Self-Immobilizing Biocatalysts Maximize Space–Time Yields in Flow Reactors

Abstract

Maximizing space–time yields (STY) of biocatalytic flow processes is essential for the establishment of a circular biobased economy. We present a comparative study in which different biocatalytic flow reactor concepts were tested with the same enzyme, the (R)-selective alcohol dehydrogenase from Lactobacillus brevis (LbADH), that was used for stereoselective reduction of 5-nitrononane-2,8-dione. The LbADH contained a genetically encoded streptavidin (STV)-binding peptide to enable self-immobilization on STV-coated surfaces. The purified enzyme was immobilized by physisorption or chemisorption as monolayers on the flow channel walls, on magnetic microbeads in a packed-bed format, or as self-assembled all-enzyme hydrogels. Moreover, a multilayer biofilm with cytosolic-expressed LbADH served as a whole-cell biocatalyst. To enable cross-platform comparison, STY values were determined for the various reactor modules. While mono- and multilayer coatings of the reactor surface led to STY < 10, higher productivity was achieved with packed-bed reactors (STY ≈ 100) and the densely packed hydrogels (STY > 450). The latter modules could be operated for prolonged times (>6 days). Given that our approach should be transferable to other enzymes, we anticipate that compartmentalized microfluidic reaction modules equipped with self-immobilizing biocatalysts would be of great utility for numerous biocatalytic and even chemo-enzymatic cascade reactions under continuous flow conditions.

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.…”

The rise of continuous flow biocatalysis – fundamentals, very recent developments and future perspectives

“…The total turnover number (TTN) refers to NADP + consumption. 91 To regenerate the cofactor NADPH, the authors used three different strategies within their study (cf. Scheme 1i)-iii)).…”

“…5). 93 91 The NADPH cofactor regeneration can be implemented (i) as substrate coupled cofactor regeneration utilizing the same enzyme by the oxidation of isopropanol to acetone, (ii) within the host's native cellular metabolisms in the case of whole-cell biocatalysis or (iii) as enzyme coupled cofactor regeneration using a second enzyme e.g. in this case glucose dehydrogenase (GDH).…”

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

“…Given this volume and a flow rate of 1 μL min -1 , the typical residence time was 7 min. Although the performance of packed-bed reactors depends on the particle packing and liquid flow patterns, we used the maximum reactor volume of 10 μL to calculate the space-time yield (STY) for a simplified approach and to allow comparison with earlier work [24,48]. The filled channels were connected with short PTFE tubing (internal diameter 0.5 mm) using Mini Luer plugs (microfluidic ChipShop).…”