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- ItemDiffusive Motion of Linear Microgel Assemblies in Solution(Basel : MDPI, 2016) Schürings, Marco-Philipp; Nevskyi, Oleksii; Eliasch, Kamill; Michel, Ann-Katrin; Liu, Bing; Pich, Andrij; Böker, Alexander; Von Plessen, Gero; Wöll, DominikDue to the ability of microgels to rapidly contract and expand in response to external stimuli, assemblies of interconnected microgels are promising for actuation applications, e.g., as contracting fibers for artificial muscles. Among the properties determining the suitability of microgel assemblies for actuation are mechanical parameters such as bending stiffness and mobility. Here, we study the properties of linear, one-dimensional chains of poly(N-vinylcaprolactam) microgels dispersed in water. They were fabricated by utilizing wrinkled surfaces as templates and UV-cross-linking the microgels. We image the shapes of the chains on surfaces and in solution using atomic force microscopy (AFM) and fluorescence microscopy, respectively. In solution, the chains are observed to execute translational and rotational diffusive motions. Evaluation of the motions yields translational and rotational diffusion coefficients and, from the translational diffusion coefficient, the chain mobility. The microgel chains show no perceptible bending, which yields a lower limit on their bending stiffness
- ItemCaLB Catalyzed Conversion of ε-Caprolactone in Aqueous Medium. Part 1: Immobilization of CaLB to Microgels(Basel : MDPI, 2016) Engel, Stefan; Höck, Heidi; Bocola, Marco; Keul, Helmut; Schwaneberg, Ulrich; Möller, MartinThe enzymatic ring-opening polymerization of lactones is a method of increasing interest for the synthesis of biodegradable and biocompatible polymers. In the past it was shown that immobilization of Candida antarctica lipase B (CaLB) and the reaction medium play an important role in the polymerization ability especially of medium ring size lactones like ε-caprolactone (ε-CL). We investigated a route for the preparation of compartmentalized microgels based on poly(glycidol) in which CaLB was immobilized to increase its esterification ability. To find the ideal environment for CaLB, we investigated the acceptable water concentration and the accessibility for the monomer in model polymerizations in toluene and analyzed the obtained oligomers/polymers by NMR and SEC. We observed a sufficient accessibility for ε-CL to a toluene like hydrophobic phase imitating a hydrophobic microgel. Comparing free CaLB and Novozym® 435 we found that not the monomer concentration but rather the solubility of the enzyme, as well as the water concentration, strongly influences the equilibrium of esterification and hydrolysis. On the basis of these investigations, microgels of different polarity were prepared and successfully loaded with CaLB by physical entrapment. By comparison of immobilized and free CaLB, we demonstrated an effect of the hydrophobicity of the microenvironment of CaLB on its enzymatic activity.
- ItemDisulfide Bond Engineering of an Endoglucanase from Penicillium verruculosum to Improve Its Thermostability(Basel : Molecular Diversity Preservation International (MDPI), 2019) Bashirova, Anna; Pramanik, Subrata; Volkov, Pavel; Rozhkova, Aleksandra; Nemashkalov, Vitaly; Zorov, Ivan; Gusakov, Alexander; Sinitsyn, Arkady; Schwaneberg, Ulrich; Davari, Mehdi D.Endoglucanases (EGLs) are important components of multienzyme cocktails used in the production of a wide variety of fine and bulk chemicals from lignocellulosic feedstocks. However, a low thermostability and the loss of catalytic performance of EGLs at industrially required temperatures limit their commercial applications. A structure-based disulfide bond (DSB) engineering was carried out in order to improve the thermostability of EGLII from Penicillium verruculosum. Based on in silico prediction, two improved enzyme variants, S127C-A165C (DSB2) and Y171C-L201C (DSB3), were obtained. Both engineered enzymes displayed a 15–21% increase in specific activity against carboxymethylcellulose and β-glucan compared to the wild-type EGLII (EGLII-wt). After incubation at 70 °C for 2 h, they retained 52–58% of their activity, while EGLII-wt retained only 38% of its activity. At 80 °C, the enzyme-engineered forms retained 15–22% of their activity after 2 h, whereas EGLII-wt was completely inactivated after the same incubation time. Molecular dynamics simulations revealed that the introduced DSB rigidified a global structure of DSB2 and DSB3 variants, thus enhancing their thermostability. In conclusion, this work provides an insight into DSB protein engineering as a potential rational design strategy that might be applicable for improving the stability of other enzymes for industrial applications.
