Optimized Spatial Configuration of Heterogeneous Biocatalysts Maximizes Cell-Free Biosynthesis of ω-Hydroxy and ω-Amino Acids

Cell-free biocatalysis is increasingly being adopted for the production of value-added chemicals, particularly through stepwise reaction cascades. However, the use of free enzymes in industrial settings is often associated with compromised stability of enzyme cascades. In this study, a stable multifunctional heterogeneous biocatalyst was developed by co-immobilizing five enzymes on microparticles for the transformation of 1,ω-diols into 1,ω-hydroxy acids. Operational efficiency and stability of the heterogeneous biocatalyst were enhanced by fine-tuning enzyme loading and spatial organization. Stability limitations were addressed through the application of a post-immobilization polymer coating.

The general applicability of the heterogeneous biocatalyst was demonstrated through scale-up in both batch and packed bed reactors, achieving product yields greater than 80%. The continuous process was supplied with H₂O₂ as the oxygen source, resulting in a Space-Time Yield (STY) of 0.76 g·L⁻¹·h⁻¹, which was maintained for the first 12 hours. Finally, the flow system was connected in a telescoped configuration to a second plug-flow reactor packed with a different heterogeneous biocatalyst. As a result, the 6-enzyme, 2-reactor system enabled the sequential transformation of 1,ω-diols into 1,ω-amino acids, while in situ recycling of NAD⁺ was achieved, along with depletion of H₂O₂ and generation of O₂.

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