Universität Stuttgart
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Item Open Access Impact of remote mutations on metallo-beta-lactamase substrate specificity : implications for the evolution of antibiotic resistance(2005) Ölschläger, Peter; Mayo, Stephen L.; Pleiss, JürgenMetallo-beta-lactamases have raised concerns due to their ability to hydrolyze a broad spectrum of beta-lactam antibiotics. The G262S point mutation distinguishing the metallo-beta-lactamase IMP 1 from IMP 6 has no effect on the hydrolysis of the drugs cephalothin and cefotaxime, but significantly improves catalytic efficiency toward cephaloridine, ceftazidime, benzylpenicillin, ampicillin, and imipenem. This change in specificity occurs even though residue 262 is remote from the active site. We investigated the substrate specificities of five other point mutants resulting from single nucleotide substitutions at positions near residue 262: G262A, G262V, S121G, F218Y and F218I. The results suggest two types of substrates: type I (nitrocefin, cephalothin and cefotaxime), which are converted equally well by IMP-6, IMP-1, and G262A, but even more efficiently by the other mutants, and type II (ceftazidime, benzylpenicillin, ampicillin, and imipenem), which are hydrolyzed much less efficiently by all the mutants, with IMP-1 being the most active. G262V, S121G, F218Y, and F218I improve conversion of type I substrates, whereas G262A and IMP-1 improve conversion of type II substrates, indicating two distinct evolutionary adaptations from IMP-6. Substrate structure may explain the catalytic efficiencies observed. Type I substrates have R2 electron donors, which may stabilize the substrate intermediate in the binding pocket and lead to enhanced activity. In contrast, the absence of these stabilizing interactions with type II substrates may result in poor conversion and increased sensitivity to mutations. This observation may assist future drug design. As the G262A and F218Y mutants confer effective resistance to Escherichia coli BL21(DE3) cells (high minimal inhibitory concentrations), they are likely to evolve naturally.Item Open Access A model of the pressure dependence of the enantioselectivity of Candida rugosa lipase towards (±)-menthol(2001) Kahlow, Ulrich; Schmid, Rolf D.; Pleiss, JürgenTransesterification of (±)-menthol using propionic acid anhydride and Candida rugosa lipase was performed in chloroform and water at different pressures (1, 10, 50, and 100 bar) to study the pressure dependence of enantioselectivity E. As a result, E significantly decreased with increasing pressure from E=55 (1 bar) to E=47 (10 bar), E=37 (50 bar), and E=9 (100 bar). In order to rationalize the experimental findings, molecular dynamics simulations of Candida rugosa lipase were carried out. Analyzing the lipase geometry at 1, 10, 50, and 100 bar revealed a cavity in the Candida rugosa lipase. The cavity leads from a position on the surface distinct from the substrate binding site to the core towards the active site and is limited by F415 and the catalytic H449. In the crystal structure of the Candida rugosa lipase, this cavity is filled with 6 water molecules. The number of water molecules in this cavity gradually increased with increasing pressure: 6 molecules in the simulation at 1 bar, 10 molecules at 10 bar, 12 molecules at 50 bar, and 13 molecules at 100 bar. Likewise, the volume of the cavity progressively increased from about 1864 ų in the simulation at 1 bar to 2529 ų at 10 bar, 2526 ų at 50 bar, and 2617 ų at 100 bar. At 100 bar, one water molecule slipped between F415 and H449, displacing the catalytic histidine side chain and thus opening the cavity to form a continuous water channel. The rotation of the side chain leads to a decreased distance between the H449-N and the (+)-menthyl-oxygen (non-preferred enantiomer) in the acyl enzyme intermediate, a factor determining the enantioselectivity of the lipase. While the geometry of the preferred enantiomer is similar in all simulations, the geometry of the non-preferred enantiomer gets gradually more reactive. This observation correlates with the gradually decreasing enantioselectivity E.Item Open Access Kapitel 7, Stofftransport im Grundwasser(1987) Kobus, Helmut; Geldner, PeterDie Nutzung des Grundwassers ist in der BRD vorranging der Trinkwasserversorgung vorbehalten. Die Zielvorstellung der Wasserversorgung ist es, Grundwasser möglichst naturbelassen und ohne Aufbereitung