Design, characterization and application of nanobiocatalysts of lipase a from Candida antarctica

Abstract

In this communication, lipases from Candida antarctica were immobilized onto magnetite or halloysite through chemical of physical adsorption. Firstly, lipase A from Candida antarctica (CALA) was immobilized by covalent bonding onto magnetite (Fe3O4) functionalized with chitosan (CHI) and activated with glutaraldehyde (GLU), labelled Fe3O4@CHI-GLU-CALA or CALA-MNPC for short, (84.1% ± 1.0 for immobilization yield and 208.0 ± 3.0 U/g for derivative activity). CALA-MNPC was characterized by X-Ray Diffraction (XRD), Fourier Transform Infrared (FTIR) spectroscopy, Thermogravimetry (TG) and Scanning Electron Microscope (SEM), indicating the incorporation of magnetite and the immobilization of CALA onto the chitosan matrix. At 85 °C, CALA-MNPC showed a half-life 8–11 times higher than that of CALA at pH 5–9. For CALA, the highest activity was at pH 7, whereas for CALA-MNPC, the highest activity was at pH 10. CALA-MNPC was applied to the production of a biolubricant ester from Tilapia oil, maintaining half of its activity after 7 consecutive cycles of esterification. Then, CALA or lipase B from Candida antarctica (CALB) were immobilized by covalent bonding onto Fe3O4 funcionalized with 3-aminopropyltriethoxysilane (APTES) and activated with GLU, labelled Fe3O4@APTES-GLU-CALA (CALA-MNPA) or Fe3O4@APTES-GLU-CALB (CALB-MNPA), respectively, (100 ± 1.2% and 57.6 ± 3.8% for immobilization yield; 198.3 ± 2.7 U/g and 52.9 ± 1.7 U/g for derivative activity, respectively). XRD and Raman spectroscopy analysis indicated the production of a magnetic nanomaterial with a diameter of 13.0 nm, whereas FTIR indicated functionalization, activation and enzyme immobilization. CALA-MNPA and CALB-MNPC were applied to the synthesis of ethyl butyrate; under optimized conditions (1:1, 45 °C and 6 h) by a Central Composite Design (CCD), it was possible to achieve 99.2 ± 0.3% of conversion for CALA-MNPA (10mg) and 97.5 ± 0.8% for CALB-MNPA (12.5mg), which retained approximately 80% of their initial activity after 10 consecutive cycles of esterification; under ultrasonic irradiation, similar conversions were achieved but at 4 h of incubation. Finally, the immobilization of CALA though ionic adsorption onto halloysite (HNT) was optimized by the Taguchi method (CALA-HNT); under optimized conditions (pH 5, 5 mM, 5 °C and 4 hours), it was possible to achieve 97.1 ± 0.1% for immobilization yield and 83.81 ± 0.5 U/g for derivative activity. Moreover, at pH 7, CALA-HNT showed a half-life 2–8 times higher than that of CALA at 50–90 °C. For CALA, the highest activity was at pH 7, whereas for CALA-HNT, the highest activity was at pH 9. HNT and CALA-HNT were be characterized by XRD, FTIR, SEM, TG, elemental analysis (CHNS) and Differential Scanning Calorimetry (DSC), proving the immobilization of CALA on HNT and maintenance of the nanotubes structure even after immobilization.