Antje Spieß
RWTH Aachen University
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Publication
Featured researches published by Antje Spieß.
Analytical Methods | 2015
Nico Anders; H. Humann; B. Langhans; Antje Spieß
A high performance anion exchange chromatography-pulsed amperometric detection (HPAEC-PAD) method using a CarboPac™ PA100 column for the simultaneous determination of 25 soluble compounds originating from all polymer classes (cellulose, hemicellulose, lignin and pectin) in biomass hydrolyzates was developed and validated. The method was optimized for resolution varying the column oven temperature (in the range of 30–50 °C) and the eluents (100 mM NaOH, 200 mM NaOH, 100 mM NaOH/500 mM NaOAc and water). The optimized method used gradient elution at 40 °C and had 70 min duration for one run. The detection limits ranged between 0.14 mg L−1 for both 2,6-dimethoxyphenol and 3,5-dimethoxy-4-hydroxybenzaldehyde and 21.9 mg L−1 for 4-methoxybenzyl alcohol, linearity was always >0.99, and sensitivity ranged between 0.0023 (nC min)/(mg L−1) for glucuronic acid and 8.4 (nC min)/(mg L−1) for 3,5-dimethoxy-4-hydroxybenzaldehyde. In contrast to the established photometric lignin measurement the HPAEC-PAD method allows for a distinction between several acid-soluble lignin compounds. The method was applied to 17 different biomass hydrolyzates and displayed the capability to quantify a wide range of soluble products covering very different hydrolyzate compositions.
ChemBioChem | 2016
Patrick C. F. Buchholz; Constantin Vogel; Waldemar Reusch; Martina Pohl; Dörte Rother; Antje Spieß; Jürgen Pleiss
The development of novel enzymes for biocatalytic processes requires knowledge on substrate profile and selectivity; this can be derived from databases and from publications. Often, these sources lack time‐course data for the substrate or product, and an unambiguous link between experiment and enzyme sequence. The lack of integrated, original data hampers the comprehensive analysis of enzyme kinetics and the evaluation of sequence–function relationships. In order to accelerate enzyme engineering, BioCatNet integrates protein sequence, protein structure, and experimental data for a given enzyme family. BioCatNet explicitly assigns the enzyme sequence to the experimental data, which consists of information on reaction conditions and time‐course data. BioCatNet facilitates the consistent documentation of reaction conditions, the archiving of time‐course data, and the efficient exchange of experimental data among collaborators. Data integration is demonstrated for three case studies by using the TEED (Thiamine diphosphate‐dependent Enzymes Engineering Database).
Biotechnology Progress | 2011
Robert Huber; Helene Wulfhorst; Lukas Maksym; Regina Stehr; Martin Pöhnlein; Gernot Jäger; Antje Spieß; Jochen Büchs
New screening techniques for improved enzyme variants in turbid media are urgently required in many industries such as the detergent and food industry. Here, a new method is presented to measure enzyme activity in different types of substrate suspensions. This method allows a semiquantitative determination of protease activity using native protein substrates. Unlike conventional techniques for measurement of enzyme activity, the BioLector technology enables online monitoring of scattered light intensity and fluorescence signals during the continuous shaking of samples in microtiter plates. The BioLector technique is hereby used to monitor the hydrolysis of an insoluble protein substrate by measuring the decrease of scattered light. The kinetic parameters for the enzyme reaction (Vmax,app and Km,app) are determined from the scattered light curves. Moreover, the influence of pH on the protease activity is investigated. The optimal pH value for protease activity was determined to be between pH 8 to 11 and the activities of five subtilisin serine proteases with variations in the amino acid sequence were compared. The presented method enables proteases from genetically modified strains to be easily characterized and compared. Moreover, this method can be applied to other enzyme systems that catalyze various reactions such as cellulose decomposition.
