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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.
[{ReN(PMe2Ph)3}{ReO3N}]2 – Structural Evidence for the Nitridotrioxorhenate(VII) Anion, [ReO3N]2−
(2005)
Mice that have been genetically humanized for proteins involved in drug metabolism and toxicity and mice engrafted with human hepatocytes are emerging and promising in vivo models for an improved prediction of the pharmacokinetic, drug–drug interaction and safety characteristics of compounds in humans. The specific advantages and disadvantages of these models should be carefully considered when using them for studies in drug discovery and development. Here, an overview on the corresponding genetically humanized and chimeric liver humanized mouse models described to date is provided and illustrated with examples of their utility in drug metabolism and toxicity studies. We compare the strength and weaknesses of the two different approaches, give guidance for the selection of the appropriate model for various applications and discuss future trends and perspectives.
An improved and convenient ninhydrin assay for aminoacylase activity measurements was developed using the commercial EZ Nin™ reagent. Alternative reagents from literature were also evaluated and compared. The addition of DMSO to the reagent enhanced the solubility of Ruhemann's purple (RP). Furthermore, we found that the use of a basic, aqueous buffer enhances stability of RP. An acidic protocol for the quantification of lysine was developed by addition of glacial acetic acid. The assay allows for parallel processing in a 96-well format with measurements microtiter plates.
A High-Throughput Functional Complementation Assay for Classification of BRCA1 Missense Variants
(2013)
BACKGROUND
Currently, several techniques exist for the downstream processing of protein, phytic acid and sinapic acid from rapeseed and rapeseed meal, but no technique has been developed to separate all of the components in one process. In this work, two new downstream processing strategies focusing on recovering sinapic acid, phytic acid and protein from rapeseed meal were established.
RESULTS
The sinapic acid content was enhanced by a factor of 4.5 with one method and 5.1 with the other. The isolation of sinapic acid was accomplished using a zeolite-based adsorbent with high adsorptive and optimal desorption characteristics. Phytic acid was isolated using the anion-exchange resin Purolite A200®. In addition, the processes resulted in two separated protein fractions. The ratios of globulin and albumin ratio to the total protein were 59.2% and 40.1%, respectively. The steps were then combined in two different ways: (a) a ‘sequential process’ using the zeolite and A200 in batch processes; and (b) a ‘parallel process’ using only A200 in a chromatographic system to separate all of the compounds.
CONCLUSIONS
It can be concluded that isolation of all three components was possible in both processes. These could enhance the added value of current processes using rapeseed meal as a protein source. © 2015 Society of Chemical Industry
A platform technology for the automated reaction control in magnetizable micro-fluidic droplets
(2014)
An immunochromatographic lateral flow dipstick assay for the fast detection of microcystin-LR was developed. Colloid gold particles with diameters of 40 nm were used as red-colored antibody labels for the visual detection of the antigen. The new dipstick sensor is capable of detecting down to 5 µg·l−1 (ppb; total inversion of the color signal) or 1 ppb (observation of color grading) of microcystin-LR. The course of the labeling reaction was observed via spectrometric wave shifts caused by the change of particle size during the binding of antibodies. Different stabilizing reagents showed that especially bovine serum albumin (BSA) and casein increase the assays sensitivity and the conjugate stability. Performance of the dipsticks was quantified by pattern processing of capture zone CCD images. Storage stability of dipsticks and conjugate suspensions over 115 days under different conditions were monitored. The ready-to-use dipsticks were successfully tested with microcystin-LR-spiked samples of outdoor drinking- and salt water and applied to the tissue of microcystin-fed mussels.
Biopharmaceuticals such as antibodies are produced in cultivated mammalian cells, which must be monitored to comply with good manufacturing practice. We, therefore, developed a fully automated system comprising a specific exhaust gas analyzer, inline analytics and a corresponding algorithm to precisely determine the oxygen uptake rate, carbon dioxide evolution rate, carbon dioxide transfer rate, transfer quotient and respiratory quotient without interrupting the ongoing cultivation, in order to assess its reproducibility. The system was verified using chemical simulation experiments and was able to measure the respiratory activity of hybridoma cells and DG44 cells (derived from Chinese hamster ovary cells) with satisfactory results at a minimum viable cell density of ~2.0 × 10⁵ cells ml⁻¹. The system was suitable for both batch and fed-batch cultivations in bubble-aerated and membrane-aerated reactors, with and without the control of pH and dissolved oxygen.
