Co-immobilization and compartmentalization of cholesterol oxidase, glucose oxidase and horseradish peroxidase for improved thermal and H₂O₂ stability

Glucose oxidase (GO_X) and cholesterol oxidase (CO_X) are enzymes with numerous practical applications in medicine and industry. Although various methods for improving of enzyme stability have been proposed, we present novel approach for enzyme compartmentalization using polyelectrolytes layers deposited on electrospun fibers made of poly (D,L-lactide-co-glycolide) and commercial membrane UFX5. Compartmentalization has a series of advantages over simple co-immobilization offering providing of suitable microenvironment for the immobilized enzymes, ensuring proper substrate/product channeling and improving stability and reusability of biomolecules. The biosystems produced via compartmentalization, resulted in higher conversion efficiency of model reaction at 45 °C and 65 °C compared to free enzymes as well as negative effect of hydrogen peroxide was minimalized after immobilization. Moreover, after 5 catalytic cycles, biosystems based on UFX5 membrane and HRP or GO_X or CO_X immobilized by compartmentalization reached efficiencies of 89% and 34%, respectively, with less than 10% enzyme elution from the support. Enzymes compartmentalized using ultrafiltration membrane showed also significant improvement of their biocatalytic productivity over repeated use, resulting in possible costs reduction of the desired process. Use of biosystems with electrospun fibers and co-immobilized enzymes resulted in enzyme elution of over 90% and very limited conversion efficiency.