Purification of ethyl docosahexaenoate by selective alcoholysis of fatty acid ethyl esters with immobilized Rhizomucor miehei lipase

Ethyl docosahexaenoate (E-DHA) is efficiently enriched by the selective alcoholysis of ethyl esters originating from tuna oil with lauryl alcohol using immobilized lipase. Alcoholysis of ethyl esters by immobilized Rhizopus delemar lipase raised the E-DHA content in the unreacted ethyl ester fraction from 23 to 49 mol% in 90% yield. However, the content of ethyl eicosapentaenoate (E-EPA) was higher than the initial content. Hence we attempted to screen for a suitable lipase to decrease the E-EPA content, and chose Rhizomucor miehei lipase. Several factors affecting the alcoholysis of ethyl esters were investigated, and the reaction conditions were determined. When alcoholysis was performed at 30 degrees C with shaking in a mixture containing ethyl esters/lauryl alcohol (1:3, mol/mol) and 4 wt% of the immobilized R. miehei lipase, the E-DHA content in the ethyl ester fraction was increased and the E-EPA content was decreased. By alcoholyzing ethyl esters in which the E-DHA content was 45 mol% (E-tuna-45) for 26 h, the E-DHA content was increased to 74 mol% in 71% yield and the E-EPA content was decreased from 12 to 6.2 mol%. To investigate the stability of the immobilized lipase, batch reactions were carried out continually by replacing the reaction mixture with fresh E-tuna-45/lauryl alcohol (1:3, mol/mol) every 24 h. The decrease in the alcoholysis extent was only 17% even after 100 cycles of reaction. It was found that increasing the proportion df lauryl alcohol increased the conversion of E-EPA to lauryl-EPA. When an ethyl ester mixture in which the E-DHA content was 60 mol% (E-tuna-60) was alcoholyzed for 24 h with 7 molar equivalents of lauryl alcohol, the E-DHA content was raised to 93 mol% with 74% yield and the E-EPA content was reduced from 8.6 to 2.9 mol%.