Harnessing Non‐Covalent Protein–Protein Interaction Domains for Production of Biocatalytic Materials Systems

Industrial biocatalysis has the potential to enable sustainable and environmentally friendly processes in the chemical industry. In continuous production systems, immobilized enzymes are required as catalysts, and providing them in a carrier-free form is preferable to maximize effective reactor space usage. This study explores the use of non-covalent protein interaction domains for fabricating catalytically active all-enzyme hydrogel (AEH) materials. For this, enzymes of a binary interacting cascade - an alcohol dehydrogenase and a glucose-1-dehydrogenase for cofactor regeneration - are genetically fused to various non-covalent binding motifs (PDZ, GBD, SH3), enabling self-assembly into non-covalent AEHs. The resulting materials are generated both in living E. coli cells and from purified enzymes as monodisperse enzyme foams. While in-cell AEHs show no significant advantages over non-assembled cascades in continuous flow reactors, the AEH foams demonstrated excellent performance. Optimized systems based on SH3 and PDZ domains, despite a 100-fold difference in binding affinity, achieved stable conversions above 70% over 30 h. Notably, drying the enzyme foams significantly improved both mechanical stability and catalytic activity. ... mehrThe successful application of this approach to a different enzyme system based on phenolic acid decarboxylase further underscores its versatility and suitability for developing robust biocatalytic materials for sustainable industrial processes.