123 results on '"Schwaneberg, Ulrich"'
Search Results
2. In Silico and Experimental ADAM17 Kinetic Modeling as Basis for Future Screening System for Modulators
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Bienstein, Marian, Minond, Dmitriy, Schwaneberg, Ulrich, Davari, Mehdi D., and Yildiz, Daniela
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biocatalysis ,QH301-705.5 ,Drug Evaluation, Preclinical ,ADAM17 Protein ,metalloproteinases ,Substrate Specificity ,Catalytic Domain ,Humans ,Computer Simulation ,Protease Inhibitors ,Biology (General) ,QD1-999 ,inhibitor design ,ADAM17 ,Binding Sites ,molecular docking ,exosite inhibitors ,High-Throughput Screening Assays ,ADAM Proteins ,Kinetics ,Chemistry ,HEK293 Cells ,ddc:540 ,kinetic modelling ,reaction mechanism - Abstract
International journal of molecular sciences 23(3), 1368 (2022). doi:10.3390/ijms23031368 special issue: "Special Issue "Natural, Designed and Engineered Metalloenzymes: Structure, Catalytic Mechanisms and Applications" / Special Issue Editors: Prof. Dr. Angela Lombardi, Guest Editor; Prof. Flavia Nastri, Guest Editor; Dr. Linda Leone, Guest Editor", Published by Molecular Diversity Preservation International, Basel
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- 2022
3. Optimized Hemolysin Type 1 Secretion System in Escherichia coli by Directed Evolution of the Hly Enhancer Fragment and Including a Terminator Region
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Pourhassan N., Zohreh, Cui, Haiyang, Khosa, Sakshi, Davari, Mehdi D., Jaeger, Karl-Erich, Smits, Sander H. J., Schwaneberg, Ulrich, and Schmitt, Lutz
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Terminator Regions, Genetic ,Hemolysin Proteins ,Type I Secretion Systems ,Escherichia coli Proteins ,Organic Chemistry ,ddc:540 ,Escherichia coli ,Molecular Medicine ,Protein Engineering ,Molecular Biology ,Biochemistry - Abstract
ChemBioChem : a European journal of chemical biology e202100702 (2022). doi:10.1002/cbic.202100702, Published by Wiley-VCH, Weinheim
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- 2022
4. Critical assessment of structure-based approaches to improve protein resistance in aqueous ionic liquids by enzyme-wide saturation mutagenesis
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El Harrar, Till, Davari, Mehdi D., Jaeger, Karl-Erich, Schwaneberg, Ulrich, and Gohlke, Holger
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ddc:570 ,Protein stability ,Site-saturation mutagenesis ,Protein engineering ,Bacillus subtilis lipase A ,TP248.13-248.65 ,ComputingMethodologies_COMPUTERGRAPHICS ,Research Article ,Ionic liquids ,Biotechnology - Abstract
Graphical abstract, Ionic liquids (IL) and aqueous ionic liquids (aIL) are attractive (co-)solvents for green industrial processes involving biocatalysts, but often reduce enzyme activity. Experimental and computational methods are applied to predict favorable substitution sites and, most often, subsequent site-directed surface charge modifications are introduced to enhance enzyme resistance towards aIL. However, almost no studies evaluate the prediction precision with random mutagenesis or the application of simple data-driven filtering processes. Here, we systematically and rigorously evaluated the performance of 22 previously described structure-based approaches to increase enzyme resistance to aIL based on an experimental complete site-saturation mutagenesis library of Bacillus subtilis Lipase A (BsLipA) screened against four aIL. We show that, surprisingly, most of the approaches yield low gain-in-precision (GiP) values, particularly for predicting relevant positions: 14 approaches perform worse than random mutagenesis. Encouragingly, exploiting experimental information on the thermostability of BsLipA or structural weak spots of BsLipA predicted by rigidity theory yields GiP = 3.03 and 2.39 for relevant variants and GiP = 1.61 and 1.41 for relevant positions. Combining five simple-to-compute physicochemical and evolutionary properties substantially increases the precision of predicting relevant variants and positions, yielding GiP = 3.35 and 1.29. Finally, combining these properties with predictions of structural weak spots identified by rigidity theory additionally improves GiP for relevant variants up to 4-fold to ∼10 and sustains or increases GiP for relevant positions, resulting in a prediction precision of ∼90% compared to ∼9% in random mutagenesis. This combination should be applicable to other enzyme systems for guiding protein engineering approaches towards improved aIL resistance.
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- 2022
5. Kill&Repel Coatings: The Marriage of Antifouling and Bactericidal Properties to Mitigate and Treat Wound Infections
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Garay-Sarmiento, Manuela, Witzdam, Lena, Vorobii, Mariia, Simons, Christian, Herrmann, Niklas, de los Santos Pereira, Andres, Heine, Elisabeth, El-Awaad, Islam, Lütticken, Rudolf, Jakob, Felix, Schwaneberg, Ulrich, and Rodriguez-Emmenegger, Cesar
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ddc:530 - Abstract
Advanced functional materials 32(9), 2106656 (2021). doi:10.1002/adfm.202106656, Published by Wiley-VCH, Weinheim
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- 2022
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6. A novel metagenome-derived viral RNA polymerase and its application in a cell-free expression system for metagenome screening
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Han, Yuchen, Kinfu, Birhanu M, Blombach, Fabian, Cackett, Gwenny, Zhang, Hongli, Pérez-García, Pablo, Krohn, Ines, Salomon, Jesper, Besirlioglu, Volkan, Mirzaeigarakani, Tayebeh, Schwaneberg, Ulrich, Chow, Jennifer, Werner, Finn, and Streit, Wolfgang R
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Multidisciplinary ,Cell-Free System ,Escherichia coli ,Cell-free ,Metagenome ,RNA, Viral ,Viral Replicase Complex Proteins ,DNA-Directed RNA Polymerases ,Metagenomics ,Enzyme production ,ddc:600 - Abstract
Scientific reports 12, 17882 (2022). doi:10.1038/s41598-022-22383-x, Published by Macmillan Publishers Limited, part of Springer Nature, [London]
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- 2022
7. Natural Product Diversification by One-Step Biocatalysis using Human P450 3A4
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Fessner, Nico D., Grimm, Christopher, Srdi��, Matic, Weber, Hansj��rg, Kroutil, Wolfgang, Schwaneberg, Ulrich, and Glieder, Anton
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ddc:540 - Abstract
ChemCatChem 14(1), e202101564 (2021). doi:10.1002/cctc.202101564, Published by WILEY-VCH Verlag, Weinheim
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- 2022
8. Aqueous ionic liquids redistribute local enzyme stability via long-range perturbation pathways
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El Harrar, Till, Frieg, Benedikt, Davari, Mehdi D., Jaeger, Karl-Erich, Schwaneberg, Ulrich, and Gohlke, Holger
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Molecular dynamics simulations ,ddc:570 ,Protein stability ,Biocatalysis ,Protein engineering ,Allostery ,TP248.13-248.65 ,ComputingMethodologies_COMPUTERGRAPHICS ,Research Article ,Biotechnology ,Ionic liquids - Abstract
Graphical abstract, Ionic liquids (IL) and aqueous ionic liquids (aIL) are attractive (co–)solvents for biocatalysis due to their unique properties. On the other hand, the incubation of enzymes in IL or aIL often reduces enzyme activity. Recent studies proposed various aIL-induced effects to explain the reduction, classified as direct effects, e.g., local dehydration or competitive inhibition, and indirect effects, e.g., structural perturbations or disturbed catalytic site integrity. However, the molecular origin of indirect effects has largely remained elusive. Here we show by multi-μs long molecular dynamics simulations, free energy computations, and rigidity analyses that aIL favorably interact with specific residues of Bacillus subtilis Lipase A (BsLipA) and modify the local structural stability of this model enzyme by inducing long-range perturbations of noncovalent interactions. The perturbations percolate over neighboring residues and eventually affect the catalytic site and the buried protein core. Validation against a complete experimental site saturation mutagenesis library of BsLipA (3620 variants) reveals that the residues of the perturbation pathways are distinguished sequence positions where substitutions highly likely yield significantly improved residual activity. Our results demonstrate that identifying these perturbation pathways and specific IL ion-residue interactions there effectively predicts focused variant libraries with improved aIL tolerance.
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- 2021
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9. A Photoclick-Based High-Throughput Screening for the Directed Evolution of Decarboxylase OleT
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Markel, Ulrich, Lanvers, Pia, Sauer, Daniel F., Wittwer, Malte, Dhoke, Gaurao V., Davari, Mehdi D., Schiffels, Johannes, and Schwaneberg, Ulrich
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decarboxylase ,directed evolution ,photoclick chemistry ,high-throughput screening ,P450 - Abstract
Enzymatic oxidative decarboxylation is an up-and-coming reaction yet lacking efficient screening methods for the directed evolution of decarboxylases. Here, we describe a simple photoclick assay for the detection of decarboxylation products and its application in a proof-of-principle directed evolution study on the decarboxylase OleT. The assay was compatible with two frequently used OleT operation modes (directly using hydrogen peroxide as the enzyme's co-substrate or using a reductase partner) and the screening of saturation mutagenesis libraries identified two enzyme variants shifting the enzyme's substrate preference from long chain fatty acids toward styrene derivatives. Overall, this photoclick assay holds promise to speed-up the directed evolution of OleT and other decarboxylases. © 2020 The Authors. Published by Wiley-VCH GmbH
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- 2021
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10. Directed Evolution of a Cp*RhIII‐Linked Biohybrid Catalyst Based on a Screening Platform with Affinity Purification
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Kato, Shunsuke, Onoda, Akira, Taniguchi, Naomasa, Schwaneberg, Ulrich, and Hayashi, Takashi
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ddc:540 - Abstract
ChemBioChem : a European journal of chemical biology (2020). doi:10.1002/cbic.202000681, Published by Wiley-VCH, Weinheim
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- 2021
11. Expression and Refolding of the Plant Chitinase From Drosera capensis for Applications as a Sustainable and Integrated Pest Management
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Sinelnikov, Igor G., Siedhoff, Niklas E., Chulkin, Andrey M., Zorov, Ivan N., Schwaneberg, Ulrich, Davari, Mehdi D., Sinitsyna, Olga A., Shcherbakova, Larisa A., Sinitsyn, Arkady P., and Rozhkova, Aleksandra M.
