Selective concentration of eicosapentaenoic acid and docosahexaenoic acid from fish oil with immobilized/stabilized preparations of Rhizopus oryzae lipase

Abstract

The effect of simultaneous use of orientation and covalent/multipoint covalent immobilization of Rhizopus oryzae lipase (ROL) on its selectivity and stability is reported. For this purpose, the epoxy-functionalized silica and silica nanoparticles (SBA-15 and MCM-41) were subjected to partial modification using iminodiacetic acid and CuSO4 to obtain heterofunctional supports with ability to coordinate with histidine residues of ROL. For oriented and covalent immobilization of ROL, first the enzyme was quickly adsorbed on the supports by its most accessible histidine residue (His 134) via metal chelate affinity interaction. Then further incubation of the oriented enzyme led to irreversible covalent attachment via the reaction of the nucleophilic groups of ROL and the remaining epoxy groups of the supports. In the other approach in order to perform oriented/multipoint covalent immobilization, ROL was modified by chemical amination to introduce new amino groups with lower pkb values. After the initially adsorption of the aminated enzyme on the heterofunctional supports, the oriented enzyme was further incubated at pH 9.2 to promote additional covalent linkages between the chemically introduced amino groups and the supports. The results showed higher thermal/co-solvent stability of the preparations of aminated ROL compared to the immobilized derivatives of not-aminated ROL. The immobilized derivatives were used in selective hydrolysis of fish oil in order to investigate the effect of immobilization procedure on selectivity of ROL. All the derivatives discriminated between cis-5,8,11,14,17-eicosapentaenoic acid (EPA) and cis-4,7,10,13,16,19-docosahexaenoic acid (DHA) in favor of EPA. Considerable improvement in selectivity (16.1:1) was obtained by oriented/multipoint immobilization of aminated ROL on SBA-15.