Quantitative nuclear magnetic resonance (qNMR) is considered as a powerful tool for multicomponent mixture analysis as well as for the purity determination of single compounds. Special attention is currently paid to the training of operators and study directors involved in qNMR testing. To assure that only qualified personnel are used for sample preparation at our GxP-accredited laboratory, weighing test was proposed. Sixteen participants performed six-fold weighing of the binary mixture of dibutylated hydroxytoluene (BHT) and 1,2,4,5-tetrachloro-3-nitrobenzene (TCNB). To evaluate the quality of data analysis, all spectra were evaluated manually by a qNMR expert and using in-house developed automated routine. The results revealed that mean values are comparable and both evaluation approaches are free of systematic error. However, automated evaluation resulted in an approximately 20% increase in precision. The same findings were revealed for qNMR analysis of 32 compounds used in pharmaceutical industry. Weighing test by six-fold determination in binary mixtures and automated qNMR methodology can be recommended as efficient tools for evaluating staff proficiency. The automated qNMR method significantly increases throughput and precision of qNMR for routine measurements and extends application scope of qNMR.
The invention relates to a system for the implementation of chemical, biological or physical reactions, consisting of - one or more magnetic micro-reactors, each comprising a shell made of hydrophobic magnetic nanoparticles encapsulating an aqueous core, - a plane platform comprising a surface to receive the micro-reactors, - a source that generates a magnetic field above or underneath the platform for manipulating the one or more hydrophobic magnetic micro-reactors, or for moving them along the surface of the platform from one position to another position, characterized in that the aqueous core of the one or more magnetic micro-reactors contains a reaction solution or buffer, and wherein the magnetic field generated by the source correlates to a defined position on the surface of the platform.
Die Lösungsmittelherstellung durch Clostridien konnte wirtschaftlich nicht mit der chemischen Synthese von Lösungsmitteln auf Erdölbasis konkurrieren und wurde in den frühen 1960er Jahren nahezu vollständig eingestellt. Das Interesse an nachwachsenden Rohstoffen hat in den letzten Jahren zu einem Wiederaufleben der ABE-Fermentation geführt. Aufgrund seiner höheren Energiedichte im Vergleich zu Ethanol ist Biobutanol als Energieträgerbesonders interessant und bietet sich z. B. als Produkt einer Bioraffinerie der 2. Generation an. Für die beschriebenen Experimente wird durch das Organosolv-Verfahren aufgeschlossenes Buchenholz verwendet. Der Faserstoff wird mithilfe von CTec2-Enzymen hydrolysiert, wobei der erhaltene Überstand eine Glucosekonzentration von 66 g L⁻¹ aufweist. Auf der Basis dieses Materials können mit Clostridium acetobutylicum Butanol-Ausbeuten erzielt werden, die mit denen unter Verwendung von reinen Zuckern vergleichbar sind. Dem Problem der hohen Produktinhibierung wird mit einer In-situ-Produktaufarbeitung begegnet. Mithilfe von Lösungsmittelimprägnierten Partikeln (SIPs) kann die Produktausbeute drastisch gesteigert werden, indem die gebildeten Lösungsmittel durch das auf dem Partikel imprägnierte Lösungsmittel während der Fermentation extrahiert werden. Zudem wird hierdurch die weitere Produktaufarbeitungstark vereinfacht.
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−.
In der Molkeverarbeitung dominieren Membranfiltrationsverfahren die Prozessführung. Hierbei werden üblicherweise Aufkonzentrierungen der Proteine und deren Trennung von dem Milchzucker Lactose durchgeführt. Der Prozess der adsorptiven Aufreinigung soll als kostengünstige Alternative zu den bisher gebräuchlichen Verfahren dienen. Weiterhin eröffnet sich durch das Verfahren die Möglichkeit, einzelne Proteinfraktionen während der Verarbeitung anzureichern. Als Proteinquellen wurden für die Untersuchungen Modellproteine, Lösungen aus Molkenproteinisolat, Dünnmolke und Molkekonzentrat verwendet. Die Eignung zur Proteinbindung wurden an Tonmaterialien, Silicaten und y-Aluminiumoxiden in Pulverform, in Form von Granulaten sowie Extrudaten als auch sphärischen Partikeln überprüft. Adsorbentien aus Bentonit/Silica und c-Aluminiumoxid können sowohl a-Lactalbumin (aLA) als auch b-Lactoglobulin (bLG) binden, wohingegen Materialien aus Siliciumoxid lediglich ein starkes Adsorptionsverhalten gegenüber bLG zeigen. Mischmaterialien aus Siliciumoxid und a-Aluminiumoxid zeigen dasselbe Verhalten wie Materialien aus Siliciumoxid, weisen jedoch eine geringere Kapazität auf. Die Materialen wurden hinsichtlich ihres Einsatzes in chromatographischen Verfahren und Batch-Prozessen untersucht und ein Prozessentwurf für einen zweistufigen Batch-Prozess im Rührkessel erarbeitet.
