Continuous flowbiocatalysis is an emerging field of industrial biotechnology that uses enzymes immobilized in flowchannels for the production of value-added chemicals.We describe the construction of self-assembling all-enzyme hydrogels that are comprised of two tetrameric enzymes.T he stereoselective dehydrogenase LbADH and the cofactorregenerating glucose 1-dehydrogenase GDH were genetically fused with aS pyTago rS pyCatcher domain, respectively,t o generate two complementary homo-tetrameric building blocks that polymerizeu nder physiological conditions into porous hydrogels.M ounted in microfluidic reactors,t he gels show excellent stereoselectivity with near quantitative conversion in the reduction of prochiral ketones along with high robustness under process and storage conditions.T he gels function as compartment that retains intermediates thus enabling high total turnover numbers of the expensive cofactor NADP(H).
All-enzyme hydrogels are efficient reagents for continuous flow biocatalysis.
Carrier-free enzyme immobilization techniques are an important development in the field of efficient and streamlined continuous synthetic processes using microreactors. Here, the use of monolithic, self-assembling all-enzyme hydrogels is expanded to phenolic acid decarboxylases. This provides access to the continuous flow production of p-hydroxystyrene from p-coumaric acid for more than 10 h with conversions ≥98% and space time yields of 57.7 g·(d·L)−1. Furthermore, modulation of the degree of crosslinking in the hydrogels resulted in a defined variation of the rheological behavior in terms of elasticity and mesh size of the corresponding materials. This work is addressing the demand of sustainable strategies for defunctionalization of renewable feedstocks.
The industrial implementation of enzymes in flow biocatalysis microreactors is expected to be essential for the emergence of a bio‐based circular economy. Major challenges concern the efficient immobilization of delicate enzymes inside miniaturized reactors without compromising their catalytic activity. We describe the exploitation of the widely used His‐tag system in a microfluidic packed‐bed reactor that contains ketoreductase‐functionalized magnetic beads. In a continuous process, these reactors produced highly stereoselective (R)‐configured alcohols (d.r. 99:1) with an average conversion of > 90 % for more than 4 days. We believe that such miniaturized flow reactors can be of great utility for future sustainable production processes.
Maximizing space–time yields (STY) of biocatalytic flow processes is essential for the establishment of a circular biobased economy. We present a comparative study in which different biocatalytic flow reactor concepts were tested with the same enzyme, the (R)-selective alcohol dehydrogenase from Lactobacillus brevis (LbADH), that was used for stereoselective reduction of 5-nitrononane-2,8-dione. The LbADH contained a genetically encoded streptavidin (STV)-binding peptide to enable self-immobilization on STV-coated surfaces. The purified enzyme was immobilized by physisorption or chemisorption as monolayers on the flow channel walls, on magnetic microbeads in a packed-bed format, or as self-assembled all-enzyme hydrogels. Moreover, a multilayer biofilm with cytosolic-expressed LbADH served as a whole-cell biocatalyst. To enable cross-platform comparison, STY values were determined for the various reactor modules. While mono- and multilayer coatings of the reactor surface led to STY < 10, higher productivity was achieved with packed-bed reactors (STY ≈ 100) and the densely packed hydrogels (STY > 450). The latter modules could be operated for prolonged times (>6 days). Given that our approach should be transferable to other enzymes, we anticipate that compartmentalized microfluidic reaction modules equipped with self-immobilizing biocatalysts would be of great utility for numerous biocatalytic and even chemo-enzymatic cascade reactions under continuous flow conditions.
All-enzyme hydrogels are biocatalytic materials, with which various enzymes can be immobilized in microreactors in a simple, mild, and efficient manner to be used for continuous flow processes. Here we present the construction and application of a cofactor regenerating hydrogel based on the imine reductase GF3546 from Streptomyces sp. combined with the cofactor regenerating glucose-1-dehydrogenase from Bacillus subtilis. The resulting hydrogel materials were characterized in terms of binding kinetics and viscoelastic properties. The materials were formed by rapid covalent crosslinking in less than 5 min, and they showed a typical mesh size of 67 ± 2 nm. The gels were applied for continuous flow biocatalysis. In a microfluidic reactor setup, the hydrogels showed excellent conversions of imines to amines for up to 40 h in continuous flow mode. Variation of flow rates led to a process where the gels showed a maximum space-time-yield of 150 g·(L·day)−1 at 100 μL/min.
Continuous flowbiocatalysis is an emerging field of industrial biotechnology that uses enzymes immobilized in flowchannels for the production of value-added chemicals.We describe the construction of self-assembling all-enzyme hydrogels that are comprised of two tetrameric enzymes.T he stereoselective dehydrogenase LbADH and the cofactorregenerating glucose 1-dehydrogenase GDH were genetically fused with aS pyTago rS pyCatcher domain, respectively,t o generate two complementary homo-tetrameric building blocks that polymerizeu nder physiological conditions into porous hydrogels.M ounted in microfluidic reactors,t he gels show excellent stereoselectivity with near quantitative conversion in the reduction of prochiral ketones along with high robustness under process and storage conditions.T he gels function as compartment that retains intermediates thus enabling high total turnover numbers of the expensive cofactor NADP(H).
All-enzyme hydrogel (AEH) particles with a hydrodynamic diameter of up to 120 nm were produced intracellularly with an Escherichia coli-based in vivo system. The inCell-AEH nanoparticles were generated from polycistronic vectors enabling simultaneous expression of two interacting enzymes, the Lactobacillus brevis alcohol dehydrogenase (ADH) and the Bacillus subtilis glucose-1-dehydrogenase (GDH), fused with a SpyCatcher or SpyTag, respectively. Formation of inCell-AEH was analyzed by dynamic light scattering and atomic force microscopy. Using the stereo-selective two-step reduction of a prochiral diketone substrate, we show that the inCell-AEH approach can be advantageously used in whole-cell flow biocatalysis, by which flow reactors could be operated for > 4 days under constant substrate perfusion. More importantly, the inCell-AEH concept enables the recovery of efficient catalyst materials for stable flow bioreactors in a simple and economical one-step procedure from crude bacterial lysates. We believe that our method will contribute to further optimization of sustainable biocatalytic processes.
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