Self‐Immobilizing Oxidoreductases for Flow Biocatalysis in Miniaturized Packed‐Bed Reactors

Abstract

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.

Efficient Nicotinamide Adenine Dinucleotide Phosphate [NADP(H)] Recycling in Closed‐Loop Continuous Flow Biocatalysis

“…Different cofactor regeneration methods are used, but since it is not recirculated but washed out, the cofactor consumption and costs, respectively, will scale proportional to the substrate amount. [7] The second concept towards continuous cofactor regeneration (Scheme 1.C) utilises (co-)immobilised or (co-)entrapped cofactor, allowing a stream of substrate, buffer etc. to pass by and perform several reaction cycles.…”

Semi-Continuous Flow Biocatalysis with Affinity Co-Immobilized Ketoreductase and Glucose Dehydrogenase

“…Peschke and colleagues [ 34 ] constructed a compartmentalised microfluidic packed-bed reactor loaded with enzyme-functionalised magnetic beads (MB). The beads were functionalised with cobalt (Co 2+ ), for selective binding of the His-tagged enzymes: ( R )-selective alcohol dehydrogenase and glucose dehydrogenase via formation of a coordination (affinity) bond.…”

“…So, this type of coordination bond can be categorized in moderate affinity within K d range between 10 −4 and 10 −7 M. The typical values of K d for antibodies are in the nM (10 −9 M) ranges. Despite a few drawbacks, such a relatively moderate affinity ( K d ~1 µM) [ 31 ], low specificity [ 32 ], and possible antagonistic effects on the activity of enzymes that contain divalent ions [ 33 ] His-tag can still be used for direct enzyme immobilization from crude cell extracts [ 34 ]. In principle, we cannot exclude the possibility of the affinity tag interfering with protein activity [ 35 ], although the relatively small size and charge of poly-His tag ensure that the protein is rarely affected.…”

The Hitchhiker's guide to biocatalysis: recent advances in the use of enzymes in organic synthesis

“…251 Another important technological development is the use of ow chemistry and microuidics. [252][253][254][255] This will facilitate the design of biocatalytic cascade reactions as these can easily be performed in series in a ow reactor. Moreover, the protective and low shear environment of micro-reactors will mean that less effort will be required for increasing the robustness of the enzymes.…”