Optimized Spatial Configuration of Heterogeneous Biocatalysts Maximizes Cell-Free Biosynthesis of ω-Hydroxy and ω-Amino Acids
Javier Santiago‐Arcos, Susana Velasco‐Lozano, Eleftheria Diamanti, Ana I. Benítez‐Mateos, Daniel Grajales‐Hernández, Francesca Paradisi, Fernando López‐Gallego
Abstract
High Resolution Image Download MS PowerPoint Slide Cell-free biocatalysis is gaining momentum in producing value-added chemicals, particularly in stepwise reaction cascades. However, the stability of enzyme cascades in industrial settings is often compromised when free enzymes are involved. In this study, we have developed a stable multifunctional heterogeneous biocatalyst coimmobilizing 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 the enzyme loading and spatial organization. Stability issues are overcome through postimmobilization 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 of >80%. The continuous process is fed with H 2 O 2 as the oxygen source, reaching a space-time yield (STY) of 0.76 g·L –1 ·h –1, maintained for the first 12 h. Finally, this flow system is telescoped with a second plug-flow reactor packed with a different heterogeneous biocatalyst integrating an additional transaminase. As a result, this 6-enzyme 2-reactor system sequentially transforms 1,ω-diols into 1,ω-amino acids while in situ recycling NAD +, depleting H 2 O 2, and generating O 2 .