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ddc:570 - Abstract
Frontiers in Bioengineering and Biotechnology 9, 728501 (2021). doi:10.3389/fbioe.2021.728501, Published by Frontiers Media, Lausanne
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- 2021
12. Additional file 1 of Tunnel engineering for modulating the substrate preference in cytochrome P450BsβHI
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Shuaiqi Meng, Ruipeng An, Zhongyu Li, Schwaneberg, Ulrich, Ji, Yu, Davari, Mehdi D., Wang, Fang, Wang, Meng, Qin, Meng, Nie, Kaili, and Liu, Luo
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Data_FILES - Abstract
Additional file 1. Additional Tables and Figures.
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- 2021
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13. Can constraint network analysis guide the identification phase of KnowVolution? A case study on improved thermostability of an endo-��-glucanase
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Contreras, Francisca, Nutschel, Christina, Beust, Laura, Davari, Mehdi D., Gohlke, Holger, and Schwaneberg, Ulrich
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Cellulase ,onstraint network analysis ,Protein engineering ,Thermostability ,GH5 endoglucanase ,KnowVolution - Abstract
Cellulases are industrially important enzymes, e.g., in the production of bioethanol, in pulp and paper industry, feedstock, and textile. Thermostability is often a prerequisite for high process stability and improving thermostability without affecting specific activities at lower temperatures is challenging and often time-consuming. Protein engineering strategies that combine experimental and computational are emerging in order to reduce experimental screening efforts and speed up enzyme engineering campaigns. Constraint Network Analysis (CNA) is a promising computational method that identifies beneficial positions in enzymes to improve thermostability. In this study, we compare CNA and directed evolution in the identification of beneficial positions in order to evaluate the potential of CNA in protein engineering campaigns (e.g., in the identification phase of KnowVolution). We engineered the industrially relevant endoglucanase EGLII from Penicillium verruculosum towards increased thermostability. From the CNA approach, six variants were obtained with an up to 2-fold improvement in thermostability. The overall experimental burden was reduced to 40% utilizing the CNA method in comparison to directed evolution. On a variant level, the success rate was similar for both strategies, with 0.27% and 0.18% improved variants in the epPCR and CNA-guided library, respectively. In essence, CNA is an effective method for identification of positions that improve thermostability.
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- 2021
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14. ChemCatChem / Natural Product Diversification by One-Step Biocatalysis using Human P450 3A4
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Fessner, Nico D., Grimm, Christopher, Srdič, Matic, Weber, Hansjörg, Kroutil, Wolfgang, Schwaneberg, Ulrich, and Glieder, Anton
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cytochrome P450 enzymes ,rugdiscovery ,biocatalysi ,450 3A4 ,natural product diversification - Abstract
Efficient synthetic techniques for the diversification of natural products are incremental for drug discovery processes of the pharmaceutical industry because these complex bioactive compounds often require an adjustment of properties. Human liver P450 3A4, key player of the body's detoxification system and decisive factor of a drug's metabolic fate, is renowned for its broad substrate scope including many natural products. In this study, we investigated the synthetic potential of human P450 3A4 for the diversification of natural product classes and isolated the produced metabolites of six selected natural products at a preparative 100-mg scale. Aided by efficient expression levels in P. pastoris, this whole-cell biocatalyst was found to be highly effective at the intended job allowing the identification of a total of 31 authentic human metabolites, many of them for the first time. By revealing an unprecedented degree of diversification, this study extends the synthetic repertoire for efficient enzymatic natural product modification in a one-step fashion and adds a completely new view to an old enzyme traditionally used for inhibition and toxicology studies. Europäische Kommission 722390 Version of record
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- 2021
15. Can constraint network analysis guide the identification phase of KnowVolution? A case study on improved thermostability of an endo-β-glucanase
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Contreras Leiva, Francisca, Nutschel, Christina, Beust, Laura, Davari, Mehdi D., Gohlke, Holger, and Schwaneberg, Ulrich
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MTP, 96-well microtiter plates ,CMC, carboxymethyl cellulose ,SSM, site-saturation mutagenesis ,AU, absorbance units ,HTS, high-throughput screening ,Constraint network analysis ,MD, molecular dynamics ,GH5 endoglucanase ,CNA, Constraint Network Analysis ,EGLII, endoglucanase II ,PCR, polymerase chain reaction ,Cellulase ,ddc:570 ,Protein engineering ,Thermostability ,TP248.13-248.65 ,Biotechnology ,ComputingMethodologies_COMPUTERGRAPHICS ,Research Article ,KnowVolution - Abstract
Graphical abstract, Cellulases are industrially important enzymes, e.g., in the production of bioethanol, in pulp and paper industry, feedstock, and textile. Thermostability is often a prerequisite for high process stability and improving thermostability without affecting specific activities at lower temperatures is challenging and often time-consuming. Protein engineering strategies that combine experimental and computational are emerging in order to reduce experimental screening efforts and speed up enzyme engineering campaigns. Constraint Network Analysis (CNA) is a promising computational method that identifies beneficial positions in enzymes to improve thermostability. In this study, we compare CNA and directed evolution in the identification of beneficial positions in order to evaluate the potential of CNA in protein engineering campaigns (e.g., in the identification phase of KnowVolution). We engineered the industrially relevant endoglucanase EGLII from Penicillium verruculosum towards increased thermostability. From the CNA approach, six variants were obtained with an up to 2-fold improvement in thermostability. The overall experimental burden was reduced to 40% utilizing the CNA method in comparison to directed evolution. On a variant level, the success rate was similar for both strategies, with 0.27% and 0.18% improved variants in the epPCR and CNA-guided library, respectively. In essence, CNA is an effective method for identification of positions that improve thermostability.
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- 2020
16. How to engineer organic solvent resistant enzymes : Insights from combined molecular dynamics and directed evolution study
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Cui, Haiyang, Stadtmüller, Tom Heinrich, Jiang, Qianjia, Jaeger, Karl-Erich, Schwaneberg, Ulrich, and Davari, Mehdi D.
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molecular dynamics simulation ,biocatalysis ,ddc:540 ,Bacillus subtilis Lipase A ,organic solvent ,directed evolution - Abstract
ChemCatChem 12(16), 4073-4083 (2020). doi:10.1002/cctc.202000422, Published by WILEY-VCH Verlag, Weinheim
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- 2020
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17. ValuePP - Phosphatrecycling aus Raps mit gekoppelter Valorisierung zu Polyphospha
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J J Christ, Herrmann, Kevin R., Ruff, Anna Joelle, Schoenauer, David, Cardinier, Kerstin, Buchold, Daniel, Schnee, Rainer, Schwaneberg, Ulrich, and Blank, Lars M.
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- 2019
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18. MOESM1 of Improved microscale cultivation of Pichia pastoris for clonal screening
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Eck, Alexander, Schmidt, Matthias, Hamer, Stefanie, Ruff, Anna, Fรถrster, Jan, Schwaneberg, Ulrich, Blank, Lars, Wiechert, Wolfgang, and Oldiges, Marco
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Additional file 1. Supplemental material.
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- 2018
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19. MOESM1 of Exploring the full natural diversity of single amino acid exchange reveals that 40â 60% of BSLA positions improve organic solvents resistance
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Frauenkron-Machedjou, Victorine, Fulton, Alexander, Zhao, Jing, Weber, Lina, Karl-Erich Jaeger, Schwaneberg, Ulrich, and Leilei Zhu
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Data_FILES - Abstract
Additional file 1. Supplymentary information.
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- 2018
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20. KnowVolution of the Polymer-Binding Peptide LCI for Improved Polypropylene Binding
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Rübsam, Kristin, Davari Dolatabadi, Mehdi, Jakob, Felix, and Schwaneberg, Ulrich
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lcsh:QD241-441 ,polymer-binding peptides ,lcsh:Organic chemistry ,ddc:540 ,immobilization ,anchor peptides ,directed evolution ,Article ,surface functionalization - Abstract
Polymers 10(4), 423 (2018). doi:10.18154/RWTH-2018-226349, Published by MDPI, Basel
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- 2018
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21. Improved microscale cultivation of Pichia pastoris for clonal screening
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Eck, Alexander, Schmidt, Matthias, Hamer, Stefanie Nicole, Ruff, Anna Joelle, Förster, Jan, Schwaneberg, Ulrich, Blank, Lars Mathias, Wiechert, Wolfgang, and Oldiges, Marco
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Pichia pastoris ,Research ,Fed-batch ,lcsh:Biotechnology ,lcsh:TP248.13-248.65 ,High throughput ,Screening ,ddc:630 ,Microbioreactor ,Phytase ,Bioprocess development - Abstract
Fungal Biology and Biotechnology 5(1), 8 (2018). doi:10.1186/s40694-018-0053-6, Published by BioMed Central, London
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- 2018
22. Are Directed Evolution Approaches Efficient in Exploring Nature’s Potential to Stabilize a Lipase in Organic Cosolvents?
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Schwaneberg, Ulrich Markel, Leilei Zhu, Victorine Frauenkron-Machedjou, Jing Zhao, Marco Bocola, Mehdi Davari, Karl-Erich Jaeger, and Ulrich
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protein engineering ,directed evolution ,gene saturation ,site-saturation mutagenesis ,lipase ,organic solvent resistance ,mutational diversity - Abstract
Despite the significant advances in the field of protein engineering, general design principles to improve organic cosolvent resistance of enzymes still remain undiscovered. Previous studies drew conclusions to engineer enzymes for their use in water-miscible organic solvents based on few amino acid substitutions. In this study, we conduct a comparison of a Bacillus subtilis lipase A (BSLA) library—covering the full natural diversity of single amino acid substitutions at all 181 positions of BSLA—with three state of the art random mutagenesis methods: error-prone PCR (epPCR) with low and high mutagenesis frequency (epPCR-low and high) as well as a transversion-enriched Sequence Saturation Mutagenesis (SeSaM-Tv P/P) method. Libraries were searched for amino acid substitutions that increase the enzyme’s resistance to the water-miscible organic cosolvents 1,4-dioxane (DOX), 2,2,2-trifluoroethanol (TFE), and dimethyl sulfoxide (DMSO). Our analysis revealed that 5%–11% of all possible single substitutions (BSLA site-saturation mutagenesis (SSM) library) contribute to improved cosolvent resistance. However, only a fraction of these substitutions (7%–12%) could be detected in the three random mutagenesis libraries. To our knowledge, this is the first study that quantifies the capability of these diversity generation methods generally employed in directed evolution campaigns and compares them to the entire natural diversity with a single substitution. Additionally, the investigation of the BSLA SSM library revealed only few common beneficial substitutions for all three cosolvents as well as the importance of introducing surface charges for organic cosolvent resistance—most likely due to a stronger attraction of water molecules.
