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

Abstract Cell-free biocatalysis is gaining momentum in the production of value-added chemicals, particularly in stepwise reaction cascades. However, the stability of enzyme cascades in industrial settings is often compromised when using free enzymes. In this study, we have developed a stable multifunctional heterogeneous biocatalyst co-immobilizing five enzymes on microparticles to transform 1,ω-diols into 1,ω-hydroxy acids. We improved the operational efficiency and stability of the heterogeneous biocatalyst by fine-tuning enzyme loading and spatial organization. Stability issues are overcome through post-immoblization polymer coating. The general applicability of this heterogeneous biocatalyst is demonstrated by its scale-up in both batch and packed bed reactors, allowing a product yield > 80%. The continuous process is fed with H2O2 as oxygen source, reaching a Space-Time Yield (STY) of 0.76 g·L− 1·h− 1, maintained for the first 12 hours. Finally, this flow system is telescoped with a second plug-flow reactor packed with a different heterogeneous biocatalyst. As a result, this 6-enzyme 2-reactor system sequentially transforms 1,ω-diols into 1,ω-aminoacids while in-situ recycling NAD+, depleting H2O2 and generating O2.