the esterification in orga nic solvent of a commercial preparation of CLA free fatty acids containing at least 12 CLA isomers. In this case, G. candidum lipase B generated a met hyl ester fraction that contained >98% cis-9,trans-11-18:2. Geotrichum cand idum lipases B and GC-4 also demonstrated high selectivity in the esterific ation of CLA with ethanol, generating ethyl ester fractions containing 96 a nd 80%, respectively, of the cis-9, trans-11 isomer. In a second set of exp eriments, CLA synthesized from pure linoleic acid, composed essentially of two isomers, cis-9,trans-11 and trans-10,cis-12, was utilized. This was sub jected to esterification with octanol in an aqueous reaction system using A mano CC-4 lipase as catalyst. The resulting ester fraction contained up to 97% of the cis-9,trans-ll isomer. After adjustment of the reaction conditio ns, a concentration of 85% trans-10,cis-12-18:2 could be obtained in the un reacted free fatty acid fraction. These lipase-catalyzed reactions provide a means for the preparative-scale production of high-purity cis-9,trans-11- 18:2, and a corresponding CLA fraction depleted of this isomer.