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- 2017
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23. Are Directed Evolution Approaches Efficient in Exploring Nature’s Potential to Stabilize a Lipase in Organic Cosolvents?
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Markel, Ulrich, Zhu, Leilei, Frauenkron-Machedjou, Victorine Josiane, Zhao, Jing, Bocola, Marco, Davari, Mehdi, Jaeger, Karl-Erich, and Schwaneberg, Ulrich
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ddc:540 - Abstract
Catalysts : open access journal 7(5), 142 (1-18) (2017). doi:10.18154/RWTH-2017-04328 special issue: "Special Issue "Catalysis in Innovative Solvents" / Guest Editor Prof. Dr. Montserrat Gómez Université de Toulouse 3 - Paul Sabatier, Laboratoire Hétérochimie Fondamentale et Appliquée, Guest Editor Dr. Daniel Pla Université de Toulouse 3 - Paul Sabatier, Laboratoire Hétérochimie Fondamentale et Appliquée", Published by MDPI, Basel
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- 2017
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24. 2-Methyl-2,4-pentanediol (MPD) boosts as detergent-substitute the performance of ß-barrel hybrid catalyst for phenylacetylene polymerization
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Bocola, Marco, Arlt, Marcus, Garakani, Tayebeh Mirzaei, Thiel, Andreas, Beckerle, Klaus, Polen, Tino, Okuda, Jun, and Schwaneberg, Ulrich
- Abstract
Beilstein journal of organic chemistry 13, 1498-1506 (2017). doi:10.3762/bjoc.13.148, Published by Beilstein-Institut zur Förderung der Chemischen Wissenschaften, Frankfurt, Main
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- 2017
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25. Grafting PNIPAAm from v-barrel shaped transmembrane nanopores
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Charan, Himanshu, Kinzel, Julia, Glebe, Ulrich, Anand, Deepak, Garakani, Tayebeh Mirzaei, Zhu, Leilei, Bocola, Marco, Schwaneberg, Ulrich, Böker, Alexander, and Publica
- Abstract
The research on protein-polymer conjugates by grafting from the surface of proteins has gained significant interest in the last decade. While there are many studies with globular proteins, membrane proteins have remained untouched to the best of our knowledge. In this study, we established the conjugate formation with a class of transmembrane proteins and grow polymer chains from the ferric hydroxamate uptake protein component A (FhuA; a v-barrel transmembrane protein of Escherichia coli). As the lysine residues of naturally occurring FhuA are distributed over the whole protein, FhuA was reengineered to have up to 11 lysines, distributed symmetrically in a rim on the membrane exposed side (outside) of the protein channel and exclusively above the hydrophobic region. Reengineering of FhuA ensures a polymer growth only on the outside of the v-barrel and prevents blockage of the channel as a result of the polymerization. A water-soluble initiator for controlled radical polymerization (CRP) was consecutively linked to the lysine residues of FhuA and N-isopropylacrylamide (NIPAAm) polymerized under copper-mediated CRP conditions. The conjugate formation was analyzed by using MALDI-ToF mass spectrometry, SDS-PAGE, circular dichroism spectroscopy, analytical ultracentrifugation, dynamic light scattering, transmission electron microscopy and size exclusion chromatography. Such conjugates combine the specific functions of the transmembrane proteins, like maintaining membrane potential gradients or translocation of substrates with the unique properties of synthetic polymers such as temperature and pH stimuli handles. FhuA-PNIPAAm conjugates will serve as functional nanosized building blocks for applications in targeted drug delivery, self-assembly systems, functional membranes and transmembrane protein gated nanoreactors.
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- 2016
26. Additional file 1: Figure S1. of Substrate thiophosphorylation by Arabidopsis mitogen-activated protein kinases
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Leissing, Franz, Nomoto, Mika, Bocola, Marco, Schwaneberg, Ulrich, Yasuomi Tada, Conrath, Uwe, and Beckers, Gerold
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Protein sequences of selected MPK3/6 substrates. Table S1. List of primers used for cloning. Table S2. List of primers used for site-directed mutagenesis. (DOCX 21Â kb)
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- 2016
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27. MOESM1 of Combinatorial optimization of synthetic operons for the microbial production of p-coumaryl alcohol with Escherichia coli
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Philana Van Summeren-Wesenhagen, Voges, Raphael, Dennig, Alexander, Sokolowsky, Sascha, Noack, Stephan, Schwaneberg, Ulrich, and Marienhagen, Jan
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InformationSystems_INFORMATIONSTORAGEANDRETRIEVAL ,ComputingMethodologies_DOCUMENTANDTEXTPROCESSING ,ComputingMilieux_COMPUTERSANDEDUCATION ,Data_FILES ,ComputerApplications_COMPUTERSINOTHERSYSTEMS - Abstract
Additional file 1. Supplementary Information.
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- 2015
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28. Combinatorial optimization of synthetic operons for the microbial production of p-coumaryl alcohol with Escherichia coli
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van Summeren-Wesenhagen, Philana V., Voges, Raphael, Dennig, Alexander, Sokolowsky, Sascha, Noack, Stephan, Schwaneberg, Ulrich, and Marienhagen, Jan
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Coumaric Acids ,Bioengineering ,Plant natural products ,Applied Microbiology and Biotechnology ,p-Coumaryl alcohol ,Synthetic operon ,Balanced gene expression ,Metabolic Engineering ,Operon ,Escherichia coli ,Phosphorothioate ,ddc:610 ,Propionates ,Technical Notes ,Biotechnology - Abstract
Microbial cell factories 14, 79 (2015). doi:10.1186/s12934-015-0274-9, Published by Biomed Central, London
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- 2015
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29. MIXed plastics biodegradation and UPcycling using microbial communities : EU Horizon 2020 project MIX-UP started January 2020
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Ballerstedt, Hendrik, Tiso, Till, Wierckx, Nick, Wei, Ren, Averous, Luc, Bornscheuer, Uwe, O���Connor, Kevin, Floehr, Tilman, Jupke, Andreas, Klankermayer, J��rgen, Liu, Luo, de Lorenzo, Victor, Narancic, Tanja, Nogales, Juan, Perrin, R��mi, Pollet, Eric, Prieto, Auxiliadora, Casey, William, Haarmann, Thomas, Sarbu, Alexandru, Schwaneberg, Ulrich, Xin, Fengxue, Dong, Weiliang, Xing, Jiamin, Chen, Guo-Qiang, Tan, Tianwei, Jiang, Min, and Blank, Lars M.
- Subjects
13. Climate action ,7. Clean energy ,12. Responsible consumption - Abstract
Environmental sciences Europe 33(1), 99 (2021). doi:10.1186/s12302-021-00536-5, Published by Springer, Berlin ; Heidelberg
30. Performance-enhanced Protease Variants
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Wieland Susanne, Siegert Petra, Connell Timothy, O., Maurer Karl-heinz, Ronny Martinez, Schwaneberg Ulrich, and Hellmuth Hendrik
31. Directed evolution of VanR biosensor specificity in yeast
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D'Ambrosio, Vasil, Pramanik, Subrata, Goroncy, Kati, Jakočiūnas, Tadas, Schönauer, David, Davari, Mehdi D., Schwaneberg, Ulrich, Keasling, Jay D., and Jensen, Michael K.
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13. Climate action
32. Cellulolytic RoboLector – towards an automated high-throughput screening platform for recombinant cellulase expression
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Mühlmann, Martina Julia, Kunze, Martin, Ribeiro, Joaquim, Geinitz, Bertram Michael, Lehmann, Christian, Schwaneberg, Ulrich, Commandeur, Ulrich Heinrich, and Büchs, Jochen
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Automation ,Environmental Engineering ,Cellulase ,ddc:570 ,Research ,High-throughput screening ,Microtiter plate ,Biomedical Engineering ,On-line monitoring ,Cell Biology ,Molecular Biology - Abstract
Background Cellulases are key player in the hydrolyzation of cellulose. Unfortunately, this reaction is slow and a bottleneck in the process chain from biomass to intermediates and biofuels due to low activities of the enzymes. To overcome this draw back, a lot of effort is put into the area of protein engineering, to modify these enzymes by directed evolution or rational design. Huge clone libraries are constructed and have to be screened for improved variants. High-throughput screening is the method of choice to tackle this experimental effort, but up to now only a few process steps are adapted to automated platforms and little attention has been turned to the reproducibility of clone rankings. Results In this study, an extended robotic platform is presented to conduct automated high-throughput screenings of clone libraries including preculture synchronization and biomass specific induction. Automated upstream, downstream and analytical process steps are described and evaluated using E. coli and K. lactis as model organisms. Conventional protocols for media preparation, cell lysis, Azo-CMC assay and PAHBAH assay are successfully adapted to automatable high-throughput protocols. Finally, a recombinant E. coli celA2 clone library was screened and a reliable clone ranking could be realized. Conclusion The RoboLector device is a suitable platform to perform all process steps of an automated high-throughput clone library screening for improved cellulases. On-line biomass growth measurement controlling liquid handling actions enables fair comparison of clone variants. Electronic supplementary material The online version of this article (doi:10.1186/s13036-016-0043-2) contains supplementary material, which is available to authorized users.
