Chemical amination of Rhizopus oryzae lipase for multipoint covalent immobilization on epoxy-functionalized supports: Modulation of stability and selectivity

• New amino groups were introduced to the surface of ROL via chemical amination.
• Multipoint immobilization of ROL was performed on epoxy-functionalized supports.
• Stability of ROL was greatly improved by multipoint covalent immobilization.
• The soluble enzyme and immobilized derivatives discriminate between EPA and DHA.
• Immobilization caused to improve the selectivity of ROL in fish oil hydrolysis.

Multipoint covalent attachment of Rhizopus oryzae lipase (ROL) on epoxy-functionalized silica and silica nanoparticles (MCM-41 and SBA-15) is reported. Multipoint immobilization of enzymes on these supports usually performs by the reaction between several epoxy groups of the support and several Lys residues on the external surface of the enzyme molecules at pH 10. However, this standard immobilization procedure is unsuitable for ROL due to the low stability of ROL at pH 10. Introducing new amino groups with lower pKb to the surface of ROL by using chemical amination strategy permits immobilization of the enzyme at lower pH values. Immobilization/stabilization of aminated ROL was performed in two steps. First the enzyme is covalently immobilized at pH 7.0 and then the already immobilized enzyme is further incubated at pH 9.2 to promote the formation of further covalent linkages between the immobilized ROL and the support. The results showed higher thermal and co-solvent stability for immobilized derivatives of aminated ROL compared to the results obtained for the derivatives of not-aminated ROL and free ROL. Influence of the immobilization procedure on selectivity of the immobilized preparations was studied in selective hydrolysis of fish oil at three different conditions. The selectivity and reusability of ROL was greatly improved after immobilization. All the derivatives discriminate 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.

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