Universität Stuttgart
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Item Open Access Activity of lipases and esterases towards tertiary alcohols : new insights into structure-function relationships(2002) Henke, Erik; Pleiss, Jürgen; Bornscheuer, Uwe TheoHydrolytic enzymes are versatile biocatalysts and find increasing applications in organic synthesis and a considerable number of industrial processes using these enzymes have been commercialized. Within this class, lipases (E.C. 3.1.1.3) and carboxyl esterases (E.C. 3.1.1.1) are frequently used as they accept a broad range of non-natural substrates, are usually very stable in organic solvents and exhibit good to excellent stereoselectivity.Item Open Access Analysis of the SPH interpolation moments matrix with regard to the influences of the discretization error in adaptive simulations(2021) Heinzelmann, Pascal; Spreng, Fabian; Sollich, Daniel; Eberhard, Peter; Williams, John R.Item Open Access At ease with your warnings: the principles of the salutogenesis model applied to automatic static analysis(2016) Ostberg, Jan-Peter; Wagner, StefanThe results of an automatic static analysis run can be overwhelming, especially for beginners. The overflow of information and the resulting need for many decisions is mentally tiring and can cause stress symptoms. There are several models in health care which are designed to fight stress. One of these is the salutogenesis model created by Aaron Antonovsky. In this paper, we will present an idea on how to transfer this model into a triage and recommendation model for static analysis tools and give an example of how this can be implemented in FindBugs, a static analysis tool for Java.Item Open Access An automatic safety-based test case generation approach based on systems-theoretic process analysis(2016) Abdulkhaleq, Asim; Wagner, StefanSoftware safety remains one of the essential and vital aspects in today’s systems. Software is becoming responsible for most of the critical functions of systems. Therefore, the software components in the systems need to be tested extensively against their safety requirements to ensure a high level of system safety. However, performing testing exhaustively to test all software behaviours is impossible. Numerous testing approaches exist. However, they do not directly concern the information derived during the safety analysis. STPA (Systems-Theoretic Process Analysis) is a unique safety analysis approach based on system and control theory, and was developed to identify unsafe scenarios of a complex system including software. In this paper, we present a testing approach based on STPA to automatically generate test cases from the STPA safety analysis results to help software and safety engineers to recognize and reduce the associated software risks. We also provide an open-source safety-based testing tool called STPA TCGenerator to support the proposed approach. We illustrate the proposed approach with a prototype of a software of the Adaptive Cruise Control System (ACC) with a stop-and-go function with a Lego-Mindstorms EV3 robot.Item Open Access Biocatalysts for the epoxidation and hydroxylation of fatty acids and fatty alcohols(2005) Maurer, Steffen Christian; Schmid, Rolf D.Whereas most products of the chemical industry are based on petrochemical feedstocks, considerable efforts have been made during the past few decades to use renewable resources as industrial raw materials. Besides polysaccharides and sugars, plant oils and animal fats play an important role in such programmes because of their ready availability (present production is >100 million t/a, which could be increased on demand) of which the lion’s share is used for nutrition (~85 million t/a), whereas ~ 15 – 20 million t/a are used for the synthesis of polymers, surfactants, emollients, lubricants, bio-diesel, emulsifiers, etc.). From a chemical point of view, most natural triglycerides offer just two reactive sites, the ester group and the double bonds of unsaturated fatty acids. In fact, the chemistry of fats and oils is largely focused on the ester group which can be hydrolyzed or catalytically reduced, leading to glycerol and fatty acids or fatty alcohols, respectively. Reactions involving the alkyl chain or double bonds of triglycerides, fatty acids, fatty alcohols or their