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Cell-free gene expression systems derived from bacterial lysates enable the expression of biosynthetic pathways from inexpensive and easily prepared DNA templates. These systems hold great promise for modular and on-demand bioproduction of valuable small molecules in resource-limited settings but are constrained in their long-term stability, reusability, and deployability. In this work, we demonstrate that multiple cell-free expressed enzymes can be co-immobilized in biocompatible hydrogels made from poly(ethylene glycol) diacrylate (PEGDA) with added glycerol for enhanced gel integrity. Using small-angle X-ray scattering (SAXS), we show that the mesh size of PEGDA-glycerol hydrogels is comparable to the globular sizes of many proteins and enzymes, which could be used for protein entrapment. We found that the combination between entrapment and chemical ligation of the enzymes was effective to retain proteins. By employing a method for direct fluorescence measurement from hydrogels, we found that proteins can be retained in PEGDA-glycerol for at least a week. By separating the cell-free enzyme expression from the immobilization step, we successfully fabricated enzyme-laden hydrogels with three heterologous cell-free enzymes for the bioconversion of pyruvic acid to malic acid, an industrially valuable and versatile precursor chemical. Both heterologous and endogenous enzymes from the lysate remain functional in photo-cross-linked hydrogels and can be reused for multiple biocatalytic cycles. Moreover, we also found that the immobilized enzymes exhibit up to 1.6-fold higher activity and 2-fold longer lifetimes than free enzymes in liquid reactions. These results could advance the deployment of cell-free synthetic biology because they show that reusable, stable, and durable multienzyme systems can be created using readily available materials and fabrication techniques.
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This content will become publicly available on February 3, 2026
Unveiling the Critical Role of Spatial Organization on Enzymatic Cascade Reactions in a Crystalline Framework with Hierarchical Porosity
Enzymatic cascades play a crucial role in energy conversion and chemical transformations in living organisms. Due to enzymes’ high selectivity in catalysis and eco-friendly nature, operating enzymatic cascades in cell-free systems has the potential to improve existing chemical transformations. However, applications involving enzymes are often limited by their poor stability in cell-free environments, and the impact of immobilization on the kinetics of enzymatic cascades remains relatively underexplored, largely due to challenges in determining the support structure and controlling the enzyme immobilization process. In this work, we developed NU-1510-Cr, a chromium-based mesoporous metal–organic framework (MOF) with the mtn topology to encapsulate an enzyme cascade that oxidizes ethanol to acetaldehyde and subsequently to acetic acid. The crystalline hierarchical pores provide spatial control of the enzymes within the host, where the impact of the spatial organization on the cascaded reaction kinetics was assessed. The findings offer valuable insights for those aiming to immobilize and compartmentalize biocatalytic or chemocatalytic cascade systems and develop efficient microscale and nanoscale bioreactors.
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- Award ID(s):
- 2119433
- PAR ID:
- 10648214
- Publisher / Repository:
- American Chemical Society
- Date Published:
- Journal Name:
- ACS Materials Letters
- Volume:
- 7
- Issue:
- 2
- ISSN:
- 2639-4979
- Page Range / eLocation ID:
- 409 to 416
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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