Self‐Assembling All‐Enzyme Hydrogels for Flow Biocatalysis

Abstract

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).

A Versatile Toolkit for Controllable and Highly Selective Multifunctionalization of Bacterial Magnetic Nanoparticles

“…For instance, the use of immobilized enzymes (on hydrogels or microbeads) in biocatalytic flow‐reactor concepts was investigated, revealing the need for appropriate methods for the immobilization of enzymes. [ 48,49 ] Likewise, magnetosomes might serve as a versatile scaffold for the spatial organization of different catalytic activities, thereby enabling cascade reactions through the close proximity of the constituting enzyme species and balanced substrate channeling rates. [ 50 ] The feasibility to use functionalized magnetosomes as reusable, multimodal catalysts could be demonstrated in this study by incorporating MamA‐mEGFP_MamG‐GOx_MamF‐GusA_MamC‐RBP magnetosomes into mCherry‐equipped hydrogel‐based matrices (Figure 5), which resulted in a model magnetic composite material with different catalytic activities.…”

Characterization and evaluation of immobilized enzymes for applications in flow reactors

“…Contrarily, irreversible immobilization can guarantee stable binding without enzyme leaching, although recyclability of the reactor/material might be problematic. Orthogonal or self-immobilizing techniques using Spy, Halo and streptavidin protein motifs, and formylglycine-generating enzymes have been used in microfluidic bed reactors or in wall-coated reactors [19][20][21][22][23]. HaloTag™ technology based on halogenases domains promotes a quick irreversible immobilization on porous beads that can be readily integrated into PBRs, with 40-65 % recovered activity and STYs of 1.58 g -1 x L -1 x h -1 [14,15].…”

“…Alternatively, carrier-free immobilization has been proven very effective to achieve high enzyme loadings. Herein, the protein aggregation can be genetically programmed by protein domains fused to the enzymes, in order to trigger the self-assembly of a 3D gel network within microreactors [19][20][21]. Such packed-bed retains several dehydrogenases and NADH to continuously asymmetrically reduce prochiral ketones [20].…”

Status of Biocatalysis in the Production of 2,5-Furandicarboxylic Acid

“…As compared with batch reactions, the traditional approach for biocatalytic processes, continuous flow microreactors offer superior mass transfer and control and thus may serve to significantly accelerate biotransformations . Furthermore, continuous flow compartmentalized microfluidic reactors with immobilized enzymes may be an advantageous approach in enzyme cascade reactions. − …”