TY - JOUR A1 - Pilas, Johanna A1 - Yazici, Yasemen A1 - Selmer, Thorsten A1 - Keusgen, Michael A1 - Schöning, Michael Josef T1 - Optimization of an amperometric biosensor array for simultaneous measurement of ethanol, formate, d- and l-lactate JF - Electrochimica Acta N2 - The immobilization of NAD+-dependent dehydrogenases, in combination with a diaphorase, enables the facile development of multiparametric sensing devices. In this work, an amperometric biosensor array for simultaneous determination of ethanol, formate, d- and l-lactate is presented. Enzyme immobilization on platinum thin-film electrodes was realized by chemical cross-linking with glutaraldehyde. The optimization of the sensor performance was investigated with regard to enzyme loading, glutaraldehyde concentration, pH, cofactor concentration and temperature. Under optimal working conditions (potassium phosphate buffer with pH 7.5, 2.5 mmol L-1 NAD+, 2.0 mmol L-1 ferricyanide, 25 °C and 0.4% glutaraldehyde) the linear working range and sensitivity of the four sensor elements was improved. Simultaneous and cross-talk free measurements of four different metabolic parameters were performed successfully. The reliable analytical performance of the biosensor array was demonstrated by application in a clarified sample of inoculum sludge. Thereby, a promising approach for on-site monitoring of fermentation processes is provided. KW - Simultaneous determination KW - Enzymatic biosensor KW - Diaphorase KW - Dehydrogenase Y1 - 2017 U6 - https://doi.org/10.1016/j.electacta.2017.07.119 SN - 0013-4686 VL - 251 SP - 256 EP - 262 PB - Elsevier CY - Amsterdam ER - TY - JOUR A1 - Röhlen, Desiree A1 - Pilas, Johanna A1 - Schöning, Michael Josef A1 - Selmer, Thorsten T1 - Development of an amperometric biosensor platform for the combined determination of l-Malic, Fumaric, and l-Aspartic acid JF - Applied Biochemistry and Biotechnology N2 - Three amperometric biosensors have been developed for the detection of L-malic acid, fumaric acid, and L -aspartic acid, all based on the combination of a malate-specific dehydrogenase (MDH, EC 1.1.1.37) and diaphorase (DIA, EC 1.8.1.4). The stepwise expansion of the malate platform with the enzymes fumarate hydratase (FH, EC 4.2.1.2) and aspartate ammonia-lyase (ASPA, EC 4.3.1.1) resulted in multi-enzyme reaction cascades and, thus, augmentation of the substrate spectrum of the sensors. Electrochemical measurements were carried out in presence of the cofactor β-nicotinamide adenine dinucleotide (NAD+) and the redox mediator hexacyanoferrate (III) (HCFIII). The amperometric detection is mediated by oxidation of hexacyanoferrate (II) (HCFII) at an applied potential of + 0.3 V vs. Ag/AgCl. For each biosensor, optimum working conditions were defined by adjustment of cofactor concentrations, buffer pH, and immobilization procedure. Under these improved conditions, amperometric responses were linear up to 3.0 mM for L-malate and fumarate, respectively, with a corresponding sensitivity of 0.7 μA mM−1 (L-malate biosensor) and 0.4 μA mM−1 (fumarate biosensor). The L-aspartate detection system displayed a linear range of 1.0–10.0 mM with a sensitivity of 0.09 μA mM−1. The sensor characteristics suggest that the developed platform provides a promising method for the detection and differentiation of the three substrates. Y1 - 2017 U6 - https://doi.org/10.1007/s12010-017-2578-1 SN - 1559-0291 VL - 183 SP - 566 EP - 581 PB - Springer CY - Berlin ER - TY - JOUR A1 - Muschallik, Lukas A1 - Molinnus, Denise A1 - Bongaerts, Johannes A1 - Pohl, Martina A1 - Wagner, Torsten A1 - Schöning, Michael Josef A1 - Siegert, Petra A1 - Selmer, Thorsten T1 - (R,R)-Butane-2,3-diol Dehydrogenase from Bacillus clausii DSM 8716T: Cloning and Expression of the bdhA-Gene, and Initial Characterization of Enzyme JF - Journal of Biotechnology N2 - The gene encoding a putative (R,R)-butane-2,3-diol dehydrogenase (bdhA) from Bacillus clausii DSM 8716T was isolated, sequenced and expressed in Escherichia coli. The amino acid sequence of the encoded protein is only distantly related to previously studied enzymes (identity 33–43%) and exhibited some uncharted peculiarities. An N-terminally StrepII-tagged enzyme variant was purified and initially characterized. The isolated enzyme catalyzed the (R)-specific oxidation of (R,R)- and meso-butane-2,3-diol to (R)- and (S)-acetoin with specific activities of 12 U/mg and 23 U/mg, respectively. Likewise, racemic acetoin was reduced with a specific activity of up to 115 U/mg yielding a mixture of (R,R)- and meso-butane-2,3-diol, while the enzyme reduced butane-2,3-dione (Vmax 74 U/mg) solely to (R,R)-butane-2,3-diol via (R)-acetoin. For these reactions only activity with the co-substrates NADH/NAD+ was observed. The enzyme accepted a selection of vicinal