Immobilization Strategies for Compartmentalized Reactions in Flow Reactors

Keywords: 3D printing, directed evolution, enzymes, flow catalysis, hydrogels

The compartmentalization of chemical reactions is a basic principle in nature, which can be implemented in technical processes by performing reaction cascades with physically separated enzymes. For the development of novel approaches in biocatalysis this principle is a major source for innovations and is therefore mimicked in several ways. The immobilization of biocatalysts in a fluidic setup is one way to achieve compartmentalization and thus precise control over artificial reaction cascades. Many established state-of-the-art technologies to arrange enzymes for sequential reactions require chemical modifications of the target enzymes, which can negatively influence the activities or specificities of the immobilized enzymes. We recently demonstrated the encapsulation of unmodified thermostable enzymes in a 3D printed, agarose-based thermoreversible hydrogel [1,2] and the site-selective immobilization of enzymes on beads.[3]



Figure 1. Schematic manufacturing process for encapsulation of unmodified enzymes in 3D printed hydrogel structures.

Both immobilization strategies allow implementation in continuous flow systems and have been successfully utilized for multi-step sequential biotransformations. ... mehrTo test the feasibility of the encapsulation strategy, we used an esterase and an alcohol dehydrogenase from thermophilic organisms as well as a ketoisovalerate decarboxylase from a mesophilic organism as exemplary biocatalysts. The latter was thermostabilized by rational protein engineering and directed protein evolution.[1,4] After the successful prove-of-concept study, we further expanded the scope of this system by integrating encapsulated phenacrylate decarboxylases into a chemoenzymatic workflow.[2] As an alternative, the site-selective immobilization strategy was employed to combine alcohol dehydrogenases and a cofactor regenerating glucose 1-dehydrogenase in continuously operated chip microreactors to produce meso diols from ketones.[3] Currently we are expanding the scope of this approach by integrating a novel thermostable benzaldehyde lyase into a custom-made packed-bed reactor for the flow production of α-hydroxy ketones.