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33. Directed Evolution of P450 BM3 towards Functionalization of Aromatic O-Heterocycles
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Santos, Gustavo De Almeida, Dhoke, Gaurao V., Davari, Mehdi D., Ruff, Anna Joëlle, and Schwaneberg, Ulrich
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3. Good health - Abstract
International journal of molecular sciences 20(13), 3353 - (2019). doi:10.3390/ijms20133353, Published by Molecular Diversity Preservation International, Basel
34. Lipases With Increased Thermostability
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Herbst Daniela, Connell Timothy, O., Mussmann Nina, Schwaneberg Ulrich, Ronny Martinez, Lehmann Christian, Besirioglu Volkan, and Vojcic Ljubica
35. A hydroquinone-specific screening system for directed P450 evolution
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Weingartner, Alexandra M., Sauer, Daniel F., Dhoke, Gaurao V., Davari, Mehdi D., Ruff, Anna Joëlle, and Schwaneberg, Ulrich
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3. Good health - Abstract
Applied microbiology and biotechnology 102(22), 9657 - 9667 (2018). doi:10.1007/s00253-018-9328-3, Published by Springer, New York
36. Mutants Of Hydantoinase
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Osswald Steffen, Schuster Heiko, Roos Jurgen, Karau Andreas, Schwaneberg Ulrich, Ronny Martinez, Mundhada Hemanshu, and Holter Ursula
37. MuteinDB : the mutein database linking substrates, products and enzymatic reactions directly with genetic variants of enzymes
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Braun, Andreas, Halwachs, Bettina, Geier, Martina, Weinhandl, Katrin, Guggemos, Michael, Marienhagen, Jan, Ruff, Anna Joëlle, Schwaneberg, Ulrich, Rabin, Vincent, Torres Pazmiño, Daniel E., Thallinger, Gerhard G., and Glieder, Anton
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3. Good health - Abstract
Database : the journal of biological databases and curation bas028, (2012). doi:10.1093/database/bas028, Published by Oxford Univ. Press, Oxford
38. MIXed plastics biodegradation and UPcycling using microbial communities : EU Horizon 2020 project MIX-UP started January 2020
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William Casey, Eric Pollet, Auxiliadora Prieto, Luc Avérous, Ulrich Schwaneberg, Luo Liu, Nick Wierckx, Lars M. Blank, Min Jiang, Till Tiso, Uwe T. Bornscheuer, Juan Nogales, Fengxue Xin, Kevin E. O’Connor, Tianwei Tan, Ren Wei, Weiliang Dong, Hendrik Ballerstedt, Guo Qiang Chen, Jiamin Xing, Tanja Narancic, Sarbu Alexandru, Tilman Floehr, Andreas Jupke, Thomas Haarmann, Rémi Perrin, Víctor de Lorenzo, Jürgen Klankermayer, European Commission, National Natural Science Foundation of China, Ballerstedt, Hendrik, Tiso, Till, Wierckx, Nick, Avérous, L., Bornscheuer, Uwe T., Jupke, Andreas, Klankermayer, Jürgen, Lorenzo, Víctor de, Narancic, Tanja, Nogales, Juan, Perrin, Rémi, Pollet, Eric, Prieto, María Auxiliadora, Haarmann, Thomas, Sarbu, Alexandru, Schwaneberg, Ulrich, Xin, Fengxue, Dong, Weiliang, Xing, Jiamin, Chen, Guo‑Qiang, Jiang, Min, Blank, Lars M., Ballerstedt, Hendrik [0000-0001-5729-1724], Tiso, Till [0000-0003-4420-5609], Wierckx, Nick [0000-0002-1590-1210], Avérous, L. [0000-0002-2797-226X], Bornscheuer, Uwe T. [0000-0003-0685-2696], Jupke, Andreas [0000-0001-6551-5695], Klankermayer, Jürgen [0000-0003-2143-9402], Lorenzo, Víctor de [0000-0002-6041-2731], Narancic, Tanja [0000-0003-3269-2200], Nogales, Juan [0000-0002-4961-0833], Perrin, Rémi [0000-0001-5917-8266], Pollet, Eric [0000-0002-4920-7024], Prieto, María Auxiliadora [0000-0002-8038-1223], Haarmann, Thomas [0000-0001-6033-8140], Sarbu, Alexandru [0000-0002-0810-4037], Schwaneberg, Ulrich [0000-0003-4026-701X], Xin, Fengxue [0000-0002-0758-8340], Dong, Weiliang [0000-0002-8556-5689], Xing, Jiamin [0000-0003-3245-7198], Chen, Guo‑Qiang [0000-0002-7226-1782], Jiang, Min [0000-0001-9470-7346], and Blank, Lars M. [0000-0003-0961-4976]
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Plastic recycling ,PHA ,Biomass ,010501 environmental sciences ,Raw material ,Polyhydroxyalkanoate ,7. Clean energy ,01 natural sciences ,Bioplastic ,Polyhydroxyalkanoates ,12. Responsible consumption ,03 medical and health sciences ,ddc:610 ,Microbial biodegradation ,Synthetic biology ,030304 developmental biology ,0105 earth and related environmental sciences ,Discussion ,0303 health sciences ,Waste management ,Mixed plastics valorisation ,Biodegradation ,Depolymerisation ,Pollution ,Biobased plastic ,Upcycling ,13. Climate action ,Microbial consortia ,Environmental science ,Plastic crisis ,Metabolic engineering - Abstract
9 p.-2 fig., This article introduces the EU Horizon 2020 research project MIX-UP, "Mixed plastics biodegradation and upcycling using microbial communities". The project focuses on changing the traditional linear value chain of plastics to a sustainable, biodegradable based one. Plastic mixtures contain five of the top six fossil-based recalcitrant plastics [polyethylene (PE), polyurethane (PUR), polypropylene (PP), polyethylene terephthalate (PET), polystyrene (PS)], along with upcoming bioplastics polyhydroxyalkanoate (PHA) and polylactate (PLA) will be used as feedstock for microbial transformations. Consecutive controlled enzymatic and microbial degradation of mechanically pre-treated plastics wastes combined with subsequent microbial conversion to polymers and value-added chemicals by mixed cultures. Known plastic-degrading enzymes will be optimised by integrated protein engineering to achieve high specific binding capacities, stability, and catalytic efficacy towards a broad spectrum of plastic polymers under high salt and temperature conditions. Another focus lies in the search and isolation of novel enzymes active on recalcitrant polymers. MIX-UP will formulate enzyme cocktails tailored to specific waste streams and strives to enhance enzyme production significantly. In vivo and in vitro application of these cocktails enable stable, self-sustaining microbiomes to convert the released plastic monomers selectively into value-added products, key building blocks, and biomass. Any remaining material recalcitrant to the enzymatic activities will be recirculated into the process by physicochemical treatment. The Chinese–European MIX-UP consortium is multidisciplinary and industry-participating to address the market need for novel sustainable routes to valorise plastic waste streams. The project's new workflow realises a circular (bio)plastic economy and adds value to present poorly recycled plastic wastes where mechanical and chemical plastic recycling show limits., This project has received funding from the European Union's Horizon 2020 research and innovation programme under Grant Agreement No. 870294. In addition, this project is funded by the National Natural Science Foundation of China (Grant Numbers 31961133017, 31961133018, 31961133019).
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- 2021
39. The analysis of fluorescence fluctuations by means of the mean single-molecule rate (mSMR)
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Sparrenberg, Lorenz Tim, Schwaneberg, Ulrich, and Berlage, Thomas
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ddc:570 ,fluorescence fluctuation spectroscopy , FCS , confocal microscopy , Monte Carlo simulation , DNA ,FCS ,DNA ,confocal microscopy ,Monte Carlo simulation ,fluorescence fluctuation spectroscopy - Abstract
Dissertation, RWTH Aachen University, 2023; Aachen : RWTH Aachen University 1 Online-Ressource : Illustrationen, Diagramme (2023). = Dissertation, RWTH Aachen University, 2023, Fluorescence fluctuation spectroscopy (FFS) is an important tool for the analysis of biological systems at the single-molecule level. FFS methods can be roughly divided into two categories. Methods of the first category examine fluctuations in the time domain and include the well-known fluorescence correlation spectroscopy (FCS) and its variations. Methods of the other category analyze fluctuations in the amplitude domain and include the photon counting histogram and related methods. In this thesis, the mean single-molecule rate (mSMR) is introduced as a new method, which uses information from both the time and amplitude domain. The mSMR is based on Mandel’s Q parameter, which can be calculated from the first two cumulants of a fluorescence trace. The cumulants can be expressed for arbitrary sampling times of a fluorescence trace, which yields the Q parameter as a sampling time-dependent quantity. By normalizing the Q parameter to its corresponding sampling time, data curves are obtained which show great similarities to the autocorrelation curves in FCS analysis and enable a comparable interpretation of the data. The model definition based on cumulants allows direct correction of common detector artefacts such as afterpulsing or dead time. For evaluation, the mSMR is subjected to a series of systematic analyses. Firstly, it was applied to simulated fluorescence traces since the simulation enables precisely adjustable parameters. It was shown that the mSMR model accurately reproduces the input parameters of the simulation both in the absence and presence of noise and detector artefacts. Secondly, the mSMR was used to analyze fluorescence traces of the dye Alexa Fluor 488 recorded with a home-built confocal plate reader. Our reader automatically conducts FFS measurements in a microtiter plate, thus enabling easy and repeatable measurements with low hands-on time. A visual and statistical comparison between the mSMR and the established FCS showed that the mSMR provides generally comparable results to the FCS method. At low excitation powers and low concentrations, however, the mSMR provides more plausible results on short time scales. This is of particular importance for the analysis of photokinetic effects. Thirdly, to show the relevance of the mSMR for biological systems, measurements were performed on DNA mixtures of defined fragment length composition. Here, too, the mSMR retrieved precise results that are in line with theoretical expectations. Based on these findings, libraries for DNA sequencing were characterized and mass concentration, mean fragment length and molarity of the libraries were determined. In just one measurement, the mSMR could provide the same results as a commonly used multistep procedure consisting of fluorescence spectroscopy and capillary gel electrophoresis. The mSMR represents a meaningful extension of previous FFS methods. The findings of this work suggest that especially for measurements with few photon events, e.g., at low excitation powers and concentrations, the mSMR is a robust and reliable method. In combination with the correction of detector artefacts, the mSMR can resolve fluctuation events on very short time scales and permits high-precision analyses of fluorescence fluctuations. This provides new insights into the analysis of photokinetic effects., Published by RWTH Aachen University, Aachen
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- 2023
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40. Accelerated production and screening of catalytically active inclusion body libraries via automated workflows
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Küsters, Kira, Oldiges, Marco, Schwaneberg, Ulrich, and Wiechert, Wolfgang
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ddc:570 - Abstract