Biotechnology Journal | 2016
Jan-Hendrik Grosch; Michaela Sieben; Clemens Lattermann; Kira Kauffmann; Jochen Büchs; Antje Spieß
Microtiter plates (MTP) and automatized techniques are increasingly applied in the field of biotechnology. However, the susceptibility of MTPs to edge effects such as thermal gradients can lead to high variation of measured enzyme activities. In an effort to enhance experimental reliability, to quantify, and to minimize instrument-caused deviations in enzyme kinetics between two MTP-readers, we comprehensively quantified temperature distribution in 96-well MTPs. We demonstrated the robust application of the absorbance dye cresol red as easily applicable temperature indicator in cuvettes and MTPs and determined its accuracy to ±0.16°C. We then quantified temperature distributions in 96-well MTPs revealing temperature deviations over single MTP of up to 2.2°C and different patterns in two commercial devices (BioTek Synergy 4 and Synergy Mx). The obtained liquid temperature was shown to be substantially controlled by evaporation. The temperature-induced enzyme activity variation within MTPs amounted to about 20 %. Activity deviations between MTPs and to those in cuvettes were determined to 40 % due to deviations from the set temperature in MTPs. In conclusion, we propose a better control of experimental conditions in MTPs or alternative experimental systems for reliable determination of kinetic parameters for bioprocess development.
Bioorganic Chemistry | 2009
Thomas Schmidt; Michael Zavrel; Antje Spieß; Marion B. Ansorge-Schumacher
Benzaldehyde lyase from Pseudomonas fluorescens (BAL, EC 4.1.2.38) is a versatile catalyst for stereoselective carboligations. Nevertheless, rather inconsistent data about its biochemical properties are reported in literature. In this study, the dependency of BAL activity on ionic strength, pH, and concentration of DMSO was for the first time systematically investigated and interpreted. It was found that the activity of BAL strongly depends on all three parameters, and a correlation exists between the dependency on pH and DMSO concentration. This correlation could be explained by an interaction of DMSO with an ionic amino acid in the catalytic site. A model-based analysis indicated that the pK(a) of this residue shifts to the alkaline milieu upon addition of DMSO. Consequently, the optimum pH also shifts to alkaline values when DMSO is present. Potentiometric experiments confirmed that the pK(a) can most probably be attributed to Glu50 which governs the activity increase of BAL on the acidic limb of its pH-activity profile. With these findings, the apparently contradicting data from literature become comprehensible and optimal reaction conditions for synthesis can easily be deduced.
Biotechnology Progress | 2017
Jan-Hendrik Grosch; David Wagner; Vasilios Nistelkas; Antje Spieß
The reaction medium has major impact on biocatalytic reaction systems and on their economic significance. To allow for tailored medium engineering, thermodynamic phenomena, intrinsic enzyme kinetics, and enzyme–solvent interactions have to be discriminated. To this end, enzyme reaction kinetic modeling was coupled with thermodynamic calculations based on investigations of the alcohol dehydrogenase from Lactobacillus brevis (LbADH) in monophasic water/methyl tert‐butyl ether (MTBE) mixtures as a model solvent. Substrate concentrations and substrate thermodynamic activities were varied separately to identify the individual thermodynamic and kinetic effects on the enzyme activity. Microkinetic parameters based on concentration and thermodynamic activity were derived to successfully identify a positive effect of MTBE on the availability of the substrate to the enzyme, but a negative effect on the enzyme performance. In conclusion, thermodynamic activity‐based kinetic modeling might be a suitable tool to initially curtail the type of enzyme–solvent interactions and thus, a powerful first step to potentially understand the phenomena that occur in nonconventional media in more detail.
Chemical Communications | 2012
Alexander Dose; Jan Oliver Jost; Antje Spieß; Petra Henklein; Michael Beyermann; Dirk Schwarzer
Here we report a simple procedure for generating colorimetric histone deacetylase (HDAC) substrates by solid-phase peptide synthesis based on racemization-free couplings of amino acid chlorides. We demonstrate the applicability of these substrates in HDAC assays.
