Combi-Clea for Reactions in Continuous Flow Supercritical CO2

The continuous production of pharmaceuticals in a ‘green' way is a concern in the recent years. In this work, we have examined a combi-enzymatic reaction in a continuous flow supercritical CO2 system using a combi-CLEA (cross linked enzyme aggregate). We have combined the oxidation of cholesterol and the reduction of H2O2. Cholesterol oxidase catalyses the oxidation of cholesterol by molecular oxygen to 4-cholest-3-one, a precursor of interest in the pharmaceutical industry, and catalase reduces H2O2 produced in during the oxidation to water and oxygen. The elimination of H2O2 is important as it can inhibit the first reaction. The reactions were performed using a combi-CLEA of cholesterol oxidase and catalase. The co-immobilisation of the two enzymes was performed by the cross-linked enzyme aggregate (CLEA®) technique (precipitation of enzymes followed by cross linking of the physical aggregated of the eznyme molecules). The optimisation of the CLEA preparation was performed using glutaraldehyde and dextran polyaldehyde as cross-linking agents. A kinetic study of the reaction as the base for the modeling of the system is also presented.