Dissertation, RWTH Aachen University, 2022; Aachen : RWTH Aachen University 1 Online-Ressource : Illustrationen, Diagramme (2022). = Dissertation, RWTH Aachen University, 2022, In recent years, the production of inclusion bodies that retained substantial catalytic activity was demonstrated. These catalytically active inclusion bodies (CatIBs) were formed by genetic fusion of an aggregation inducing tag to a gene of interest via short linker polypeptides and overproduction of the resulting gene fusion in Escherichia coli. The resulting CatIBs are known for their simple and cost efficient production, recyclability as well as high stability and provide an alternative, purely biological technology for enzyme immobilization. Due to their ability to self aggregate in a carrier-free, biodegradable form, no further laborious immobilization steps or additional reagents are needed. These advantages put CatIBs in a beneficial position in comparison to traditional immobilization techniques. Recent studies outlined the impact of cooperative effects of the linker and aggregation inducing tag on the activity level of CatIBs. However, no a priori prediction is possible to indicate the best linker/aggregation inducing tag combination. So, testing of many variations is required to find the best performing CatIB variant. Therefore, an accelerated CatIB processing is needed to allow a fast construction and screening of CatIB libraries. In a proof of concept study, a set of lysine decarboxylase CatIBs from E. coli (EcLDCc) were generated via Golden Gate Assembly and processed with an optimized purification protocol that would allow automation of the workflow in further processes. A total of ten EcLDCc variants consisting of combinations of two linker and five aggregation inducing tag sequences were generated. A flexible Serine/Glycine (SG)- as well as a rigid Proline/Threonine (PT)-Linker were tested in combination with artificial peptides (18AWT, L6KD and GFIL8) or coiled-coil domains (TDoT and 3HAMP) as aggregation inducing tags. Interestingly, most of the combinations with the rigid PT-Linker showed the highest conversions. EcLDCc-PT-L6KD was identified as the best of all variants allowing a specific volumetric productivity of 256 gDAP L 1 d 1 gCatIB-1. Furthermore, we demonstrated that microscopic analysis can serve as a tool to identify CatIB producing strains and thus allow for prescreening at an early stage to save time and resources. To accelerate the microscopy process, an automated microscopy workflow was implemented and used to observe the CatIB formation process. The automated and manual evaluated results showed that the CatIB formation started after 27 h. After the proof of concept study with the EcLDCc, a semi-automated cloning workflow was implemented to allow fast CatIB library construction with a time reduction of 83 %, resulting in only 11 h of manual work for the construction of 96 CatIB variants. The cloning workflow was used to generate 14 CgAHAS (acetolactate synthase of Corynebacterium glutamicum)-, 60 LbADH (alcohol dehydrogenase of Lactobacillus brevis)-, 63 BsGDH (glucose dehydrogenase of Bacillus subtilis)- and 30 BmMO (monooxygenase of Bacillus megaterium)-CatIB variants successfully. To test the constructed CatIB variants in parallel an acceleration of the manual CatIB analysis was needed. Therefore, a miniaturization of the cultivation and CatIB purification from a 10 mL shake flask scale to a 1 mL FlowerPlate® scale was realized. Furthermore, the enzymatic assay was performed in a microtiter plate (250 µL) instead of reaction tubes (1 mL). The accelerated workflow was used to screen the CgAHAS- and LbADH-CatIB libraries, which revealed 2 out of 14 (CgAHAS) and 8 out of 60 (LbADH) successful CatIB candidates. After process acceleration, the next step was to automate the CatIB screening workflow. The first step was to implement a robust manual workflow, which was transferred to a robotic platform subsequently. Therefore, 14 C-terminally tagged BsGDH variants were generated and analyzed in a manual approach. Similar as in the EcLDC study, SG- or PT-Linker were combined with one of eight aggregation inducing tags. Besides the five tested aggregation inducing tags, 18AWT, L6KD, GFIL8, TDoT and 3HAMP, the influence of ELK16, TorA and CBDCell on the CatIB activity were analyzed. The enzymatic assay showed that the highest conversions were again reached with PT-Linker variants. The most promising variant was BsGDH-PT-CBDCell with a specific volumetric productivity of 55.8 gNADH L 1 d 1 gCatIB-1. This variant was characterized in more detail including long-term storage at 20 °C as well as NADH cofactor regeneration performance in repetitive batch experiments with CatIB recycling. After freezing, BsGDH PT CBDCell CatIBs only lost approx. 10 % activity after 8 weeks of storage. Furthermore, after 11 CatIB recycling cycles in repetitive batch operation, 80 % of the activity was still present. Not only NADH recycling was analyzed with BsGDH-CatIB, but also NADPH recycling. BmMO-CatIBs as NADPH consumer were constructed and used to prove the NADPH recyclability of the BsGDH. 13 out of 30 BmMO-CatIBs revealed activity in combination with NADPH recycling. After realizing the manual screening, BsGDH-PT-CBDCell was used to implement, optimize and validate the automated CatIB screening workflow to enhance the analysis of several CatIB candidates. Important optimization steps were for example the exclusion of a position effect in the BioLector® by enhancing the cultivation temperature to 25 °C and the provision of pipetting reproducibility by improving pipetting settings like velocity, mixing, air gaps and height. The validation of the optimized workflow with a relative standard deviation of 1.9 % revealed a high reproducibility for the whole workflow. After validation, the workflow was performed in combination with Thompson sampling, as a decision tool for strain selection, to analyze 63 BsGDH-CatIB variants. Three rounds with 48 cultivations in parallel each were realized. The screening showed that 24 BsGDH-CatIB out of 63 were successful CatIB variants. Already in the first screening round TDoT-PT-BsGDH was a promising CatIB candidate, resulting in a high likelihood of being the best BsGDH-CatIB performer. Therefore, the Thompson sampling algorithm has selected this variant in 50 biological replicates during the three screening rounds. With the implemented semi-automated CatIB construction, processing and screening workflow the manual work time could be reduced from 59 h to 7 h for 48 variants (-88 %). Moreover, an upscaling as shake flask cultivations of the best three BsGDH-CatIB variants as a validation study for the Thompson sampling revealed similar results as compared to the FlowerPlate® cultivation. These results showed that the Thompson sampling was successful for the BsGDH screening. Although the analysis of all CatIB enzymes revealed that for example the PT-Linker was more likely to form successful CatIB variants compared to the SG-Linker, still no a priori prediction is possible, because every enzyme requires different linker/aggregation inducing tag combinations for highest performance. Therefore, using the automated workflow is an important tool to enhance and simplify the screening to find the best CatIB producer from large CatIB libraries., Published by RWTH Aachen University, Aachen
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- 2022
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41. Ultrasound-mediated activation of drugs
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Shi, Zhiyuan, Herrmann, Andreas, and Schwaneberg, Ulrich
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theranostics ,release kinetic ,disulfide-bond scission ,ddc:540 ,drug delivery ,polymer chemistry ,polymer chemistry , mechanochemistry , drug delivery , disulfide-bond scission , theranostics , release kinetic ,mechanochemistry - Abstract
Dissertation, RWTH Aachen University, 2021; Aachen : RWTH Aachen University 1 Online-Ressource : Illustrationen, Diagramme (2021). = Dissertation, RWTH Aachen University, 2021, Treatment using pharmaceutical drugs is arguably the most important medical therapy warranting the health of every human. However, the systemic application of drugs often has severe side effects due to the intrinsic lack of drug selectivity. It is thus desirable and necessary to develop control mechanisms gating where and when drugs exhibit activity, regardless of the selected target, and yield a more effective and accurate treatment. Ultrasound as a stimulus with high spatiotemporal and energetic resolution together with its non-invasive character outperforms the physicochemical stimuli for controlled drug release, such as light, temperature, pH or redox reactions. In this work, we present two individual approaches, both culminating in the release of small drug molecules from their respective inactive precursors by ultrasound-induced site-specific scission of disulfide bonds. The first example is the intermolecular disulfide bond scission induced drug release system, capable of successively releasing furan-containing small molecules by ultrasound-induced selective scission of a disulfide mechanophore embedded at the center of a polymer chain. Upon disulfide scission, thiyl radicals were generated and protonated in aqueous media yielding thiols. These, in turn, underwent a Michael-type addition to Diels-Alder (DA) adducts of furylated drugs and acetylenedicarboxylate, which ultimately induced a retro DA reaction releasing the corresponding small molecule. To outline the conceptual scope of this system, we successively released the furylated fluorophore dansyl and the drugs furosemide as well as furylated doxorubicin. The second example is the intramolecular mechanochemical disulfide bond scission induced drug release system. Here, we employ a disulfide-centered polymer carrying the drug bound via a carbonate/carbamate linker on the β-carbon. These polymers were designed such that ultrasonication induced central bond scission in aqueous medium would lead to the generation of free thiols and subsequently a kinetically favored 5-exo-trig cyclization would extrude the target molecule from the carbonate/carbamate linker. The results for this intramolecular drug release system were then split into three subchapters, and we describe the unprecedented use of mechanical force for the activation of seven different prodrugs along with three reference molecules. To prove the concept, we first activated the latent fluorophore umbelliferone to optimize the experimental conditions. Afterward, we successfully activated a pharmacologically relevant drug molecule camptothecin. Subsequently, the intramolecular drug release was extended for the mechanochemical activation of disulfide-based multifunctional constructs for theranostic drug release, which allows direct and fluorescence-based monitoring of the released drugs. We show two ultrasound-responsive multifunctional constructs capable of releasing either naphthalimide or coumarin as the fluorescent reporter and camptothecin or gemcitabine as the anticancer agent from its disulfide polymer. In principle, this theranostic approach would allow the drug delivery and release to be monitored directly in a non-invasive manner with sub-molecular precision. Furthermore, we systematically investigated the effects of linkage bond on the release kinetics of the disulfide polymer for the development of novel mechanochemically sensitive disulfide conjugates to achieve tailored and controllable release. For this purpose, we employed three naphthalimides to display colorimetric and green fluorescence off-on changes upon release, which allow the analytical description of the release process. We believe that this work may serve as a novel blueprint for many more approaches employing ultrasound to release different functional molecules for a manifold of other applications in the field of medicine and beyond., Published by RWTH Aachen University, Aachen
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- 2021
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42. Engineering of biocatalytic microgels and bifunctional peptides for biohybrid systems
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N��th, Maximilian Wolfgang Stefan, Schwaneberg, Ulrich, and Pich, Andrij
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Materialfunktionalisierung ,Mikrogel ,P450 BM3 Monooxygenase ,Enzymimmobilisierung ,Materialbindepeptid ,Klickchemie ,P450 BM3 monooxygenase ,enzyme immobilization ,microgel ,material functionalization ,material binding peptide ,click chemistry ,ddc:570 - Abstract
