Enzyme Immobilization on Synthesized Nanoporous Silica Particles and their Application in a Bi‐enzymatic Reaction

The application of enzymes presents a great advantage regarding highly selective reactions; however, it involves also challenges due to their sensitivity. Immobilization offers one strategy to overcome those challenges enabling enzyme stabilization, as well as retention. In the present study, covalent attachment on hydrophilic amino‐functionalized carriers is found to be the most promising immobilization method for the investigated reaction system. To achieve this, a novel method for preparation of silica particles with subsequent amino‐functionalization is developed to prepare spherical carriers for enzyme immobilization, whereby high porosities are obtained based on polymerization. With these particles, immobilization of an alcohol dehydrogenase and a formate dehydrogenase is realized with residual activities of 70 and 80 % after 12 consecutive batches, respectively. The two immobilized enzymes are used in the reduction of cinnamyl aldehyde with in situ cofactor regeneration, obtaining a conversion of 100 % and up to 10‐fold higher turnover numbers compared to the free enzyme. Different immobilization methods were evaluated for application of two biocatalysts in a cofactor dependent reaction. Promising immobilization and activity yields were obtained by covalent immobilization on synthesized hydrophilic silica particles. High reaction stabilities for the two immobilized enzymes resulted in a 10‐fold increase of the turnover number.