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).

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

“…The in‐depth comparison of the enzymatic activities in solution revealed that the His‐LbADH variant showed a 13 % lower activity than a previously described LbADH variant with a 39‐amino acid SBP‐tag and a 30 % higher activity than an LbADH variant that contained the 113‐amino acid SpyCatcher domain for specific immobilization. The purified GDH‐His enzyme variant showed a more than threefold higher specific activity than a previously described GDH‐SBP variant ; however, the activity was still 20 % lower than that of a variant fused with the SpyTag (13‐amino acid) peptide . Although all enzyme activities found here are in the same order of magnitude, the slight changes nevertheless clearly emphasize that genetic fusion is often accompanied by changes in the specific activity.…”

“…The His‐tagged LbADH and GDH enzymes were cloned, heterologously overexpressed in E. coli and purified to homogeneity using IMAC as previously described . The purity of the enzymes was found to be > 95 %, as determined by SDS‐PAGE with subsequent Coomassie staining (Fig.…”

“…Expression, purification, and characterization of the proteins were done as previously described [24,25]. In brief, Escherichia coli BL21(DE3) cells were transformed with the corresponding expression vectors using electroporation.…”

“…In comparison to other NRE systems, such as formate/ lactate decarboxylases, GDH does not produce CO 2 gas bubbles, which are detrimental for microfluidic processes. The His-tagged LbADH and GDH enzymes were cloned, heterologously overexpressed in E. coli and purified to homogeneity using IMAC as previously described [25]. The purity of the enzymes was found to be > 95 %, as determined by SDS-PAGE with subsequent Coomassie staining (Fig.…”

3D‐Printed Phenacrylate Decarboxylase Flow Reactors for the Chemoenzymatic Synthesis of 4‐Hydroxystilbene

“…[3a, 4] For the realization of such compartmentalized biocatalytic processes in continuous flow,t he fast and simple immobilization of enzymes inside the microstructured flow reactorsi sa ne ssential step to ensure efficients patials eparation of different reactions. [5] To enable the simple rapid prototyping of biocatalytic reaction modules for compartmentalized flow microreactors, we have recently established af abrication process using direct 3D printing of inexpensive biodegradable agarose-based hydrogel bioinks containingt hermostable enzymes( Figure 1). [6] 3D printing( also known as additive manufacturing) is af abrication process employing layer-by-layer deposition of materials to produce complex 3D structures with ah igh flexibility in designa nd minimal waste of material, [7] which has found applications ranging from the aerospace and constructioni ndustry to smart materials, medicine and biotechnology.…”