Publications and patentsBiotec
We are experts in protein engineering with a focus on directed evolution approaches and semirational or rational design. Our research includes empirical and computational method development, and the application of the latter to ultimately generate enzyme variants with tailored characteristics. Computational methods further serve to solve structure/function relationships of targeted enzymes. The research output of the group is reflected by over 180 peer-reviewed articles and over 20 filed patents.
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High interfacial activity of a transmembrane protein leads to nano-thin walled micro-compartmentsRoyal Society of Chemistry
Protein-polymer conjugates have several applications in the field of biotechnology, medicine and nanotechnology. One example of conjugates is based on the transmembrane protein ferric hydroxamate uptake protein component A, FhuA, an outer membrane protein of Escherichia coli. These conjugates present properties such as high interfacial activity, which can be utilized for generation of Pickering emulsions. Instead of surfactant molecules, the conjugate particles are able to stabilize emulsions. We generated Pickering emulsions for the first time with the transmembrane protein FhuA. Stable micro-compartments made of these Pickering emulsions were later crosslinked by a newly synthesized UV-crosslinkable monomer, 3,4-dimethyl maleic imidobutyl acrylate, DMMIBA, to polymer chains. Characterization of the micro-compartments with scanning force microscopy determined membrane thickness of 11.1 ± 0.6 nm and fluorescent microscopy revealed the stability of the micro-compartments even after treatment with ethanol. These micro-compartments have potential application for drug delivery systems and bring us closer to the possibility of protein-polymer membrane generation, within which properties of membranes can be altered by temperature and pH stimuli.
Charan H., Glebe U., Anand D., Kinzel J., Zhu L., Bocola M., Mirzaei Garakani T., Schwaneberg U., Böker A. (2017) Nano-thin walled micro-compartments from transmembrane protein-polymer conjugates, Soft Matter 13, 2866-2875. [DOI: 10.1039/C6SM02520J.]
Are Directed Evolution Approaches Efficient in Exploring Nature’s Potential to Stabilize a Lipase in Organic CosolventsMDPI AG
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 compare 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 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, 2,2,2-trifluoroethanol, and dimethyl sulfoxide. Our analysis revealed that although a significant amount of all possible single substitutions contributes to improved cosolvent resistance, only a fraction of these substitutions could be detected in the three random mutagenesis libraries. 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, our investigation of the BSLA SSM library indicates the importance of introducing surface charges for organic cosolvent resistance.
Markel, U., Zhu, L., Frauenkron-Machedjou, V. J., Zhao, J., Bocola, M., Davari, M. D., Jaeger, K.-E., Schwaneberg, U. (2017). Are Directed Evolution Approaches Efficient in Exploring Nature’s Potential to Stabilize a Lipase in Organic Cosolvents? Catalysts, 7, 142.
2-Methyl-2,4-pentanediol boosts as detergent-substitute the performance of ß-barrel hybrid catalyst for phenylacetylene polymerizationACS Publications
Transmembrane proteins have a hydrophobic middle part due to their presence in natural lipid bilayer. Therefore, stabilizing agents are essentially used to cover hydrophobic regions to solubilize purified transmembrane proteins for usage in aqueous media. 2-Methyl-2,4-pentanediol -MPD- a small molecule was applied for stabilization of ferric hydroxamate uptake protein component A, FhuA, which is a transmembrane protein found in the outer membrane of Escherichia coli. The β-barrel shaped protein was used for hosting a rhodium catalyst to perform a polymerization reaction of phenylacetylene as proof of concept. MPD does not form micelles compared to other commonly used detergents such as sodium dodecylsulfate and polyethylene polyethyleneglycol leading to higher polymer product yield and polymer masses. Computer-based simulations supported the suitability of the amphiphilic MPD molecules for successful stabilization of the transmembrane protein FhuA and enabling the functionality of the protein channel.
Kinzel, J., Sauer, D. F., Bocola, M., Arlt, M., Mirzaei Garakani, T., Thiel, A., Beckerle, K., Polen, T., Okuda, J., Schwaneberg, U.; 2-Methyl-2,4-pentanediol (MPD) boosts as detergent-substitute the performance of ß-barrel hybrid catalyst for phenylacetylene polymerization; Beilstein J. Org. Chem. 2017, 13, 1498-1506 [DOI: 10.3762/bjoc.13.148.]
Green and versatile functionalization of polypropylene by anchor peptidesBio VI
Polypropylene is a wide spread commodity polymer used in medicine, textiles or packaging. Surface functionalization of polypropylene is performed to increase wettability, adhesion, or dyeing behavior, but it is challenging due to absent functional groups. Functionalization using peptides naturally evolved for surface interaction represents an attractive alternative to conventional modification techniques.
In this study polypropylene binding peptides were identified, characterized and used to successfully functionalize polypropylene surfaces. Anchor peptides bind to polypropylene at room temperature in water and show stability against treatment with surfactants. The presented anchor peptide toolbox enables easy to handle, green functionalization of polypropylene based materials.
Rübsam, K., Stomps, B., Jakob, F., Böker, A., Schwaneberg, U.; Anchor peptides: A green and versatile method for polypropylene functionalization; Polymer, 2017