All-Enzyme Hydrogels for Flow-Biocatalysis

Flow biocatalysis offers a powerful approach for enhancing reaction control and efficiency in chemical syntheses by integrating enzymes into interconnected, continuously perfused reaction chambers. To enable such concepts for industrial use, enzymes and enzyme cascades must be efficiently and stably immobilized into biocatalytically active materials. To address this challenge, we have established all enzyme hydrogels (AEHs) that autocatalytically self-assemble through site-specific enzyme conjugation using the SpyCatcher/SpyTag technology.[1] AEHs were successfully developed by fusing the SpyCatcher (SC) or SpyTag (ST) with various enzymes, including phenolic acid decarboxylase (PAD),[2] alcohol dehydrogenase (ADH) with glucose 1 dehydrogenase (GDH),[1] and xylose reductase (XR) with galactitol dehydrogenase (GalDH).[3] The site-specific mediated enzyme conjugation allowed the formulation of biomaterials with high stability and catalytic efficiency while consisting almost exclusively of enzymes, in contrast to traditional carrier-based immobilization techniques. Additionally, AEH-based bienzymatic cascades were formulated directly inside cells,[4] and recently developed as a novel foam formulation which further increased material storage and process stability.[3] The new enzyme-foam materials were extensively characterized using dynamic light scattering (DLS), scanning electron microscopy (SEM), atomic force microscopy (AFM), X-ray tomography, fluorescence microscopy, laser scanning microscopy (LSM), IR spectroscopy, and thermogravimetry. ... mehrContinuous flow experiments demonstrated excellent stereoselectivity, stable conversion rates, and high space-time yields (STY), with cofactor retention for more than four days.[5 8] Inline benchtop NMR analysis of acquired from the reaction solution in real time allowed the automated self-optimization of the biocatalytic microreactors by autonomously adjusting flow rates.[9] AEHs, particularly in their foam formulation, offer a promising platform for sustainable and industrially relevant biocatalysis. Their adaptability across enzyme classes, high catalytic performance, and extended stability make them an attractive alternative for continuous biotransformations. Ongoing advancements in AEH formulations and inline NMR analysis will further expand their potential for bioprocessing applications.