als Trinkwasser abzugeben. Hieraus resultiert die ungeschriebene Forderung, daß der Rohstoff Grundwasser die Qualitätsanforderungen an Trinkwasser erfüllen soll. Eine vorrangige Aufgabe der Grundwasserwirtschaft ist darin zu sehen, anthropogene Beeinträchtigungen der Grundwasserqualität zu erfassen und zu beurteilen. Die Frage nach den Auswirkungen von Schadensfällen setzt das Verständnis des Transports von Schadstoffen im Untergrund voraus. Dies ist umso wichtiger, wenn wasserwirtschaftliche Maßnahmen zur Schadensbegrenzung oder Schadensanierung geplant werden.Item Open Access Rapid detection of neurotoxic insecticides in food using disposable acetylcholinesterase-biosensors and simple solvent extraction(2002) Schulze, Holger; Schmid, Rolf D.; Bachmann, Till T.The extensive use of pesticides to protect agricultural crops necessitates reliable tools for the detection of residues in food and water, thus ensuring environmental protection and consumer safety. Neuroinhibitors such as organophosphates and carbamates in particular, represent a potential hazard to human health. These compounds are frequently found in food but conventional methods of analysis are limited as they are either time consuming or not sufficiently sensitive. As a result, a rapid and sensitive biosensor test based on AChE-inhibition was developed. The disposable AChE-biosensor was directly applied in solvent extracts of food samples using isooctane as extraction solvent. A complete assay could be performed in less than 2 hours. Recovery rates of 84 % were obtained in tests with spiked orange juice samples. Tests in food samples with a lower water content resulted in reduced recovery rates (44 % for peach pap baby food). Phosphorothionate insecticides could be detected after direct oxidation in food with N-bromosuccinimide and solvent extraction. The assay displayed a detection limit of 2 μg/kg paraoxon which was sufficient for the monitoring of maximum residue limits in food according to EU regulations.Item Open Access Nachhaltigkeit in der Energieversorgung - relevante Stromerzeugungstechniken auf dem Prüfstand(1998) Voß, AlfredDas Leitbild einer "Nachhaltigen Entwicklung" (sustainable development) hat in den letzten Jahren eine erstaunliche Karriere gemacht. Seit der Konferenz der Vereinten Nationen über Umwelt und Entwicklung (UNCED) in Rio de Janeiro 1992 ist das Ziel einer nachhaltigen Entwicklung das zentrale Leitbild der internationalen umwelt-, wirtschafts- und entwicklungspolitischen Diskussion, das wirtschaftliche Entwicklung zur Überwindung von Hunger und Armut, und die Schaffung humaner Lebensbedingungen sowie den Erhalt der natürlichen Lebensgrundlagen miteinander verbinden will. Auch in die energiepolitische und energiewirtschaftliche Diskussion hat das Leitbild einer nachhaltigen Entwicklung mittlerweile verstärkt Eingang gefunden. Obwohl festzustellen ist, daß das Leitbild einer nachhaltigen Entwicklung auch über die verschiedenen gesellschaftlichen Gruppen hinweg eine breite prinzipielle Zustimmung findet, so spannen doch die Vorstellungen und Interpretationen des Leitbildes, sowohl hinsichtlich ihrer normativen bzw. theoretisch-naturwissenschaftlichen Fundierung als auch hinsichtlich ihrer abgeleiteten Handlungsziele bzw. Handlungsanweisungen - dies gilt gerade für den Energiebereich - eine große Bandbreite auf. Dies birgt nicht nur die Gefahr, daß dieses Leitbild von verschiedenen Interessengruppen instrumentalisiert wird, sondern auch, daß falsche Weichenstellungen vorgenommen werden. Aus diesem Grund erscheint es notwendig, auch wegen der essentiellen Bedeutung, die der Energieversorgung für eine nachhaltige Entwicklung zukommt, sich über die Konkretisierung des Leitbildes zu verständigen, um die Energieversorgungsoptionen, aber auch die energiepolitischen Vorstellungen, diesbezüglich einordnen zu können.Item Open Access ERA - Energy-based reliability analysis - Energiebasierte Zuverlässigkeitsanalyse(2014) Kemmler, Stefan; Koller, Oliver; Bertsche, BerndDa die Wechselwirkungen zwischen mechatronischen Komponenten in Systemen eine entscheidende Rolle auf ihre Belastung einnehmen, ist die Betrachtung dieser Wechselwirkungen un- verzichtbar. Zur Identifikation solcher Wechselwirkungen ist eine ergänzende Methode zur den bisher klassischen Systemanalysen von Nöten. Dies wird bei der vorgestellten energiebasierten Zuverlässigkeitsanalyse (engl. Energy-based Reliability Analysis - ERA) berücksichtigt, indem