Cross-Linking of Lipases Adsorbed on Hydrophobic Supports: Highly Selective Hydrolysis of Fish Oil Catalyzed by RML
- 257 Downloads
Organic cosolvents may improve the properties of lipases (e.g., selectivity); however, organic cosolvents also promote the desorption of the enzyme from its hydrophobic supports. In this study, adsorbed lipase molecules were cross-linked with polyfunctional polymers, such as aldehyde-dextrans, to prevent this desorption. The desorption of adsorbed lipases was greatly reduced by optimizing the polymer size, polymer/lipase ratio, and cross-linking time. More than 95% of cross-linked, immobilized Rhizomucor miehei lipase (RML) remained adsorbed on the support after washing with cosolvents or detergents. This new, immobilized RML preparation mediated the hydrolysis of sardine oil in the presence of organic cosolvents. The presence of cosolvents promoted small losses of hydrolytic activity. Interestingly, however, 50% 2-propanol also promoted increased selectivity in the release of eicosapentaenoic acid (EPA) in relation to docosahexaenoic acid (DHA). An EPA/DHA ratio of 4:1 in the absence of 2-propanol was increased to a ratio of 22:1 in the presence of 2-propanol. The new RML derivatives were relatively stable under the selected reaction conditions. Their overall half-life was 100 h, but, in a second inactivation phase (below 60% of remaining activity), it took 600 h to reach 30% of their remaining activity.
KeywordsRhizomucor miehei lipase Enzyme immobilization Polyfunctional polymers Selective release of eicosapentenoic acid Hydrolysis of sardine oil Omega-3 fatty acids
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 and Dr. Betancor.
- 2.Fernández-Lorente G, Pizarro C, López-Vela D, Betancor L, Carrascosa AC, Pessela B, Guisan M (2010) Hydrolysis of fish oil by lipases immobilized inside porous supports. J Am Oil Chem Soc (Submitted)Google Scholar
- 4.Akai S, Kita Y (2007) Recent progress on the lipase-catalyzed asymmetric syntheses. J Synth Org Chem 65:772–782Google Scholar
- 14.Palomo JM, Muñoz G, Fernández-Lorente G, Mateo C, Fuentes M, Guisan JM (2003) Modulation of Mucor miehei lipase properties via directed immobilization on different hetero-functional epoxy resins: hydrolytic resolution of (R, S)-2-butyroyl-2-phenylacetic acid. J Mol Catal B Enzym 21(4–6):201–210CrossRefGoogle Scholar
- 18.Guisán JM, Rodríguez V, Rosell CM, Soler G, Bastida A, Fernández-Lafuente R (1997) Stabilization of immobilized enzymes by chemical modification with polyfunctional macromolecules. In: Bickerstaff GF (ed) Methods in biotechnology 1 immobilization of enzymes and cells. Humana Press, NJ, pp 289–297Google Scholar
- 19.Fernandez-Lorente G, Godoy CA, Mendes AA, Lopez-Gallego F, Grazu V, de las Rivas B, Palomo JM, Hermoso J, Fernandez-Lafuente R, Guisan JM (2008) Solid-phase chemical amination of a lipase from Bacillus thermocatenulatus to improve its stabilization via covalent immobilization on highly activated glyoxyl-agarose. Biomacromolecules 9(9):2553–2561CrossRefGoogle Scholar