Immobilization of Thermostabilized Enzymes in Flow Reactors

The immobilization of biocatalysts in a fluidic setup is one way to achieve compartmentalization and thus precise control over artificial reaction cascades. We recently demonstrated the encapsulation of unmodified thermostable enzymes in a 3D printed, agarose-based thermoreversible hydrogel to enable multi-step sequential biotransformations.[1] To test the feasibility of the encapsulation strategy, we used an esterase and an alcohol dehydrogenase from thermophiles as well as a ketoisovalerate decarboxylase from a mesophile as exemplary biocatalysts. The latter was thermostabilized by rational design and directed protein evolution.[1,2] After the successful proof-of-concept study, we further expanded the scope of this system by integrating phenacrylate decarboxylases into a chemoenzymatic workflow.[3] As an alternative, the site-selective immobilization of enzymes on beads can be employed. Currently, we are expanding the scope of this approach by integrating a benzaldehyde lyase into a custom-made packed-bed reactor for the flow production of α-hydroxy ketones.[4] To this end, we employed a computational prediction tool[5] based on a machine learning model for the identification of a novel thermostable benzaldehyde lyase and homology-modelling of the protein structure to create enzyme variants with altered substrate scope.