Hydrolysis of Fish Oil by Lipases Immobilized Inside Porous Supports
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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 miehei lipase (RML) adsorbed onto octyl-Sepharose. Standard reaction conditions were: (a) an organic phase composed by 30/70 (v:v) of oil in cyclohexane, (b) an aqueous phase containing 50 mM methyl-cyclodextrin in 10 mM Tris buffer at pH 7.0. The whole reaction system was incubated at 25 °C. 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 (e.g., 0.05 mM) of released omega-3 fatty acid could be detected with a precision higher than 99%. Three different lipases adsorbed on octyl-Sepharose 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 selective towards polyunsaturated fatty acids (PUFA) versus oleic plus palmitic acids (a fourfold 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.
KeywordsEnzymatic release of omega-3 acids Selective enzymatic release of EPA Solubilization of oil by randomly methylated β-cyclodextrins
This work was sponsored by the Spanish Ministry of Science and Innovation (project AGL-2009-07526) and the Comunidad Autonoma de Madrid (Project S0505/PPQ/03449). We gratefully recognize the Spanish Ministry of Science and Innovation for the “Ramón y Cajal” contract for Dr. Fernandez-Lorente. We thank the Spanish Ministry of Science and Innovation (MICINN) grant Consolider INGENIO 2010 CSD2007-00063 FUN-C-FOOD and the Comunidad de Madrid (CAM) ALIBIRD-S2009/AGR-1469 for financial support.
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