diketones, α-hydroxy ketones and vicinal diols as alternative substrates. Although the physiological function of the enzyme in B. clausii remains elusive, the data presented herein clearly demonstrates that the encoded enzyme is a genuine (R,R)-butane-2,3-diol dehydrogenase with potential for applications in biocatalysis and sensor development. Y1 - 2017 U6 - https://doi.org/10.1016/j.jbiotec.2017.07.020 SN - 0168-1656 VL - 258 SP - 41 EP - 50 PB - Elsevier CY - Amsterdam ER - TY - JOUR A1 - Tippkötter, Nils A1 - Duwe, Anna-Maria A1 - Wiesen, Sebastian A1 - Sieker, Tim A1 - Ulber, Roland T1 - Enzymatic hydrolysis of beech wood lignocellulose at high solid contents and its utilization as substrate for the production of biobutanol and dicarboxylic acids JF - Bioresource Technology N2 - The development of a cost-effective hydrolysis for crude cellulose is an essential part of biorefinery developments. To establish such high solid hydrolysis, a new solid state reactor with static mixing is used. However, concentrations >10% (w/w) cause a rate and yield reduction of enzymatic hydrolysis. By optimizing the synergetic activity of cellulolytic enzymes at solid concentrations of 9%, 17% and 23% (w/w) of crude Organosolv cellulose, glucose concentrations of 57, 113 and 152 g L⁻¹ are reached. However, the glucose yield decreases from 0.81 to 0.72gg⁻¹ at 17% (w/w). Optimal conditions for hydrolysis scale-up under minimal enzyme addition are identified. As result, at 23% (w/w) crude cellulose the glucose yield increases from 0.29 to 0.49gg⁻¹. As proof of its applicability, biobutanol, succinic and itaconic acid are produced with the crude hydrolysate. The potential of the substrate is proven e.g. by a high butanol yield of 0.33gg⁻¹. Y1 - 2014 U6 - https://doi.org/10.1016/j.biortech.2014.06.052 VL - 167 SP - 447 EP - 455 PB - Elsevier CY - Amsterdam ER - TY - JOUR A1 - Wiesen, Sebastian A1 - Tippkötter, Nils A1 - Muffler, Kai A1 - Suck, Kirstin A1 - Sohling, Ulrich A1 - Ruf, Friedrich A1 - Ulber, Roland T1 - Adsorption of fatty acids to layered double hydroxides in aqueous systems JF - Adsorption N2 - Due to their anion exchange characteristics, layered double hydroxides (LDHs) are suitable for the detoxification of aqueous, fatty acid containing fermentation substrates. The aim of this study is to examine the adsorption mechanism, using crude glycerol from plant oil esterification as a model system. Changes in the intercalation structure in relation to the amount of fatty acids adsorbed are monitored by X-ray diffraction and infra-red spectroscopy. Additionally, calcination of LDH is investigated in order to increase the binding capacity for fatty acids. Our data propose that, at ambient temperature, fatty acids can be bound to the hydrotalcite by adsorption or in addition by intercalation, depending on fatty acid concentration. The adsorption of fatty acids from crude glycerol shows a BET-like behavior. Above a fatty acid concentration of 3.5 g L−1, intercalation of fatty acids can be shown by the appearance of an increased interlayer spacing. This observation suggests a two phase adsorption process. Calcination of LDHs allows increasing the binding capacity for fatty acids by more than six times, mainly by reduction of structural CO32−. Y1 - 2015 VL - 21 IS - 6-7 SP - 459 EP - 466 PB - Springer CY - Berlin ER - TY - JOUR A1 - Wulfhorst, Helene A1 - Duwe, Anna-Maria A1 - Merseburg, Johannes A1 - Tippkötter, Nils T1 - Compositional analysis of pretreated (beech) wood using differential scanning calorimetry and multivariate data analysis JF - Tetrahedron N2 - The composition of plant biomass varies depending on the feedstock and pre-treatment conditions and influences its processing in biorefineries. In order to ensure optimal process conditions, the quantitative proportion of the main polymeric components of the pre-treated biomass has to be determined. Current standard procedures for biomass compositional analysis are complex, the measurements are afflicted with errors and therefore often not comparable. Hence, new powerful analytical methods are urgently required to characterize biomass. In this contribution, Differential Scanning Calorimetry (DSC) was applied in combination with multivariate data analysis (MVA) to detect the cellulose content of the plant biomass pretreated by Liquid Hot Water (LHW) and Organosolv processes under various conditions. Unlike conventional techniques, the developed analytic method enables the accurate quantification of monosaccharide content of the plant biomass without any previous sample preparation. It is easy to handle and avoids errors in sample preparation. Y1 - 2016 U6 - https://doi.org/10.1016/j.tet.2016.04.029 VL - 72 IS - 46 SP - 7329 EP - 7334 PB - Elsevier CY - Amsterdam ER - TY - JOUR A1 - Al-Kaidy, Huschyar