derivatives represent far less than 10% of today’s oleochemistry, with the production of sulfonated fatty alcohols and their derivatives being a major process of this kind. Oxidation reactions at the alkyl or alkenyl chains would be highly desirable as they would lead to oleochemicals with new properties, but the methods available today lack selectivity and require harsh conditions. Notable exceptions are the epoxidation of unsaturated plant oils and the synthesis and use of a few hydroxy fatty acids.Item Open Access Biotransformation of ionones by engineered cytochrome P450 BM-3(2005) Urlacher, Vlada B.; Makhsumkhanov, Akhmadjan; Schmid, Rolf D.Wild type cytochrome P450 monooxygenase from Bacillus megaterium (P450 BM-3) has low activity for the hydroxylation of beta-ionone (>1 min-1). Substitution of phenylalanine by valine at position 87 increased the beta-ionone hydroxylation activity up to 100-fold (115 min-1). For further activity improvement methods of site-directed and random mutagenesis were applied. The R47L Y51F F87V mutant, designed by site-directed mutagenesis and the A74E F87V P386S mutant, obtained after two rounds of error-prone PCR, exhibit an increase in activity up to 300-fold compared to the wild type enzyme. All mutants converted -ionone regioselectively to 4-hydroxy-beta-ionone.Item Open Access Biotransformations using prokaryotic P450 monooxygenases(2002) Urlacher, Vlada B.; Schmid, Rolf D.Recent studies on microbial cytochrome P450 enzymes cover several new areas. Advances have been made in structure-function analysis. New non-enzymatic/electrochemical systems for the replacement of NAD(P)H have been developed. The properties of some enzymes have been re-engineered by site-directed mutagenesis or by methods of directed evolution. New P450s have been functionally expressed and characterized. A combination of these approaches is believed to facilitate the use of isolated P450 monooxygenases in biocatalysis.Item Open Access Blocking the tunnel: engineering of Candida rugosa lipase mutants with short chain length specificity(2002) Schmitt, Jutta; Brocca, Stefania; Schmid, Rolf D.; Pleiss, JürgenThe molecular basis of chain length specificity of Candida rugosa lipase 1 was investigated by molecular modelling and site-directed mutagenesis. The synthetic lip1 gene and the lipase mutants were expressed in Pichia pastoris and assayed for their chain length specificity in single substrate assays using triglycerides as well as in a competitive substrate assay using a randomized oil. Mutation of amino acids at different locations inside the tunnel (P246F, L413F, L410W, L410F/S300E, L410F/S365L) resulted in mutants with a different chain length specificity. Mutants P246F and L413F have a strong preference for short chain lengths whereas substrates longer than C10 are hardly hydrolyzed. Increasing the bulkiness of the amino acid at position 410 led to mutants that show a strong discrimination of chain lengths longer than C14. The results obtained can be explained by a simple mechanical model: the activity for a fatty acid sharply decreases as it becomes long enough to reach the mutated site. In contrast, a mutation at the entrance of the tunnel (L304F) has a strong impact on C4 and C6 substrates. This mutant is nevertheless capable to hydrolyze chain lengths longer than C8.Item Open Access Catalytic hydroxylation in biphasic systems using CYP102A1 mutants(2005) Maurer, Steffen Christian; Kühnel, Katja; Kaysser, Leonard A.; Eiben, Sabine; Schmid, Rolf D.; Urlacher, Vlada B.Cytochrome P450 monooxygenases are biocatalysts that hydroxylate or epoxidise a wide range of hydrophobic organic substrates. To date their technical application is limited to a small number of whole-cell biooxidations. The use of the isolated enzymes is believed to be impractical due to the low stability of this enzyme class, to the stochiometric need of the expensive cofactor NADPH, and due to the low solubility of most substrates in aqueous media. To overcome these problems we have investigated the application of a bacterial monooxygenase (mutants of CYP102A1) in a biphasic reaction system supported by cofactor recycling with NADP+-dependent formate dehydrogenase from Pseudomonas sp 101. Using this experimental setup, cyclohexane, octane and myristic acid were hydroxylated. To reduce the