Self-assembling all-enzyme hydrogels for biocatalytic flow processes

Peschke, Theo; Gallus, Sabrina; Bitterwolf, Patrick; Hu, Yunxia; Oelschlaeger, Claude; Willenbacher, Norbert; Rabe, Kersten S.; Cm, Niemeyer

doi:10.1101/240325

Cited by 2 publications

(1 citation statement)

References 46 publications

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“…Industrial-scale biocatalysis can be enhanced by the sequestration/immobilisation of valuable enzymes 2 in continuous flow biocatalysis, 8,9 whilst also offering potential synergistic effects through the co-localisation of specific enzymes. 10 Proteins immobilised on surfaces have also emerged as useful therapeutic agents, enhancing in vivo half-life and providing extra control over drug delivery (both temporally and spatially) 1,11 . Despite the demonstrated utility of immobilised proteins across multiple fields, the methods of immobilisation are highly diverse and typically bespoke.…”

Section: Introductionmentioning

confidence: 99%

Gigavalent display of proteins on monodisperse polyacrylamide hydrogels as a versatile modular platform for functional assays and protein engineering

Fryer

Rogers

Mellor

et al. 2021

Preprint

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The robust modularity of biological components that are assembled into complex functional systems is central to synthetic biology. Here we apply modular “plug and play” design principles to a microscale solid phase protein display system that enables protein purification and functional assays for biotherapeutics. Specifically, we capture protein molecules from cell lysates on polyacrylamide hydrogel display beads (‘PHD beads’), made in microfluidic droplet generators. These monodisperse PHD beads are decorated with predefined amounts of anchors, methacrylate-PEG-benzylguanine (BG) and methacrylate-PEG-chloroalkane (CA). Anchors form covalent bonds with fusion proteins bearing cognate tag recognition (SNAP and Halo-tags) in specific, orthogonal and stable fashion. Given that these anchors are copolymerised throughout the 3D structure of the beads, proteins are also distributed across the entire bead sphere, allowing attachment of ∼109 protein molecules per bead (Ø 20 μm). This mode of attachment reaches a higher density than possible on widely used surface-modified beads, and additionally mitigates surface effects that often complicate studies with proteins on beads. We showcase a diverse array of protein modules that enable the secondary capture of proteins, either non-covalently (IgG and SUMO-tag) or covalently (SpyCatcher, SpyTag, SnpCatcher and SnpTag). Proteins can be displayed in their monomeric forms, but also reformatted as a multivalent display (using secondary capture modules that create branches) to test the contributions of avidity and multivalency towards protein function. Finally, controlled release of modules by irradiation of light is achieved by incorporating the photocleavable protein PhoCl: irradiation severs the displayed protein from the solid support, so that functional assays can be carried out in solution. As a demonstration of the utility of valency engineering, an antibody drug screen is performed, in which an anti-TRAIL-R1 scFv protein is released into solution as monomers-hexamers, showing a ∼50-fold enhanced potency in the pentavalent format. The ease of protein purification on solid support, quantitative control over presentation and release of proteins and choice of valency make this experimental format a versatile, modular platform for large scale functional analysis of proteins, in bioassays of protein-protein interactions, enzymatic catalysis and bacteriolysis.Table of Contents Graphics

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Section: Introductionmentioning

confidence: 99%

Gigavalent display of proteins on monodisperse polyacrylamide hydrogels as a versatile modular platform for functional assays and protein engineering

Fryer

Rogers

Mellor

et al. 2021

Preprint

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Flow Process for Ketone Reduction Using a Superabsorber-Immobilized Alcohol Dehydrogenase from Lactobacillus brevis in a Packed-Bed Reactor

Adebar

Gröger

2019

Bioengineering

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Flow processes and enzyme immobilization have gained much attention over the past few years in the field of biocatalytic process design. Downstream processes and enzyme stability can be immensely simplified and improved. In this work, we report the utilization of polymer network-entrapped enzymes and their applicability in flow processes. We focused on the superabsorber-based immobilization of an alcohol dehydrogenase (ADH) from Lactobacillus brevis and its application for a reduction of acetophenone. The applicability of this immobilization technique for a biotransformation running in a packed bed reactor was then demonstrated. Towards this end, the immobilized system was intensively studied, first in a batch mode, leading to >90% conversion within 24 h under optimized conditions. A subsequent transfer of this method into a flow process was conducted, resulting in very high initial conversions of up to 67% in such a continuously running process.were described by further groups using carrier-bound ADH from Lactobacillus brevis (LB-ADH) in combination with a mobile aqueous phase [30], or magnetic nanoparticle-bound ADH [31]. In addition, Buehler et al. investigated intensively the use of ADH in aqueous/organic segmented flow systems. It was shown that emulsion formation could be eliminated, and enzymatic mass transfer-limited reactions can be enhanced [32]. Furthermore, different means of stabilization of this reaction system were investigated [33]. Very recently, our group published an improved downstream process by means of such a segmented flow process, illustrated using two different ADHs and substrates [34].In continuous processes using packed bed reactors (PBR), catalyst immobilization is a prerequisite. Also, downstream processes benefit from immobilization, as separation of the reaction mixture from the catalyst is easier, and the catalyst reusability is improved [35]. Moreover, both, operational, as well as storage stability of the catalyst can potentially be significantly increased. However, often a significant loss of enzymatic activity during the immobilization process represents a drawback, and the reproducible production of immobilized catalyst may be a further challenge [36]. Numerous techniques have been developed over the past years, which can be classified in the following three major methods: (a) binding to a solid support (carrier), (b) entrapment (encapsulation) in polymer networks, or (c) cross-linking in the form of cross-linked enzyme aggregates (CLEAs) or cross-linked enzyme crystals (CLECs) [35]. Recently, much progress in the field of enzyme immobilization has been made [37][38][39][40][41].In this work, the option of entrapment of enzymes in polymer networks has been chosen. Often for this purpose, organic polymers containing cavities, sol-gel-processed particles, or membranes are used. A special case is the use of superabsorbent polymers to immobilize the entire aqueous phase. [42,43]. In this case, the enzyme is immobilized in its native aqueous environment, whereby undesired interact...

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Self-assembling all-enzyme hydrogels for biocatalytic flow processes

Cited by 2 publications

References 46 publications

Gigavalent display of proteins on monodisperse polyacrylamide hydrogels as a versatile modular platform for functional assays and protein engineering

Gigavalent display of proteins on monodisperse polyacrylamide hydrogels as a versatile modular platform for functional assays and protein engineering

Flow Process for Ketone Reduction Using a Superabsorber-Immobilized Alcohol Dehydrogenase from Lactobacillus brevis in a Packed-Bed Reactor

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