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End date: 2024 × Start date: 2020 × DDC: 570 × Publisher: New York, NY : Wiley × WGL Institute: DWI ×

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Now showing 1 - 3 of 3
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    Display of functional nucleic acid polymerase on Escherichia coli surface and its application in directed polymerase evolution
    (New York, NY : Wiley, 2020) Chung, Mu-En; Goroncy, Kati; Kolesnikova, Alisa; Schönauer, David; Schwaneberg, Ulrich
    We report a first of its kind functional cell surface display of nucleic acid polymerase and its directed evolution to efficiently incorporate 2′-O-methyl nucleotide triphosphates (2′-OMe-NTPs). In the development of polymerase cell surface display, two autotransporter proteins (Escherichia coli adhesin involved in diffuse adherence and Pseudomonas aeruginosa esterase A [EstA]) were employed to transport and anchor the 68-kDa Klenow fragment (KF) of E. coli DNA polymerase I on the surface of E. coli. The localization and function of the displayed KF were verified by analysis of cell outer membrane fractions, immunostaining, and fluorometric detection of synthesized DNA products. The EstA cell surface display system was applied to evolve KF for the incorporation of 2′-OMe-NTPs and a KF variant with a 50.7-fold increased ability to successively incorporate 2′-OMe-NTPs was discovered. Expanding the scope of cell-surface displayable proteins to the realm of polymerases provides a novel screening tool for tailoring polymerases to diverse application demands in a polymerase chain reaction and sequencing-based biotechnological and medical applications. Especially, cell surface display enables novel polymerase screening strategies in which the heat-lysis step is bypassed and thus allows the screening of mesophilic polymerases with broad application potentials ranging from diagnostics and DNA sequencing to replication of synthetic genetic polymers. © 2020 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals LLC
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    A scalable bubble-free membrane aerator for biosurfactant production
    (New York, NY : Wiley, 2021) Bongartz, Patrick; Bator, Isabel; Baitalow, Kristina; Keller, Robert; Tiso, Till; Blank, Lars Mathias; Wessling, Matthias
    The bioeconomy is a paramount pillar in the mitigation of greenhouse gas emissions and climate change. Still, the industrialization of bioprocesses is limited by economical and technical obstacles. The synthesis of biosurfactants as advanced substitutes for crude-oil-based surfactants is often restrained by excessive foaming. We present the synergistic combination of simulations and experiments towards a reactor design of a submerged membrane module for the efficient bubble-free aeration of bioreactors. A digital twin of the combined bioreactor and membrane aeration module was created and the membrane arrangement was optimized in computational fluid dynamics studies with respect to fluid mixing. The optimized design was prototyped and tested in whole-cell biocatalysis to produce rhamnolipid biosurfactants from sugars. Without any foam formation, the new design enables a considerable higher space-time yield compared to previous studies with membrane modules. The design approach of this study is of generic nature beyond rhamnolipid production.
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    Matter-tag: A universal immobilization platform for enzymes on polymers, metals, and silicon-based materials
    (New York, NY : Wiley, 2020) Dedisch, Sarah; Wiens, Annika; Davari, Mehdi D.; Söder, Dominik; Rodriguez-Emmenegger, Cesar; Jakob, Felix; Schwaneberg, Ulrich
    Enzyme immobilization is extensively studied to improve enzyme properties in catalysis and analytical applications. Here, we introduce a simple and versatile enzyme immobilization platform based on adhesion-promoting peptides, namely Matter-tags. Matter-tags immobilize enzymes in an oriented way as a dense monolayer. The immobilization platform was established with three adhesion-promoting peptides; Cecropin A (CecA), liquid chromatography peak I (LCI), and Tachystatin A2 (TA2), that were genetically fused to enhanced green fluorescent protein and to two industrially important enzymes: a phytase (from Yersinia mollaretii) and a cellulase (CelA2 from a metagenomic library). Here, we report a universal and simple Matter-tag–based immobilization platform for enzymes on various materials including polymers (polystyrene, polypropylene, and polyethylene terephthalate), metals (stainless steel and gold), and silicon-based materials (silicon wafer). The Matter-tag–based enzyme immobilization is performed at ambient temperature within minutes (<10 min) in an aqueous solution harboring the phytase or cellulase by immersing the targeted material. The peptide LCI was identified as universal adhesion promoter; LCI immobilized both enzymes on all investigated materials. The attachment of phytase-LCI onto gold was characterized with surface plasmon resonance spectroscopy obtaining a dissociation constant value (KD) of 2.9·10−8 M and a maximal surface coverage of 504 ng/cm². © 2019 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals, Inc.