process costs a novel NADH-dependent double mutant of CYP102A1 was designed. For recycling of NADH during myristic acid hydroxylation in a biphasic system NAD+-dependent FDH was used. Stability of the monooxygenase under the reaction conditions is quite high as revealed by total turnover numbers of up to 12850 in NADPH-dependent cyclohexane hydroxylation and up to 30000 in NADH-dependent myristic acid oxidation.Item Open Access Cloning, expression, and characterization of a self-sufficient cytochrome P450 monooxygenase from Rhodococcus ruber DSM 44319(2006) Liu, Luo; Schmid, Rolf D.; Urlacher, Vlada B.A new member of class IV of cytochrome P450 monooxygenases was identified in Rhodococcus ruber strain DSM 44319. As the genome of Rhodococcus ruber has not been sequenced, a P450-like gene fragment was amplified using degenerated primers. The flanking regions of the P450-like DNA fragment were identified by directional genome walking using PCR. The primary protein structure suggests a natural self-sufficient fusion protein consisting of a ferredoxin, flavin-containing reductase and P450 monooxygenase. The only flavin found within the enzyme was FMN. The enzyme was successfully expressed in Escherichia coli and purified and characterized. In the presence of NADPH, the P450 monooxygenase showed hydroxylation activity towards polycyclic aromatic hydrocarbons naphthalene, indene, acenaphthene, toluene, fluorene, m-xylene and ethyl benzene. The conversion of naphthalene, acenaphthene and fluorene resulted in respective ring monohydroxylated metabolites. Alkyl aromatics like toluene, m-xylene and ethyl benzene were hydroxylated exclusively at the side chains. The new enzyme’s ability to oxidize such compounds makes it a potential candidate for biodegradation of pollutants and an attractive biocatalyst for synthesis.Item Open Access A comparative study of extraction and purification methods for environmental DNA from soil and sludge samples(2006) Roh, Changhyun; Villatte, Francois; Kim, Byung-Gee; Schmid, Rolf D.An important prerequisite for a successful metagenome library construction is an efficient extraction procedure for DNA out of environmental samples. In this study we compared three indirect and four direct extraction methods, including a commercial kit, in terms of DNA yield, purity and time requirement. A special focus was set on methods which are appropriate for the extraction of environmental DNA (eDNA) from very limited sample sizes (0.1 g) to enable a highly parallel approach. Direct extraction procedures yielded on average 100-fold higher DNA amounts than indirect ones. A drawback of direct extraction was the small fragment size of appr. 12 kb. The quality of the extracted DNA was evaluated by the ability of different restriction enzymes to digest the environmental DNA. Only the commercial kit and a direct extraction method using freeze-thaw cell lysis in combination with an in gel patch electrophoresis with hydroxyapatite to remove humic acid substances yielded DNA, which was completely digested by all restriction enzymes. Moreover, only DNA extracted by these two procedures could be used as template for the amplification of fragments of several 16S rDNA, 18S rDNA groups under standard PCR conditions.Item Open Access The database of epoxide hydrolases and haloalkane dehalogenases: one structure, many functions(2004) Barth, Sandra; Fischer, Markus; Schmid, Rolf D.; Pleiss, JürgenThe epoxide hydrolases and haloalkane dehalogenase database (EH/HD) integrates sequence and structure of a highly diverse protein family including mainly the Asp-hydrolases of EHs and HDs but also proteins like the Ser-hydrolases non-heme peroxidases, prolyl iminopetidases or 2-hydroxymuconic semialdehyde hydrolases. These proteins have a highly conserved structure, but display a remarkable diversity in sequence and function. 