- Item2-Methyl-2,4-pentanediol (MPD) boosts as detergent-substitute the performance of ß-barrel hybrid catalyst for phenylacetylene polymerization(Frankfurt, M. : Beilstein-Institut zur Förderung der Chemischen Wissenschaften, 2017) Kinzel, Julia; Sauer, Daniel F.; Bocola, Marco; Arlt, Marcus; Mirzaei Garakani, Tayebeh; Thiel, Andreas; Beckerle, Klaus; Polen, Tino; Okuda, Jun; Schwaneberg, UlrichCovering hydrophobic regions with stabilization agents to solubilize purified transmembrane proteins is crucial for their application in aqueous media. The small molecule 2-methyl-2,4-pentanediol (MPD) was used to stabilize the transmembrane protein Ferric hydroxamate uptake protein component A (FhuA) utilized as host for the construction of a rhodium-based biohybrid catalyst. Unlike commonly used detergents such as sodium dodecyl sulfate or polyethylene polyethyleneglycol, MPD does not form micelles in solution. Molecular dynamics simulations revealed the effect and position of stabilizing MPD molecules. The advantage of the amphiphilic MPD over micelle-forming detergents is demonstrated in the polymerization of phenylacetylene, showing a ten-fold increase in yield and increased molecular weights.
- ItemPolymer Brush-Functionalized Chitosan Hydrogels as Antifouling Implant Coatings(Columbus, Ohio : American Chemical Society, 2017) Buzzacchera, Irene; Vorobii, Mariia; Kostina, Nina Yu; de Los Santos Pereira, Andres; Riedel, Tomáš; Bruns, Michael; Ogieglo, Wojciech; Möller, Martin; Wilson, Christopher J.; Rodriguez-Emmenegger, CesarImplantable sensor devices require coatings that efficiently interface with the tissue environment to mediate biochemical analysis. In this regard, bioinspired polymer hydrogels offer an attractive and abundant source of coating materials. However, upon implantation these materials generally elicit inflammation and the foreign body reaction as a consequence of protein fouling on their surface and concomitant poor hemocompatibility. In this report we investigate a strategy to endow chitosan hydrogel coatings with antifouling properties by the grafting of polymer brushes in a "grafting-from" approach. Chitosan coatings were functionalized with polymer brushes of oligo(ethylene glycol) methyl ether methacrylate and 2-hydroxyethyl methacrylate using photoinduced single electron transfer living radical polymerization and the surfaces were thoroughly characterized by XPS, AFM, water contact angle goniometry, and in situ ellipsometry. The antifouling properties of these new bioinspired hydrogel-brush coatings were investigated by surface plasmon resonance. The influence of the modifications to the chitosan on hemocompatibility was assessed by contacting the surfaces with platelets and leukocytes. The coatings were hydrophilic and reached a thickness of up to 180 nm within 30 min of polymerization. The functionalization of the surface with polymer brushes significantly reduced the protein fouling and eliminated platelet activation and leukocyte adhesion. This methodology offers a facile route to functionalizing implantable sensor systems with antifouling coatings that improve hemocompatibility and pave the way for enhanced device integration in tissue.
- ItemComparison of Candida antarctica Lipase B Variants for Conversion of ε-Caprolactone in Aqueous Medium-Part 2(Basel : MDPI, 2018) Höck, Heidi; Engel, Stefan; Weingarten, Simone; Keul, Helmut; Schwaneberg, Ulrich; Möller, Martin; Bocola, MarcoEnzyme-catalyzed ring-opening polymerization of lactones is a method of increasing interest for the synthesis of polyesters. In the present work, we investigated which changes in the structure of Candida antarctica lipase B (CaLB) shift the catalytic equilibrium between esterification and hydrolysis towards polymerization. Therefore, we present two concepts: (i) removing the glycosylation of CaLB to increase the surface hydrophobicity; and (ii) introducing a hydrophobic lid adapted from Pseudomonas cepacia lipase (PsCL) to enhance the interaction of a growing polymer chain to the elongated lid helix. The deglycosylated CaLB (CaLB-degl) was successfully generated by site-saturation mutagenesis of asparagine 74. Furthermore, computational modeling showed that the introduction of a lid helix at position Ala148 was structurally feasible and the geometry of the active site remained intact. Via overlap extension PCR the lid was successfully inserted, and the variant was produced in large scale in Pichia pastoris with glycosylation (CaLB-lid) and without (CaLB-degl-lid). While the lid variants show a minor positive effect on the polymerization activity, CaLB-degl showed a clearly reduced hydrolytic and enhanced polymerization activity. Immobilization in a hydrophobic polyglycidol-based microgel intensified this effect such that a higher polymerization activity was achieved, compared to the “gold standard” Novozym® 435.