die stationären Energie- beziehungsweise die dynamischen Leistungsflüsse mechatronischer Systemen in Form von Energieflussdiagrammen dargestellt werden. Mit der Modellierung des Energieflusses und damit das Ansetzen des ERA-Verfahrens kann der Nutzer Wirkzusammenhänge und Schwachstellen erkennen, eine exaktere Bestimmung der Zuverlässigkeit durch Berechnung der Belastung erreichen und folglich Komponenten zuverlässigkeitsbasiert auslegen.Item Open Access Distributed cooperative deep transfer learning for industrial image recognition(2020) Maschler, Benjamin; Kamm, Simon; Nasser, Jazdi; Weyrich, MichaelIn this paper, a novel light-weight incremental class learning algorithm for live image recognition is presented. It features a dual memory architecture and is capable of learning formerly unknown classes as well as conducting its learning across multiple instances at multiple locations without storing any images. In addition to tests on the ImageNet dataset, a prototype based upon a Raspberry Pi and a webcam is used for further evaluation: The proposed algorithm successfully allows for the performant execution of image classification tasks while learning new classes at several sites simultaneously, thereby enabling its application to various industry use cases, e.g. predictive maintenance or self-optimization.Item Open Access The Lipase Engineering Database – a navigation and analysis tool for protein families(2003) Fischer, Markus; Pleiss, JürgenThe Lipase Engineering Database (http://www.led.uni-stuttgart.de) integrates information on sequence, structure, and function of lipases, esterases, and related proteins. Sequence data on 806 protein entries are assigned to 38 homologous families, which are grouped into 16 superfamilies with no global sequence similarity between each other. For each family, multisequence alignments are provided with functionally relevant residues annotated. Pre-calculated phylogenetic trees allow navigation inside superfamilies. Experimental structures of 45 proteins are superposed and consistently annotated. The Lipase Engineering Database has been applied to systematically analyze sequence-structure-function relationships of this vast and diverse enzyme class. It is a useful tool to identify functionally relevant residues apart from the active site residues, and to design mutants with desired substrate specificity.Item Open Access An industrial case study on the evaluation of a safety engineering approach for software-intensive systems in the automotive domain(2016) Abdulkhaleq, Asim; Vöst, Sebastian; Wagner, Stefan; Thomas, JohnSafety remains one of the essential and vital aspects in today's automotive systems. These systems, however, become ever more complex and dependent on software which is responsible for most of their critical functions. Therefore, the software components need to be analysed and verified appropriately in the context of software safety. The complexity of software systems makes defining software safety requirements with traditional safety analysis techniques difficult. A new technique called STPA (Systems-Theoretic Process Analysis) based on system and control theory has been developed by Leveson to cope with complex systems. Based on STPA, we have developed a comprehensive software safety engineering approach in which the software and safety engineers integrate the analysis of software risks with their verification to recognize the software-related hazards and reduce the risks to a low level. In this paper, we explore and evaluate the application of our approach to a real industrial system in the automotive domain. The case study was conducted analysing the software controller of the Active Cruise Control System (ACC) of the BMW Group.Item Open Access A non-intrusive nonlinear model reduction method for structural dynamical problems based on machine learning(2020) Kneifl, Jonas; Grunert, Dennis; Fehr, JörgThe paper uses a nonlinear non-intrusive model reduction approach, to derive efficient and accurate surrogate models for structural dynamical problems. Therefore, a combination of proper orthogonal decomposition along with regression algorithms from the field of machine learning is utilized to capture the dynamics in a reduced representation. This allows highly performant approximations of the original system. In this context, we provide a comparison of several regression algorithms based on crash simulations of a structural dynamic frame.