305 protein entries were assigned to 14 homologous families, forming two superfamilies. Annotated multisequence alignments and phylogenetic trees are provided for each homologous family and superfamily. Experimentally derived structures of 19 proteins are superposed and consistently annotated. Sequence and structure of all 305 proteins were systematically analysed. Thus, deeper insight is gained into the role of a highly conserved sequence motifs and structural elements. The EH/HD database is available at http://www.led.uni-stuttgart.de.Item Open Access Deep learning based soft sensors for industrial machinery(2020) Maschler, Benjamin; Ganssloser, Sören; Hablizel, Andreas; Weyrich, MichaelA multitude of high quality, high-resolution data is a cornerstone of the digital services associated with Industry 4.0. However, a great fraction of industrial machinery in use today features only a bare minimum of sensors and retrofitting new ones is expensive if possible at all. Instead, already existing sensors’ data streams could be utilized to virtually ‘measure’ new parameters. In this paper, a deep learning based virtual sensor for estimating a combustion parameter on a large gas engine using only the rotational speed as input is developed and evaluated. The evaluation focusses on the influence of data preprocessing compared to network type and structure regarding the estimation quality.Item Open Access Design of acetylcholinesterases for biosensor applications(2003) Schulze, Holger; Vorlová, Sandra; Villatte, Francois; Bachmann, Till T.; Schmid, Rolf D.In recent years, the use of acetylcholinesterases (AChEs) in biosensor technology has gained enormous attention, in particular with respect to insecticide detection. The principle of biosensors using AChE as a biological recognition element is based on the inhibition of the enzyme’s natural catalytic activity by the agent that is to be detected. The advanced understanding of the structure-function-relationship of AChEs serves as the basis for developing enzyme variants, which, compared to the wild type, show an increased inhibition efficiency at low insecticide concentrations and thus a higher sensitivity. This review describes different expression systems that have been used for the production of recombinant AChE. In addition, approaches to purify recombinant AChEs to a degree that is suitable for analytical applications will be elucidated as well as the various attempts that have been undertaken to increase the sensitivity of AChE to specified organophosphates and carbamates using side-directed mutagenesis and employing the enzyme in different assay formats.Item Open Access Development, validation, and application of an acetylcholinesterase-biosensor test for the direct detection of insecticide residues in infant food(2002) Schulze, Holger; Scherbaum, Ellen; Anastassiades, Michelangelo; Vorlová, Sandra; Schmid, Rolf D.; Bachmann, Till T.A highly sensitive and rapid food-screening test based on disposable screen-printed biosensors was developed, which is suitable for monitoring infant food. The exposure of infants and children to neurotoxic organophosphates and carbamates is of particular concern because of their higher susceptibility to adverse effects. The European Union has therefore set a very low limit for pesticides in infant food which must not contain concentrations exceeding 10 μg/kg for any given pesticide. The maximum residue limit (MRL) has been set to be near the determination threshold that is typically achieved for pesticides with traditional analytical methods. The biosensor method could detect levels lower than 5 μg/kg and thus clearly fulfills the demands of the EU. To substantiate these measurements, recovery rates were determined and amounted on average to 104 % in food. Matrix effects were eliminated by the introduction of a special electrode treatment. The test was compared with two traditional pesticide multiresidue analysis methods (GC/MS, LC/MS) using 26 fruit and vegetable samples from local markets and 23 samples of processed infant food from Germany, Spain, Poland and the USA. Three infant food samples exceeded the MRL of 10 μg/kg when analyzed by either biosensor test or multiresidue methods.Item Open Access Directed evolution of a bacterial alpha-amylase : towards enhanced pH-performance and higher specific activity(2003) Bessler, Cornelius; Schmitt, Jutta; Maurer, Karl-Heinz; Schmid, Rolf D.Alpha-Amylases, in particular, microbial Alpha-amylases are used widely in industrial processes such as starch liquefaction and pulp processes and more recently in detergency. Following the need for Alpha-amylases adapted to latter, we enhanced the alkali-activity of the Alpha-amylase from Bacillus amyloliquefaciens (BAA). The genes coding for the wild type BAA