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).
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.
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