Thermoswitched immobilization-a novel approach in reversible immobilization

The present work is based on the finding that the mesophilic carbohydrate-binding domain from Clostridium cellulovorans fused with thermophilic enzymes from Pyrococcus furiosus can be reversibly denaturated and renaturated by a simple switch of temperature. Modular recombinant enzymes are active and free in the reaction mixture at 80-90 degrees C and deactivated and immobilized by affinity adsorption on cellulose at 40-30 degrees C. The temperature transition between both modes is rather sharp and occurs within the range of 40-50 degrees C. Due to the elevated temperature, there is no limitation by a diffusion step, and contamination does not occur during the reaction. After the reaction, the enzymes are quickly deactivated, adsorbed on the affinity matrix, removed from the reaction mixture, and ready for use in another reaction cycle.