and the mutants BAA S201N and BAA N297D were subjected to error prone PCR and gene shuffling. For the screening of mutants we developed a novel, reliable assay suitable for high throughput screening based on the Phadebas® assay. One mutant (BAA 42) has an optimal activity at pH 7, corresponding to a shift of one pH unit compared to the wild type. BAA 42 is active over a broader pH-range than the wild type resulting in a fivefold higher activity at pH 10. In addition, the activity in periplasmic extracts and the specific activity increased 4 and 1.5 fold, respectively. Another mutant (BAA 29) possesses a wild type like pH-profile but reveals a 40-fold higher activity in periplasmic extracts and a nine fold higher specific activity. The comparison of the amino acid sequences of these two mutants with other homologous microbial Alpha-amylases revealed the mutation of the highly conserved residues W194R, S197P and A230V. In addition, three further mutations were found K406R, N414S and E356D, the latter being present in other bacterial Alpha-amylases.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 DNA isolation from soil samples for cloning in different hosts(2004) Kauffmann, Isabelle Melanie; Schmitt, Jutta; Schmid, Rolf D.Many protocols to extract DNA directly from soil samples have been developed in recent years. We employed two extraction methods which differed in the method of lysis and compared these methods with respect to yield, purity and degree of shearing. The main focus was on the specific isolation of DNA from different microorganisms, especially DNA from actinomycetes, as these cells are very difficult to lyse in contrast to non-actinomycetes. Thus, we used both methods to isolate DNA from Pseudomonas (Entcheva et al. 2001), Arthrobacter and Rhodococcus (Borneman et al. 1996) and from soil spiked with the respective microorganisms. Both methods rendered high DNA yields with a low degree of shearing but differed in the type of cells that were lysed. By one protocol (utilizing enzymatic lysis) only DNA from the Gram-negative Pseudomonas strain could be obtained whereas by the other protocol (utilizing mechanical lysis), all microorganisms that were used could be lysed and DNA from them extracted. Using a combination of both protocols, DNA from those organisms could be obtained selectively. Furthermore, one of the protocols was modified, resulting in higher DNA yield and purity.Item Open Access Effiziente Modellierung flexibler Robotersysteme zur Echtzeitsimulation am Beispiel eines Leichtbauroboters(2025) Hoschek, Sebastian; Rodegast, Philipp; Gesell, Jakob; Scheid, Jonas; Fehr, JörgDie Echtzeitsimulation mechanischer Systeme und deren digitale Zwillinge gewinnen in der Industrie zunehmend an Bedeutung. Sie ermöglichen unter anderem die Optimierung von Steuerungsalgorithmen, die Vorhersage des Systemverhaltens und die Implementierung von Regelstrategien in der Automatisierungstechnik. Ein Industriepartner entwickelt derzeit einen mobilen Leichtbauroboter für den Einsatz im Logistikbereich, bei dem die hohe Flexibilität der Struktur zu elastischen Durchbiegungen führt. Um die Genauigkeit und Leistungsfähigkeit des Roboters zu verbessern, ist eine präzise Modellierung dieser elastischen Effekte erforderlich. In dieser Arbeit werden zwei verschiedene Modellierungsansätze für die Echtzeitsimulation untersucht. Der erste basiert auf einer physikalischen White-Box-Modellierung als flexibles Mehrkörpersystem, wobei ein klassisches Finite-Elemente-Modell (FEM) durch Modellordnungsreduktion vereinfacht wird, um eine effiziente Berechnung zu ermöglichen. Der zweite Ansatz verwendet ein Finite-Segmente-Modell, das eine Parameteridentifikation erfordert, um eine realitätsgetreue Abbildung des Systemverhaltens zu gewährleisten. Beide Methoden werden auf den Leichtbauroboter angewendet und hinsichtlich ihrer Vor- und Nachteile verglichen. Wesentliche Kriterien sind dabei der Modellierungsaufwand, die Berechnungsgeschwindigkeit und die Genauigkeit der Simulationsergebnisse. Die Ergebnisse liefern eine Entscheidungsgrundlage zur Auswahl geeigneter Modellierungsmethoden in Echtzeitanwendungen.Item Open Access Electronic Publishing(2002) Scholze, Frank; Stephan, WernerBegriff und Geschichte, Definition und Arten des Electronic Publishing werden vorgestellt und erläutert.