Immobilization of human carbonic anhydrase I on glutaraldehyde modified bentonite - chitosan composite and investigation of its properties

Greenhouse gas accumulation in the atmosphere resulting from industrialization and human activities is accepted as the main cause of climate change. Increasing temperatures by greenhouse gases have severe effects on meteorological and climatic events along with reduced available water resources. Carbon dioxide (CO2) is known as the most serious anthropogenic greenhouse gas in the atmosphere. In recent years the systems prepared by immobilizing carbonic anhydrase enzymes using CO2 as a substrate on some matrices are predominantly preferred. In this study, after the bentonite clay was activated with H2SO4, a composite material was prepared by mixing it with chitosan. The hCA I purified from human erythrocytes was immobilized to the composite material by glutaraldehyde-mediated cross-linking technique. The composite material was characterized using FT-IR, XRD, FE-SEM and EDX techniques. Optimum immobilization conditions were determined for the immobilization process. Parameters such as kinetic constants (Km, Vmax, kcat, kcat/Km), thermal stability, storage stability and reusability of immobilized hCA I were determined. Immobilized hCA I showed optimum activity at pH 8.0 and 30 °C. Km value was 5.11 mM for immobilized hCA I and 1.13 mM for free hCA I. After 30 days of incubation at 4°C, immobilized hCA I retained 87.5% of its initial activity. After 12 cycles of use, the relative activity of immobilized hCA I was 89.1%. In addition to kinetic parameters, CO2 removal activity was determined by two different methods. According to the CA hydratase activity method, the hydratase activity of immobilized hCA I was 7.31 WAU/mg hCA I. According to the CaCO3 precipitation method, the CO2 removal activity of immobilized hCA I was 7.41 mg CaCO3/mg hCA I. Keywords: Bentonite, Carbonic Anhydrase, Chitosan, Composite, Immobilization