Dissertation, RWTH Aachen University, 2021; Aachen : RWTH Aachen University 1 Online-Ressource : Illustrationen, Diagramme (2021). = Dissertation, RWTH Aachen University, 2021, Biohybrid materials and systems have great potential in the fields of biocatalysis and materials science, as the combination of biological and synthetic building blocks enables the design of novel biohybrid catalyst and material concepts that convey new functionalities. New strategies for integrating biocatalytic functionalities into materials and for material and surface functionalization are needed, to develop new biohybrid materials and systems. In this thesis, two novel biohybrid systems for biocatalysis and universal material and surface functionalization were developed: in the first chapter, P450 ��-Gelzymes were established by immobilizing cytochrome P450 BM3 monooxygenase in stimuli-responsive "smart" microgels, while in the second chapter, a universal surface functionalization toolbox based on bifunctional peptides was developed for the functionalization of synthetic polymers, metals, and silicon-based materials.Enzymes are biocatalysts evolved by nature to perform (bio)chemical reactions at environmentally benign reaction conditions with impressive chemo-, regio-, and stereoselectivities. P450 monooxygenases are versatile biocatalysts with high synthetic application potential, but their application is yet challenged, amongst others, by their low operational stability (e.g., low organic solvent tolerance, enzyme inactivation after a certain reaction time). Enzyme immobilization is one the most successful strategies to improve enzyme stability and enables the release, re-immobilization, and recycling of enzymes. P450 monooxygenases are challenging to immobilize, and in the case of P450 BM3 from Bacillus megaterium, the activity of the immobilized enzyme is often deteriorated or entirely lost. Microgels have attracted attention as an innovative class of "smart" and stimuli-responsive carriers for enzyme immobilization due to their chemical and mechanical stability, tuneable architecture, biocompatibility, porosity, high water content, and the ability to achieve high enzyme loadings and provide a protective environment for the immobilized enzymes. Therefore, new strategies for integrating enzymes in microgels and new microgel systems need to be developed to harness the potential of stimuli-responsive microgels as a carrier platform for "sensitive" enzymes that are challenging to immobilize. This work reports the first pH-independent immobilization of P450 BM3 in novel poly(N-vinylcaprolactam) microgels with 1-vinyl-3-methylimidazolium as comonomer without loss of catalytic activity (biohybrid P450 ��-Gelzymes) and the first systematic study of P450 ��-Gelzyme performance. Poly(N-vinylcaprolactam) microgels were synthesized with a pH-independent, positive charge by modifying 1-vinylimidazole moieties through a quaternization reaction (1-vinyl-3-methylimidazolium). The pH-independent immobilization allowed to operate biohybrid P450 ��-Gelzyme catalysts at the pH activity optimum (pH 8). In addition, P450 ��-Gelzymes enabled ionic-strength triggered release and re-immobilization of P450 BM3 as well as biocatalyst recycling for repeated use and provided initial protective effects against organic cosolvents.The biological transformation of materials science is paving the way to novel biohybrid material concepts by combining and integrating biological and synthetic building blocks in materials (e.g., biofunctionalization of synthetic polymers, metals, and silicon-based materials). Universally applicable and specific material and surface functionalization methodologies are prerequisites for the biological transformation of materials science but pose a key challenge due to the vastly different properties and chemistries of materials and surfaces. Innovative material and surface functionalization technologies have to be developed to address this challenge. Biological surface functionalization with material binding or anchor peptides is a simple strategy to endow materials with biological and synthetic functionalities and an energy-efficient and environmentally benign alternative to chemical and physical surface functionalization strategies. Following this notion, a novel toolbox concept for universal material and surface functionalization based on bifunctional peptides was developed. Bifunctional peptides were synthesized by specific modification of the universal anchor peptide LCI from Bacillus subtilis with different functional amine moieties (e.g., reactive groups for click chemistry, fluorescent dyes, antibiotics, synthetic polymers) through sortase-mediated ligation employing sortase A from Staphylococcus aureus. Sortase-mediated ligation further enabled the purification of bifunctional peptides by a negative purification strategy using Strep���tag II affinity chromatography (purities > 90%). In general, the bifunctional peptide toolbox enabled surface functionalization either as a two or one-step strategy. In the case of the one-step strategy, the desired functionality was directly introduced to LCI, while in the case of the two-step strategy, LCI was modified with a reactive group that enabled further functionalization (e.g., employing click chemistry). For the two-step strategy, synthetic polymers (polypropylene, polyethylene terephthalate), metals (stainless steel, gold), and silicon were functionalized with reactive groups for copper-free azide-alkyne click chemistry. The one-step strategy was demonstrated by direct functionalization of polypropylene with a fluorescent dye and biotin. These results represent the first systematic combination of universal anchor peptides, like LCI, and sortase-mediated ligation in a toolbox concept for universal surface functionalization, including the first peptide-mediated functionalization of polypropylene and polyethylene terephthalate with reactive groups for copper-free azide-alkyne click chemistry., Published by RWTH Aachen University, Aachen
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- 2021
43. Phytase engineering for efficient phosphate recovery from press cakes
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Herrmann, Kevin Rico, Schwaneberg, Ulrich, and Blank, Lars M.
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inositol phosphates ,DNA recombination ,Thermostabilität ,Inositolphosphate ,thermal stability ,chimera ,DNA-Rekombination ,circular bioeconomy ,enzyme blend ,phosphate ,phosphorus recycling ,phytase ,phytate hydrolysis ,protein engineering ,PTRec ,sustainability ,valorization ,Chimäre ,zirkuläre Bioökonomie ,Enzymmischungen ,Phosphat ,Phosphor-Recycling ,Phytase ,Phytat-Hydrolyse ,Protein-Engineering ,Nachhaltigkeit ,Valorisierung ,ddc:570 - Abstract
Dissertation, RWTH Aachen University, 2021; Aachen : RWTH Aachen University 1 Online-Ressource : Illustrationen (2021). = Dissertation, RWTH Aachen University, 2021, Phosphorus (P) is one of the most essential elements and plays a central role in feeding humankind as a key nutrient in fertilizers. Higher consumption than regeneration rates, precarious mining conditions and the transition to a renewable bioeconomy create a strong social impetus to develop P recovery strategies. Main objective of this thesis is the efficient phosphate recovery from deoiled seeds and nuts using phytase enzymes for valorization of plant byproducts. Quantification of the phosphorus storage form phytate (InsP6) in globally abundant press cakes revealed phytate contents > 3 % (w/w), highlighting the hidden potential for P recovery. The development of a widely applicable and sustainable phosphate recovery process from plant biomass using phytases turned out to be very effective in recovering high amounts of phosphate. One-pot phytase-based hydrolysis of InsP6 in aqueous suspension at moderate temperatures releases phosphate amounts > 20 mg/g press cake and is applicable to more than 20 press cakes. Worldwide industrial application could provide significant amounts of up to 1 million tons, with the recovered phosphate being used to produce premium P-based products such as green fertilizer or polyphosphate as food preservative that enable a circular bioeconomy for P. A bottleneck for complete phytate hydrolysis by phytase enzymes is the drastically reduced degradation of lower phosphorylated reaction intermediates from InsP4 onwards compared to InsP6. Phytate harbors six phosphate groups which are negatively charged under reaction conditions, and the net charge is drastically reduced during hydrolysis to lower InsPs. Therefore, from a protein engineering point of view, the ability to remove all six phosphate groups is very challenging for an enzyme as it has to cope with these substantial changes. Here, the first reported engineering campaign to evolve the E. coli phytase toward improved hydrolysis of InsP4 and InsP3 was conducted. After implementation of a suitable screening setup and isolation of required substrates, KnowVolution-based engineering led to variants with up to 3.8-fold improved hydrolysis of InsP4, while InsP3 hydrolysis was increased up to 2.7-fold. Beneficial substitutions are located within the binding pocket and are involved in substrate binding and orientation. Furthermore, there is a demand for tailored phytases in a wide range of areas such as food, feed or technical applications. By DNA recombination of distantly related genes, chimeras can be generated that combine beneficial properties of the parental enzymes in one protein. A PTRec-based DNA recombination library based on sophisticated computational analysis demonstrated the potential for efficient recombination of six genes with low sequence identities of < 50 % to obtain highly functional chimeras. Two phytase chimeras with up to 32 % improved residual activity (90 °C, 60 min) and retained high specific activities of > 1100 U/mg were identified. In summary, P recycling is an important element for a circular bioeconomy and a building block can be the phytase-based process presented here for application to biomass. Protein engineering approaches offer a variety of capabilities to further tailor the phytases and thus make P recovery processes more economical., Published by RWTH Aachen University, Aachen
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- 2021
44. Copper-mediated cross-coupling reactions toward tandem-catalysis
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Thomas, Fabian, Herres-Pawlis, Sonja, and Schwaneberg, Ulrich
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copper catalysis ,tandem-catalysis ,ddc:540 ,Kupferkatalyse ,Nitrene ,cross-coupling ,C-H Aktivierung ,Tandemkatalyse ,Kreuzkupplungen ,C-H activation ,nitrene - Abstract
Dissertation, RWTH Aachen University, 2021; Aachen : RWTH Aachen University 1 Online-Ressource : Illustrationen, Diagramme (2021). = Dissertation, RWTH Aachen University, 2021, Catalytic transformations are key to the development of atom efficient processes. An important functional group in biologically, synthetically and pharmacologically relevant molecules are amines. The central intermediates in atom efficient reactions to introduce an amine function are terminal nitrenes. Terminal nitrene complexes are an elusive species and rational ligand design helps to understand connection between the structure and the reactivity. Therefore, this PhD thesis includes the synthesis of a library of different bis(pyrazolyl)methane ligands. The aim was to investigate the influence of the ligand design on the formation and activity of the generated copper nitrene complexes. This was achieved by variation of the third N donor unit of the ligand, whose electronic as well as the steric properties were varied. By the application of the nitrene generating agent SPhINTs, six novel copper nitrene complexes were synthesized at low temperature in dichloromethane. These nitrene complexes were characterized by ultra-high resolution cryo ESI mass spectrometry, density functional theory, UV/Vis and NMR spectroscopy. It was shown that terminal nitrene complexes are generated as singlet species in high yields. In addition, the thermal stability and catalytic activity was investigated. By variation of the third N donor unit copper nitrene complexes with a high stability as well as a high cata-lytic activity were obtained. At room temperature the C���H amination of different benzylic, alkylic and aromatic substrates and the aziridination of different styrene derivatives is possible. Due to the relevance of enantiomeric pure products, also chiral precursor complexes were used in asymmetric catalytic reactions and enantiomeric enriched products could be obtained. To further increase the atom efficiency of the catalytic amination reaction under avoidance of SPhINTs, also aromatic azides were used as nitrene generating agents. A first success was achieved by the combination of benzoyl azide and electron rich bis(pyrazolyl)methane ligands. Beside copper(I) complexes also iron(II) complexes were investigated as precursor complexes to generate nitrene complexes. Another approach for the development of environmentally sustainable processes are tandem reactions. By the combination of two or more catalysts in one process, a purification of the intermediates is redundant. This leads to savings in energy and solvent consumption as well as in the investment costs. Therefore, in the framework of the SFB 985 in the Subproject A1 the aim was to develop a tandem reaction between a P450 BM3 variant and a copper bis(pyrazolyl)methane complex. Two possible tandem reactions were investigated. In the first tandem reaction a combination of an aromatic hydroxylation of dimethylbenzene to yield dimethylphenol followed by a copper catalyzed C���O cross-coupling to a diphenyl ether was planned. Both reactions could be performed separately, but a combination was not successful. The second approach is the combination of a copper catalyzed benzofuran synthesis from iodophenol and alkyne followed by a hydroxylation of the benzofuran by a P450 BM3 variant. The combination of both reactions could be realized in a stepwise reaction and the addition of EDTA., Published by RWTH Aachen University, Aachen
