Hydrolysis of Fish Oil by Lipases Immobilized inside Porous Supports

A new assay was designed to measure the release of omega-3 acids (eicosapentaenoic acid (EPA) and docosahexaenoic acid ( DHA)) from the hydrolysis of sardine oil by lipases immobilized inside porous supports. A biphasic system was used containing the fish oil dissolved in the organic phase and the immobilized lipase suspended in the aqueous phase. The assay was optimized by using a very active derivative of Rhizomucor miehie lipase (RML) adsorbed onto octyl agarose. Optimal conditions were: a.an organic phase composed by 30/70 (v:v) of oil in cyclohexane , b.an aqueous phase containing 50 mM methylcyclodextrin in 10 mM TRIS buffer at pH 7.0. The whole reaction system was incubated at 25oC. Under these conditions, up to 2% of the oil is partitioned into the aqueous phase and most of the 95% of released acids were partitioned into the organic phase. The organic phase was analyzed by RP-HPLC (UV detection at 215 nm) and even very low concentrations (eg., 0.05 mM) of released omega-3 fatty acid could be detected with a precision higher than 99%. Three different lipases adsorbed on octyl-agarose were compared: Candida antarctica lipase-fraction B (CALB), Thermomyces lanuginosa lipase (TLL) and RML. The three enzyme derivatives were very active. However, most active and selectivetowards polyunsaturated fatty acids (PUFAs) versus oleic plus palmitic acids (a 4-fold factor) was CALB. On the other hand, the most selective derivatives towards EPA versus DHA (a 4.5 fold factor) were TLL and RML derivatives.