Biotechnology Progress | 2017
Jan-Hendrik Grosch; David Wagner; Niklas Knaup; Timm Keil; Antje Spieß
For the design of bioconversion processes parallel experimentation in microtiter plates is commonly applied to reduce the experimental load, although data accuracy and reproducibility are often reduced. In an effort to quantify the impact of different microscale experimental systems on the estimation of enzyme kinetic parameters from progress curves, we comprehensively evaluated the enzymatic reduction of acetophenone in both open and closed polystyrene and quartz microtiter plates as well as quartz cuvettes. Differences in conversion of up to 50% over time were observed increasing from polystyrene MTPs to quartz MTPs to quartz cuvettes. Initial reaction velocities increased systematically from polystyrene to quartz MTPs and cuvettes. The experimental errors decreased in the same order showing highest experimental error of about 20% in polystyrene. We further evaluated reasons causing the deviations within one system as well as between the systems. The choice of reaction vessel material, temperature effects and substrate cross contaminations in MTPs were shown to be of importance in the experimental results. Although the experimental data differed between the reaction vessels, no distinct trends in estimated kinetic parameters were found. While the microkinetic parameters vary up to an order of magnitude between different systems, the corresponding macrokinetic parameters lie in the same range for all systems varying by 29–118%.
Journal of Chromatography & Separation Techniques | 2016
Nico Anders; Bernd Langhans; Harald Humann; Antje Spieß
C wiring into nanoscale holes with high aspect ratio by electrodeposition is an important problem for 3-D integration in integrated circuit technology toward miniaturization of electronic devices. However, void and pinhole found in Cu wiring for the integration can cause trouble for miniature device. Cu electroplating method without void and pinhole is needed. We have proposed novel electroplating methods with supercritical carbon dioxide (sc-CO2) emulsion (EP-SCE). The electrochemical reaction is carried out in an emulsion of sc-CO2 in electrolyte with surfactants. Sc-CO2 has low viscosity and compatibility of hydrogen. Thus, this method is applicable in fine Cu wiring. The aim of this report is to examine Cu electrodeposition by using sc-CO2 emulsified electrolyte into nano-scale Cu wiring on the viewpoints of dissolution of Cu seed layer, gap-filling capability into nano-scale holes and contamination in the plated Cu. Moreover a continuous-flow reaction system is proposed and examined for filling of Cu into holes with 60 nm in diameter and aspect ratio of 2 and 5 by EP-SCE on a round-type large-area hole test element group with diameter of 300 mm, which has an integrated structure of Cu seed layer on TiN barrier layer sputtered on Si substrates.T spectroscopy has gained popularity as a promising non-invasive investigation tool in recent decades. In studies of solid-state pharmaceuticals, its usefulness is enhanced, since it enables one to distinguish diff erent polymorphic and pseudo-polymorphic forms. Terahertz absorption spectra are additive, meaning that the resulting spectrum of two or more compounds in the sample is an algebraic sum, in the linear region of the Beer’s law. For this reason, the evolving-in-time linear mixture of unknown pure components can be resolved using the blind source separation approach, where both spectral sources and their concentrations are to be estimated. Such an evolution occurs naturally in temperature-induced (pseudo) polymorphic transitions, i.e. dehydration, where the terahertz spectrum is the hydrate and dehydrates’s response with timevarying proportions. Conventionally, to estimate the kinetics of a reaction–an important physical-chemical parameter–the area of the unique spectral peak is integrated and normalized to evaluate the abundance, but a diffi culty of strongly overlapping peaks unable its application. To overcome this problem, we show, that by employing the blind source separation procedure, we can resolve a complex, multi-compound spectral mixture with signifi cant cross-bands, where the peak area method failed. We evaluate the performance of our approach in studies on dehydration of a well-known polycrystalline hydrate–α-D-glucose monohydrate. Seeing that the polymorphic transitions can completely change the properties of a pharmaceutical, our approach can fi nd application in a drug development process, where their careful characterization is of utmost importance.
Chemical Engineering and Processing | 2008
Antje Spieß; Werner Eberhard; Martina Peters; Marrit Eckstein; Lasse Greiner; Jochen Büchs