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- 2021
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45. Microgel-based Regenerative Materials and Biofunctionalization
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Gehlen, David Benedikt, De Laporte, Laura, and Schwaneberg, Ulrich
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microgels ,magnetic nanoparticles ,tissue engineering ,regenerative biomaterials ,Anisogel ,magnetic orientation ,biofunctionalization ,anisotropy ,ddc:540 - Abstract
Dissertation, RWTH Aachen University, 2021; Aachen : RWTH Aachen University 1 Online-Ressource : Illustrationen, Diagramme (2021). = Dissertation, RWTH Aachen University, 2021, Over the last decades, significant progress has been made in the field of implants and medical devices, which are capable of taking over the function of essential organs and tissues. In addition, new biomaterial systems are being designed to better understand cell behavior for basic research. However, controlling the cell-material interaction remains a crucial challenge for these systems and devices. For example, in the case of polydimethylsiloxane (PDMS), which is often used to study mechanobiology, most of the coating methods to enable stable cell adhesion, are complex, time-consuming, unstable over time, and include toxic compounds. Therefore, I developed a completely novel method using an anchor peptide with a fused cell adhesive peptide for coating PDMS surfaces in a one-step process in a robust and environmental-safe manner resulting in stable cell adherence. Importantly, one of the main goals in modern medicine remains the production and regeneration of fully functional tissues and organs. A lot of progress has been made over the last decades in designing advanced and smart materials using complex fabrication methods to generate controlled cell constructs. This scientific discipline is called tissue engineering but is still limited due to a large gap with products in clinical use. This is a consequence of the complex nature of native tissues with scales ranging from μm for individual cells, over mm for blood vessels, to cm for arrangements of tubular and other structures. Hydrogels, consisting of either natural or synthetic hydrophilic polymers, are the most promising approach to create injectable 3D scaffolds for tissue regeneration of soft and sensitive tissues. However, high porosity is missing in most synthetic hydrogels with elastic nanometer-size meshes, which is essential for cell infiltration, migration, and proliferation. To overcome this challenge, I developed an easy and rapid process to create granular hydrogel scaffolds. Crosslinked cellulose nanofibril hydrogels with calcium are crushed through a Nylon mesh, leading to homogeneous 3D cell growth in a very effective manner. For a more controlled approach, I developed a microporous scaffold using enzymatically crosslinked microgels. The combination of rod-shaped and spherical microgels results in maximized porosity. For the regeneration of functional tissues or for generating ex vivo models, it is essential to resemble their structure and hierarchical order. For example, the kidney possesses tubular structures. To resemble these tubular structures in an embedded matrix, I produced cellulose nanofibril microgels by pipetting the solution through thin needles into a stirring calcium bath and subsequently breaking them down mechanically during purification. Fibroblasts can be seeded and cultured on these microgels and embedded into synthetic PEG-based hydrogels, which allowed for the production of tubular cell structures inside of a highly controlled surrounding matrix. In vivo therapies for sensitive tissues, such as the spinal cord, require a low invasive injection to avoid implantation and additional removal of healthy tissue. Therefore, I worked on the Anisogel (Anisometric hydrogel) system, which forms an aligned structure in situ after injection with high controllability of the mechanical, biochemical, and structural properties. This is achieved by incorporating superparamagnetic nanoparticles into rod-shaped microgels with defined micron-scale diameters, allowing microgel alignment parallel to a magnetic field in a solution. Next, the solution crosslinks enzymatically to fixate the oriented microgels. Strikingly, I showed for the first time that not only the cells are aligned inside of the Anisogel but also the fibronectin produced by the cells, indicating a potential positive feedback cycle. Additional biofunctionalization of the guiding elements with cell-adhesive peptides results in high interactions with the cells and slightly increased cell-alignment but reduces the amount of fibronectin produced by the cells, which may hinder the regeneration process and replacement of the temporal artificial scaffold during its degradation. Investigating the location of the mechanosensitive protein YAP revealed that the cells are not only aligned by contact guidance but rather feel the overall mechanical anisotropy of the material, indicating a more complex mechanism behind cell alignment. To further study and characterize this, I developed a method with magnet-assemblies for rheology measurements, enabling investigation of the overall mechanical anisotropy of the Anisogel. Furthermore, controlling the Anisogel structure with different dimensions of microgels showed that the volume of guiding elements to initiate nerve alignment can be reduced by using microgels with diameters of 2.5 μm instead of 5 μm. After being able to control all three parameters inside the Anisogel system, which are the mechanical, biochemical, and structural properties, I worked on actively moving the microgels within the surrounding matrix for potential stimulation of cells. However, the embedded microgels did not move and stronger magnetic properties of the microgels are necessary for the future to generate forces in a relevant range. To expand the use of the magneto-responsive microgels for other applications, such as magnetic field-assisted bioprinting, I also developed a method to pre-program the orientation angle of the microgels in a static magnetic field. Therefore, ellipsoidal maghemite nanoparticles were aligned within the microgel network at a defined angle during their crosslinking, enabling the orientation of the final microgels in different directions in the magnetic field according to the defined angle of the pre-aligned ellipsoidal nanoparticles. In conclusion, controlling and mimicking the biochemical properties, macro-porosity, and oriented structures of native tissues results in a better understanding of 3D cell growth in artificial scaffolds, which can be further developed to stimulate and actuate cells during tissue maturation., Published by RWTH Aachen University, Aachen
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- 2021
46. Investigations into enzymatic transglycosylation reactions for the synthesis of glycoconjugates and glyco-functionalized polyphenols
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Hoffmann, Marius, Elling, Lothar, and Schwaneberg, Ulrich
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neo-glycoprotein ,glycopolymer ,catalysis ,Enzyme ,ddc:570 ,glycosidase ,glycosyltransferase ,glycosynthase - Abstract
Dissertation, RWTH Aachen University, 2021; Aachen : RWTH Aachen University 1 Online-Ressource : Illustrationen, Diagramme (2021). = Dissertation, RWTH Aachen University, 2021, With a myriad of functions, glycans constitute the most abundant of the four fundamental biological macromolecules (DNA, RNA, proteins and glycans). Besides structural and metabolical importance, they provide an essential coding platform for the transfer of information within the cellular network. Despite the arrival of glycome-related fields in mainstream science throughout the last decades, much of the glyco code remains unexplored. This is largely due to a lack of defined sample material. Enzymatic synthesis of glycans via a diverse toolbox of natural and artificial catalysts can help close this gap. This thesis explores the glycosylation of aglycon acceptor substrates for use as effector molecules with increased bioactivity or as building blocks for the assembly of glycan-presenting macromolecules. Section one addresses the demand for tailored synthetic flavonoid glycosylation. Four enzymes with different regiospecificities of plant and bacterial origin are recombinantly produced and their respective reactions analyzed. During the process, a novel analytical method based on multiplexed capillary electrophoresis suitable for the analysis of flavonoid glycosylation and related polyphenolic small molecules in a 96-well formate is validated. For the macrolide glycosyltransferase SorF from Sorangium cellulosum, a substrate screening revealed an interesting spectrum of polyphenols that indicates a potential application of this enzyme as a tool in the glycodiversification of small molecules. Section two covers the application of the hyperthermophilic glycosidase PwGly from P. furiosus in the glycosylation of acrylates and amine-functionalized linker substrates. The enzyme’s remarkable tolerance towards synthesis conditions are analyzed. The gained insights are put into practice during the synthesis of galacto functionalized acrylates on a 100 mL scale. High production yields > 80% and an efficient purification procedure enable the subsequent assembly of the galactomonomers to galactose presenting polymers with a tunable amount of incorporated sugars. The resulting glycopolymers represent suitable multivalent binding scaffolds for the lectin RCA120. PwGly’s versatility regarding transglycosylation reactions is further expored in the glycosylation of amine functionalized alkanols. Finally, the glycosynthase mutant BgaC Glu233Gly is applied in the synthesis of the azide functionalized pan-carcinomic Thomsen-Friedenreich (TF ) antigen. Subsequent coupling of the disaccharide to BSA via Cu2+-catalyzed alkyne azide cycloaddition yield tailored TF antigen presenting neo-glycoproteins whose properties as novel Gal-3 binding partners are investigated. In conclusion, the presented results highlight the role of a diverse spectrum of enzyme catalysts in synthetic glycotechnology. While the first part represents a first step towards rational-driven effector enhancement through tailored glycosylation, the second section lays the foundation for the assembly of glycan-presenting scaffolds via an efficient supply of functionalized building blocks., Published by RWTH Aachen University, Aachen
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- 2021
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47. Bacillus pumilus als Plattform für die Produktion technischer Enzyme
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Küppers, Tobias, Schwaneberg, Ulrich, and Wiechert, Wolfgang
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α-amylase ,industrial biotechnology ,protease ,strain development ,process development ,13C Metabolic Flux Analysis ,13C metabolic flux analysis ,ddc:570 - Abstract
