Abstract
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.
Highlights
Biocatalysis is emerging as a powerful approach for the production of active pharmaceutical ingredients (APIs) [1]
We have recently developed all-enzyme hydrogels that allow the entire reactor room to be filled with biocatalytic material
To demonstrate the scope of this all enzyme hydrogel concept and to open it for further biocatalytic transformations, we report here on a novel hydrogel based on the nicotinamide adenine dinucleotide phosphate (NADPH) dependent (S)-selective dimeric imine reductase (IRED) GF3546 from Streptomyces sp. [19] in combination with the NADPH cofactor regenerating homotetrameric glucose-1-dehydrogenase from Bacillus subtilis
Summary
Biocatalysis is emerging as a powerful approach for the production of active pharmaceutical ingredients (APIs) [1]. The improved mass transport between individual compartments simplifies the addition of substrates, the separation of side products, and the avoidance of undesired crosstalk between various reaction steps [10,11] This approach opens up numerous possibilities for the integration of automated in-line analysis, purification, and downstream processing. The use of carrier materials represents a compromise to increase the effective surface area for immobilization This inevitably reduces space-time yields, as the carrier material occupies a significant space in the reactor volume. To avoid this problem, we have recently developed all-enzyme hydrogels that allow the entire reactor room to be filled with biocatalytic material. Only few examples of IREDs immobilized on carrier materials have been reported so far and none have been used for continuous flow biocatalysis [22,23]
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