Adsorptive Vorbehandlung von Rohglycerin für die 1,3-Propandiol Fermentation mit Clostridium diolis
(2014)
Bei der Gewinnung von Fettsäuren aus Pflanzenölen, z. B. zur Herstellung von Biopolymeren, oder bei der Biodiesel- und Seifenproduktion, fällt Glycerin als Nebenprodukt an. Bei der Biokonversion dieses Rohstoffes zu 1,3-Propandiol wird der Produktionsorganismus Clostridium diolis durch Verunreinigungen im Rohglycerin gehemmt. Als inhibierende Substanzen konnten freie Fettsäuren identifiziert werden. Mithilfe eines adsorptiven Aufarbeitungsverfahrens ist es gelungen, die Fettsäuren zu entfernen und die Konversionseffizienz zu 1,3-Propandiol zu erhöhen.
Bacterial cellulose (BC) is a biopolymer produced by different microorganisms, but in biotechnological practice, Komagataeibacter xylinus is used. The micro- and nanofibrillar structure of BC, which forms many different-sized pores, creates prerequisites for the introduction of other polymers into it, including those synthesized by other microorganisms. The study aims to develop a cocultivation system of BC and prebiotic producers to obtain BC-based composite material with prebiotic activity. In this study, pullulan (PUL) was found to stimulate the growth of the probiotic strain Lactobacillus rhamnosus GG better than the other microbial polysaccharides gellan and xanthan. BC/PUL biocomposite with prebiotic properties was obtained by cocultivation of Komagataeibacter xylinus and Aureobasidium pullulans, BC and PUL producers respectively, on molasses medium. The inclusion of PUL in BC is proved gravimetrically by scanning electron microscopy and by Fourier transformed infrared spectroscopy. Cocultivation demonstrated a composite effect on the aggregation and binding of BC fibers, which led to a significant improvement in mechanical properties. The developed approach for “grafting” of prebiotic activity on BC allows preparation of environmentally friendly composites of better quality.
Aktiver und passiver antimikrobieller Oberflächenschutz durch funktionalisierte Mikropartikel
(2014)
Mikrobielle Verunreinigungen von Oberflächen in technischen und medizinischen Systemen sind allgegenwärtig. Sie basieren üblicherweise auf adsorptiven Oberflächenbindungen organischer Komponenten (Proteine und Fette) oder Membrankomponenten aerogener sowie wassergebundener Mikroorganismen. In laufenden Forschungsarbeiten wird eine aktive sowie passive Biomodifikation von Oberflächen zu deren Schutz vor Adsorption von Proteinen und Mikroorganismen verfolgt. Der antimikrobielle Schutz soll dabei sowohl durch die Mikrostrukturierung bzw. Rauheitsanpassung der Oberflächen durch deren Beschichtung mit Mikro-und Nanopartikeln erfolgen. Ferner werden antimikrobielle Enzyme und funktionelle Gruppen auf den Mikropartikeln gebunden, um den Oberflächenschutz zu verstärken. In ersten Versuchen wurden quartäre Ammoniumverbindungen auf eigens synthetisierten superparamagnetischen Eisenoxid-Nanopartikeln (Durchmesser 10 – 30 nm) immobilisiert und die wachstumshemmende Wirkung untersucht. Erste Ergebnisse zeigten, dass eine Konzentration von 10 mg mL⁻¹ der Ammoniumverbindung in einer Wachstumshemmung des verwendeten Gram-negativen Modell-Mikroorganismus E. coli GFPmut2 resultiert. Zurzeit werden synergistisch wirkende Kombinationen von Partikeln mit Proteasen, quartären Ammoniumverbindungen, hydrophoben Oberflächen und mikrostrukturierten Oberflächen als antimikrobieller Schutz untersucht.