Dissertation, RWTH Aachen University, 2021; Aachen : RWTH Aachen University 1 Online-Ressource : Illustrationen (2022). = Dissertation, RWTH Aachen University, 2021, The scope of this thesis is the development of an alternative production platform for the production of alkaline hydrolases, which are used in modern washing and cleaning agents. For this purpose, the strain Bacillus pumilus Jo2 was provided and should be developed to produce competitive enzyme yields in reference to a downscaled production process of Henkel’s established industrial workhorse Bacillus licheniformis E.Based on alkaline protease BL18, which was chosen as a well-characterized reporter molecule, B. pumilus Jo2 was initially developed to produce protease in reference to B. lichniformis E. Thereby, the iterative process development and adjustments, as well as genetic modifications, enabled to reach a final product titer of 114.5% in reference to B. licheniformis E. at the same scale. Further homologues promoter sequences were identified, and their use was claimed for various enzyme expressions (WO2013113689). In a final exploratory setup, the production capacity of the generate Jo2 expression platform was re-evaluated for its performance in producing non-proteolytic hydrolases. Therefore, Bacillus spec A7-7s α-amylase was chosen as a suitable reporter molecule. Besides minor pH and glucose adjustments, the developed B. pumilus Jo2 extemporaneously reached a maximum amylase titer of about 65.7% in comparison to B. licheniformis E protease titer at the same scale. This amylase titer, which is by far more than 4 g/L, indicates the outstanding potential of the developed B. pumilus platform, in due regard to its further optimization potential for amylase production. In addition to its development, the B. pumilus Jo2 platform was characterized in regard to its substrate utilization, amino acid synthesis and to identify optimization targets for further optimizations employing 13C-flux analysis and proteomics. Thus, among other results, it was shown, that in the subtilisin BL18 production process approximately 41.1 ± 4.2% of subtilisin BLl8’s carbon is derived from glucose. Based on results of amino acid pools, aspects for further media and substrate optimization are presented, as for instance precursor availability of alanine, asparagine/aspartic acid, serine and valine. As finding of the proteomic approach various optimization targets were proposed for subsequent strain improvements such as to avoid the formation of flagella protein Hag and extracellular enzymes (BglS, PelB)., Published by RWTH Aachen University, Aachen
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- 2021
48. Engineering of prodigiosin ligase pigC towards production of short-chain prodiginines
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Brands, Stefanie, Schwaneberg, Ulrich, and Jaeger, Karl-Erich
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protein engineering ,directed evolution ,natural products ,prodiginines ,high-throughput screening ,ddc:570 - Abstract
Dissertation, RWTH Aachen University, 2021; Aachen : RWTH Aachen University 1 Online-Ressource : Illustrationen (2021). = Dissertation, RWTH Aachen University, 2021, Prodigiosin synthetase PigC is an ATP-dependent membrane-associated ligase that catalyzes the final condensation step in the biosynthesis of prodigiosin in Serratia marcescens. Two pyrrolic precursor molecules, 4-methoxy-2,2’-bipyrrole-5-carbaldehyde (MBC) and 2-methyl-3-amyl-pyrrole (MAP) are linearly combined to form the deeply red tripyrrole pigment prodigiosin. As secondary metabolites, prodiginines provide a wide spectrum of bioactivities like antibiotic, antifungal, nematicidal, antimalaria as well as anticancer effects that have attracted the interest of agrobusiness, pharma and food industries. Interestingly, the side chains that decorate the tripyrrolic prodiginine scaffold modulate the bioactivity of prodiginines. The anticancer effect was especially pronounced in prodiginines with short aliphatic side chains. Prodiginine precursors with short side chains are, however, badly accepted by prodiginine ligases, which are bottleneck to biocatalytic prodiginine production. In this work, the engineering of prodigiosin ligase PigC towards acceptance of short-chain pyrroles opens up a promising biosynthetic route to short-chain prodiginines and provides a first molecular understanding on substrate binding in the active pocket of PigC. Mutagenesis libraries of pigC were heterologously generated in Pseudomonas putida KT2440, which has proved remarkably tolerant towards antibiotic prodiginine compounds. The establishment of a high-throughput screening system with low product detection limits (0.5 µM), wide linear product detection ranges (0.5-250 µM) and low standard deviations, Published by RWTH Aachen University, Aachen
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- 2021
49. Synthesis of neo-glycoproteins for binding studies and scavenging of Clostridium difficile toxin A - of microgels and biosensors
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Heine, Viktoria, Elling, Lothar, and Schwaneberg, Ulrich
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fucosyltransferases ,microgels ,neo-glycoproteins ,ddc:570 ,Clostridium difficile TcdA ,biosensors ,Clostridium difficile TcdA , neo-glycoproteins , fucosyltransferases , microgels , biosensors - Abstract
Dissertation, RWTH Aachen University, 2021; Aachen : RWTH Aachen University 1 Online-Ressource : Illustrationen (2021). = Dissertation, RWTH Aachen University, 2021, Clostridium difficile infections cause enormous costs in the health care sector. The bacterium colonizes the human intestinal tract and secretes toxins that destroy the epithelial cell layer of the colon. The toxins bind to cell-surface glycans on human intestinal cells and induce processes that eventually lead to cell death. Since toxin A comprises a carbohydrate recognition domain with combined repetitive oligopeptide domains, it can bind multiple glycan ligands at once. This feature can be exploited for increasing the interaction strength between ligand and toxin by multivalency and can be transferred to other toxins comprising carbohydrate recognition domains. To find the best possible toxin ligand for C. difficile toxin A, we established an in-plate ligand library. Multivalent neo-glycoproteins served as scaffold for complex glycosylation patterns to assemble a variety of 40 glycan structures. Screening of this library with the receptor domain of toxin A presented Lewisy-Lewisx-decorated BSA as promising toxin scavenger. Tests with the holotoxin confirmed the findings and the ligand was produced for in vitro applications. For the production of the glycan (Lewisy-Lewisx), a set of efficient fucosyltransferases was established and tested with a range of new substrates (N-acetyllactosamine tetrasaccharides), revealing unexpected fucosylation patterns. The toxin A scavenger was produced and coupled to BSA. They were further utilized for coupling to microgels. Neo-glycoproteins and neo-glycoprotein-presenting microgels were able to protect human cells from toxin A. Furthermore, the neo-glycoproteins showed a good affinity to the Cholera toxin. Hence, Lewisy-Lewisx-BSA is considered a one-for-two scavenger. To enable a deeper understanding of the binding behavior, a neo-glycoprotein biosensor was established. BSA-neo-glycoproteins carrying N-acetyllactosamine were bound to the biosensor and glycosylated on-chip. Online measurements enabled the real-time investigation of kinetic binding processes between glycan and enzyme or glycan and lectin via electrochemical impedance spectroscopy for the first time. This approach could be transferred to a variety of interaction partners. Summarizing, this work paves the way towards the treatment of bacterial-associated diseases by establishing toxin scavengers and developing novel analytical methods for toxin-ligand interactions., Published by RWTH Aachen University, Aachen
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- 2021
50. Directed evolution of polymerases and its application in sequence saturation mutagenesis
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Chung, Mu-En, Schwaneberg, Ulrich, and Elling, Lothar
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polymerase ,enzyme engineering ,ddc:570 ,cell-surface display ,directed evolution ,mutagenesis ,cell-surface display , polymerase , mutagenesis , directed evolution , enzyme engineering - Abstract
Dissertation, RWTH Aachen University, 2021; Aachen : RWTH Aachen University 1 Online-Ressource : Illustrationen, Diagramme (2021). = Dissertation, RWTH Aachen University, 2021, Directed evolution plays a crucial role in advancing modern biotechnological, medical, and industrial technologies. The importance of directed evolution was recently acknowledged by Novel prize in chemistry in 2018. Directed evolution consists of iterative steps of diversity generation and isolation of the variants with improved protein property. Each round of evolution can be seen as one step toward the “hilltop of desired property” in the protein sequence space. As the sequence space of a regular sized protein already far exceeds the throughput of existing screening methods, a gene diversification method that generates a diverse and unbiased library is crucial to an efficient walk to the “hilltop” with minimal number of steps. Sequence saturation mutagenesis (SeSaM) was reported to generate a distinct mutational spectrum unobtainable by epPCR, which is widely used for mutagenesis. However, SeSaM as well as epPCR rarely generates consecutive transversions (TvTv). TvTv mutations are particularly important for generating enzyme libraries with diverse amino acid substitutions and broad chemical diversity. 91.4% of amino acid substitutions generated by TvTv mutations are distinct, and of which 90% are chemically different. The challenge of generating TvTv mutations lies in the structure of the DNA, in which transversion mutations form bulky or wobbly mispairs that destabilize double helix formation and are structurally unfavored for DNA polymerases to read over comparing to transition mutations. In the first part of the thesis, we developed SeSaM-DV, an advanced SeSaM method which employs a new mismatch-tolerant polymerase and generated the highest amount of TvTv mutations ever reported (217% of the best reported number). In the development of SeSaM-DV, a DNA polymerase assay targeting mismatch tolerance ability was developed. Subsequently, thirteen DNA polymerases were evaluated for their ability to read over consecutive phosphorothioated mismatches, which is considered a “bottle neck” for increasing consecutive substitutions in SeSaM libraries. Employing the polymerase mismatch tolerance assay S. islandicus DNA polymerase IV (Sis. Dpo4) exhibited the best ability of extending consecutive mismatches and was therefore subjected to an additional PCR-based assay which directly validated the ability of Sis. Dpo4 to extend four types of consecutive mismatches thus generate TvTv, TvTs, TsTv, and TsTs substitutions. Finally, Sis. Dpo4 was incorporated into SeSaM step 3 with the addition of Vent exo- to achieve efficient with extension after reading over mismatches. The resulted SeSaM-DV library was benchmarked by sequencing and 10% of the identified mutations were TvTv mutations, which is 2.17-fold better than the best reported number. With the new type of TvTv-enriched mutational bias, SeSaM-DV can efficiently evolve enzymes by exploring natures sequence space in a new “non-natural” way with an expanded chemical diversity, which might be a better approach to engineer enzymes for industrial productions which often involve non-natural conditions such as organic solvents and altered pH. Microbial cell surface display (CSD) is a powerful platform to present and immobilize the protein of interest on microbial surfaces. CSD provides a genotype-phenotype linkage without the need of artificial compartmentalization of the protein and its coding gene thus enables FACS-based ultra-high throughput screening of protein libraries. Various proteins have been displayed on microbial surface, however, to the best of our knowledge, no nucleic acid (NA) polymerase has been successfully displayed on microbial surface. In the second part of the thesis, we developed 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 of E. coli DNA polymerase I (KF) 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 incorporation of 2’-OMe-NTPs and a KF variant with 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 PCR and sequencing based biotechnological and medical applications. Especially, cell surface display enables novel polymerase screening strategies in which heat-lysis step are 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., Published by RWTH Aachen University, Aachen
- Published
- 2021
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