Immobilisation and characterisation of bovine carbonic anhydrase enzyme on iron oxide magnetic nanoparticles: investigation of its potential applicability in co2 removal

In this study, the CA enzyme was purified with 35.98% yield using the sepharose 4B-L-tyrosine sulfanilamide affinity chromatography method. The purification coefficient for CA enzyme was 83.42 and the specific activity was 1122.94 μ/mg. To check the purity of the enzyme and to determine its molecular mass, 3-8% batch sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed and a single band was observed. The molecular mass was determined to be approximately 29 kDa. In the next step, two different support materials were synthesized for immobilisation. In the synthesis of the first support material (Fe3O4-NH2), the magnetic nanoparticle (MNP) was functionalised by the addition of amine group (NH2) using 3-aminopropyl)triethoxysilane (APTES). Then, bovine carbonic anhydrase (BCA) was immobilised by covalent binding method using glutaraldehyde (GA). In the synthesis of the second support material (Fe3O4@SiO2-NH2), MNP was coated with silicon oxide using tetraethyl orthosilicate (TEOS). It was then functionalized with APTES and immobilised by the same method. They were characterised by FT-IR, SEM-EDX and VSM before and after immobilisation. Activity was determined for free and immobilised enzymes in the pH range of 5–9, with an optimum pH of 9 observed. The Km and Vmax values of free BCA were 4.888 mM and 3.081 EU mL -1, while the Km and Vmax values of immobilised BCA on functionalised I. support material were found to be 9.369 mM and 1.810 EU mL-1, respectively, and for the second, the Km and Vmax values were found to be 16.160 mM and 2.195 EU mL-1, respectively. When the immobilised enzymes were reused 20 times for operational stability, the BCA activity immobilised on the first support material retained 73% of its activity, while the second retained 86% of its activity. For the storage stability study, the samples were incubated at 4 °C for 60 days. At the end of 60 days, the first support material retained 60% of the immobilised BCA activity, while the second retained 49% of its activity. Under the same conditions, only 17% of the free BCA activity was retained. In the final stage, the potential applicability of BCA in CO2 removal was determined. Keywords: Carbonic anhydrase, magnetic nanoparticles, purification, immobilization, CO2 removal