A1 - Kuthan, Kai A1 - Hering, Thomas A1 - Tippkötter, Nils T1 - Aqueous droplets used as enzymatic microreactors and their electromagnetic actuation JF - Journal of Visualized Experiments N2 - For the successful implementation of microfluidic reaction systems, such as PCR and electrophoresis, the movement of small liquid volumes is essential. In conventional lab-on-a-chip-platforms, solvents and samples are passed through defined microfluidic channels with complex flow control installations. The droplet actuation platform presented here is a promising alternative. With it, it is possible to move a liquid drop (microreactor) on a planar surface of a reaction platform (lab-in-a-drop). The actuation of microreactors on the hydrophobic surface of the platform is based on the use of magnetic forces acting on the outer shell of the liquid drops which is made of a thin layer of superhydrophobic magnetite particles. The hydrophobic surface of the platform is needed to avoid any contact between the liquid core and the surface to allow a smooth movement of the microreactor. On the platform, one or more microreactors with volumes of 10 µL can be positioned and moved simultaneously. The platform itself consists of a 3 x 3 matrix of electrical double coils which accommodate either neodymium or iron cores. The magnetic field gradients are automatically controlled. By variation of the magnetic field gradients, the microreactors' magnetic hydrophobic shell can be manipulated automatically to move the microreactor or open the shell reversibly. Reactions of substrates and corresponding enzymes can be initiated by merging the microreactors or bringing them into contact with surface immobilized catalysts. Y1 - 2016 U6 - https://doi.org/10.3791/54643 SN - 1940-087X IS - Issue 126 ER - TY - JOUR A1 - Sieker, Tim A1 - Ulber, Roland A1 - Dimitrova, Darina A1 - Bart, Hans-Jörg A1 - Neuner, Andreas A1 - Heinzle, Elmar A1 - Tippkötter, Nils T1 - Silage : Fermentationsrohstoff für die chemische Industrie? JF - labor&more N2 - In Anbetracht des zu erwartenden Rückgangs der Verfügbarkeit fossiler Rohstoffe müssen nicht nur für den Energiesektor, sondern auch für die Herstellung industrieller Produkte alternative Rohstoffe gefunden werden. Ein Beispiel für einen nicht in Nahrungsmittelkonkurrenz stehenden nachwachsenden Rohstoff ist grüne Biomasse wie Gras und Klee. Diese lassen sich in Deutschland auf großen Flächen anbauen und enthalten eine Vielzahl potenzieller Substrate für Fermentationen. Y1 - 2009 IS - 2 SP - 44 EP - 45 ER - TY - JOUR A1 - Tippkötter, Nils A1 - Wollny, S. A1 - Kampeis, P. A1 - Oster, J. A1 - Schneider, H. A1 - Ulber, Roland T1 - Magnetseparation von Proteinen : Separation von Zielmolekülen durch hochselektive Aptamere JF - GIT Labor-Fachzeitschrift N2 - Durch die Kombination von Oligonukleotid-Liganden (Aptameren) hoher Bindungsaffinitäten mit hochselektiv abtrennbaren magnetisierbaren Mikropartikeln wird eine einstufige Separation von Zielmolekülen aus mikrobiologischen Produktionsansätzen möglich. Die Aptamere werden hierfür reversibel auf den Partikeloberflächen gebunden und für die spezifische Isolierung von Bioprodukten eingesetzt. Die Abtrennung der beladenen Partikel erfolgt durch einen neuen Rotor-Stator-Separator mit Hochgradient-Magnetfeld. Y1 - 2011 VL - 55 IS - 10 SP - 666 PB - Wiley CY - Weinheim ER - TY - JOUR A1 - Kappler-Tanudyaya, Nathalie A1 - Schmitt, Heike A1 - Tippkötter, Nils A1 - Meyer, Lina A1 - Lenzen, Sigurd A1 - Ulber, Roland T1 - Combination of biotransformation and chromatography for the isolation and purification of mannoheptulose JF - Biotechnology Journal N2 - Mannoheptulose is a seven-carbon sugar. It is an inhibitor of glucose-induced insulin secretion due to its ability to selectively inhibit the enzyme glucokinase. An improved procedure for mannoheptulose isolation from avocados is described in this study (based upon the original method by La Forge). The study focuses on the combination of biotransformation and downstream processing (preparative chromatography) as an efficient method to produce a pure extract of mannoheptulose. The experiments were divided into two major phases. In the first phase, several methods and parameters were compared to optimize the mannoheptulose extraction with respect to efficiency and purity. In the second phase, a mass balance of mannoheptulose over the whole extraction process was undertaken to estimate the yield and efficiency of the total extraction process. The combination of biotransformation and preparative chromatography allowed the production of a pure mannoheptulose extract. In a biological test, the sugar inhibited the glucokinase enzyme activity efficiently. Y1 - 2007 U6 - https://doi.org/10.1002/biot.200700004 SN - 1860-7314 VL - 2 IS - 6 SP - 692 EP - 699 ER -