Publications and patents

  Publications and patents Biotec

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 190 peer-reviewed articles and over 20 filed patents.

You can also access the full publication list via the library server (please note that the data deposited there are updated by the RWTH library).

 
 

Electron transfer pathways in a light, oxygen, voltage (LOV) protein devoid of the photoactive cysteine

Blue-light absorption by the flavin chromophore in light, oxygen, voltage (LOV) photoreceptors triggers photochemical reactions that lead to the formation of a flavin-cysteine adduct. While it has long been assumed that adduct formation is essential for signaling, it was recently shown that LOV photoreceptor variants devoid of the photoactive cysteine can elicit a functional response and that flavin photoreduction to the neutral semiquinone is sufficient for signal transduction. Currently, the mechanistic basis of the underlying electron- (eT) and proton-transfer (pT) reactions is not well understood.

In this collaborative study, we reengineered pT into the naturally not photoreducible iLOV protein. A single amino acid substitution (Q489D) was sufficient to enable efficient photoreduction under aerobic conditions, which suggests that an eT pathway is naturally present in the protein. Employing this variant, we investigated the underlying eT and pT reactions using a combination of steady-state UV/Vis, transient absorption, electron paramagnetic resonance spectroscopy combined with site-directed mutagenesis. Our study provides strong evidence that several Tyrosine and Tryptophan residues, highly conserved in all LOV proteins, constitute the eT pathway for flavin photoreduction, suggesting that the propensity for photoreduction is evolutionary imprinted in all LOV domains, while efficient pT is needed to stabilize the neutral semiquinone radical.

Kopka, B., Magerl, K., Savitsky, A., Davari, M.D., Röllen, K., Bocola, M., Dick, B., Schwaneberg, U., Jaeger, K.E., Krauss, U.; Electron transfer pathways in a light, oxygen, voltage (LOV) protein devoid of the photoactive cysteine; Scientific Reports 7, Article number: 13346 (2017); doi:10.1038/s41598-017-13420-1

 
 

Engineering of Candida parapsilosis alcohol dehydrogenase for conversion of methyl 3-hydroxyalkanoates

Engineering of a Candida parapsilosis alcohol dehydrogenase Bio VI

Expanding the substrate scope of enzymes opens up new routes for the synthesis of valuable chemicals. Ketone-functionalized fatty acid derivatives and corresponding chiral alcohols are valuable building blocks for the synthesis of a variety of chemicals including pharmaceuticals.

Candida parapsilosis alcohol dehydrogenase wild type is an S-selective alcohol dehydrogenase that does not convert 3-hydroxy fatty acid methyl esters larger than methyl 3-hydroxypentanoate. Thus, methyl 3-hydroxyhexanoate and methyl (R)-3-hydroxybutyrate are not accepted as substrate by wild type Candida parapsilosis alcohol dehydrogenase. Enlarging the binding pocket of the wild type enzyme through substitution of tryptophan at position 286 by alanine led to a chain length specificity shift and oxidation of methyl 3-hydroxyhexanoate to the corresponding ketone. Furthermore, a second variant with double substitution on positions 119 and 286 exhibited inverted enantiopreference for methyl 3-hydroxybutyrate from the S-enantiomer to the R-enantiomer.

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Y. Ensari, G. V. Dhoke, M. D. Davari, M. Bocola, A. J. Ruff, U. Schwaneberg, Chem. Eur. J. 2017, 23, 12636.

 
 

Sortase-mediated functionalization of microgels with biomolecules

Sortase-mediated functionalization of microgels ACS Publications

Microgels are colloidal macromolecular networks swollen in water while forming stable dispersions. They are highly porous, soft, deformable and exhibit a stimuli-responsiveness in aqueous solutions. Due to their interesting properties, microgels gained much attention in macromolecular and biomedical research fields.

In this work we developed a sortase-mediated method for chemo-selective and efficient decoration of aqueous microgels with biomolecules. Poly(N-vinylcaprolactam) (VCL) microgels with different amounts of glycidyl methacrylate (GMA) as a comonomer incorporated in the microgel shell were synthesized and characterized regarding to their size, swelling degree and temperature-responsiveness in aqueous solutions. The surface of the PVCL/GMA microgel was modified with the specific recognition peptide sequence (LPETG) for Sortase A. Sortase-mediated conjugation of oligoglycin-tagged enhanced Green Fluorescent Protein (eGFP) to the LPETG-modified microgels was successfully performed. Sortase-mediated bioconjugation can be used as a simple and powerful technique for the targeted surface functionalization of stimuli-responsive microgels with biomolecules.

Gau, E., Mate, D. M., Zou, Z., Oppermann, A., Töpel, A., Jakob, F., Schwaneberg, U. & Pich, A. (2017). Sortase-Mediated Surface Functionalization of Stimuli-Responsive Microgels. Biomacromolecules

 
 

High interfacial activity of a transmembrane protein leads to nano-thin walled micro-compartments

Generation of micro compartiments Royal 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 Cosolvents

Directed evolution of lipase in organic solvents MDPI 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 polymerization

MPD as detergent-substitute for hybrid catalyst ACS 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 peptides

Anchor peptides Bio 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