- ItemPerforming DNA nanotechnology operations on a zebrafish(Cambridge : RSC, 2018) Yang, Jian; Meng, Zhuojun; Liu, Qing; Shimada, Yasuhito; Olsthoorn, René C. L.; Spaink, Herman P.; Herrmann, Andreas; Kros, AlexanderNanoscale engineering of surfaces is becoming an indispensable technique to modify membranes and, thus cellular behaviour. Here, such membrane engineering related was explored on the surface of a living animal using DNA nanotechnology. We demonstrate the immobilization of oligonucleotides functionalized with a membrane anchor on 2 day old zebrafish. The protruding single-stranded DNA on the skin of zebrafish served as a handle for complementary DNAs, which allowed the attachment of small molecule cargo, liposomes and dynamic relabeling by DNA hybridization protocols. Robust anchoring of the oligonucleotides was proven as DNA-based amplification processes were successfully performed on the outer membrane of the zebrafish enabling the multiplication of surface functionalities from a single DNA-anchoring unit and the dramatic improvement of fluorescent labeling of these animals. As zebrafish are becoming an alternative to animal models in drug development, toxicology and nanoparticles characterization, we believe the platform presented here allows amalgamation of DNA nanotechnology tools with live animals and this opens up yet unexplored avenues like efficient bio-barcoding as well as in vivo tracking. © The Royal Society of Chemistry.
- ItemHigh-Throughput Production of Micrometer Sized Double Emulsions and Microgel Capsules in Parallelized 3D Printed Microfluidic Devices(Basel : MDPI, 2019) Jans, Alexander; Lölsberg, Jonas; Omidinia-Anarkoli, Abdolrahman; Viermann, Robin; Möller, Martin; De Laporte, Laura; Wessling, Matthias; Kuehne, Alexander J. C.Double emulsions are useful geometries as templates for core-shell particles, hollow sphere capsules, and for the production of biomedical delivery vehicles. In microfluidics, two approaches are currently being pursued for the preparation of microfluidic double emulsion devices. The first approach utilizes soft lithography, where many identical double-flow-focusing channel geometries are produced in a hydrophobic silicone matrix. This technique requires selective surface modification of the respective channel sections to facilitate alternating wetting conditions of the channel walls to obtain monodisperse double emulsion droplets. The second technique relies on tapered glass capillaries, which are coaxially aligned, so that double emulsions are produced after flow focusing of two co-flowing streams. This technique does not require surface modification of the capillaries, as only the continuous phase is in contact with the emulsifying orifice; however, these devices cannot be fabricated in a reproducible manner, which results in polydisperse double emulsion droplets, if these capillary devices were to be parallelized. Here, we present 3D printing as a means to generate four identical and parallelized capillary device architectures, which produce monodisperse double emulsions with droplet diameters in the range of 500 µm. We demonstrate high throughput synthesis of W/O/W and O/W/O double emulsions, without the need for time-consuming surface treatment of the 3D printed microfluidic device architecture. Finally, we show that we can apply this device platform to generate hollow sphere microgels.
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- ItemNovel Antibacterial Polyglycidols: Relationship between Structure and Properties(Basel : MDPI, 2018) Marquardt, Fabian; Stöcker, Cornelia; Gartzen, Rita; Heine, Elisabeth; Keul, Helmut; Möller, MartinAntimicrobial polymers are an attractive alternative to low molecular weight biocides, because they are non-volatile, chemically stable, and can be used as non-releasing additives. Polymers with pendant quaternary ammonium groups and hydrophobic chains exhibit antimicrobial properties due to the electrostatic interaction between polymer and cell wall, and the membrane disruptive capabilities of the hydrophobic moiety. Herein, the synthesis of cationic–hydrophobic polyglycidols with varying structures by post-polymerization modification is presented. The antimicrobial properties of the prepared polyglycidols against E. coli and S. aureus are examined. Polyglycidol with statistically distributed cationic and hydrophobic groups (cationic–hydrophobic balance of 1:1) is compared to (i) polyglycidol with a hydrophilic modification at the cationic functionality; (ii) polyglycidol with both—cationic and hydrophobic groups—at every repeating unit; and (iii) polyglycidol with a cationic–hydrophobic balance of 1:2. A relationship between structure and properties is presented.