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    Impact of remote mutations on metallo-beta-lactamase substrate specificity : implications for the evolution of antibiotic resistance
    (2005) Ölschläger, Peter; Mayo, Stephen L.; Pleiss, Jürgen
    Metallo-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.
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    Kapitel 7, Stofftransport im Grundwasser
    (1987) Kobus, Helmut; Geldner, Peter
    Die 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.
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    Microbial P450 enzymes in biotechnology
    (2004) Urlacher, Vlada B.; Lutz-Wahl, Sabine; Schmid, Rolf D.
    Oxidations are key reactions in chemical syntheses. Biooxidations using fermentation processes have already conquered some niches in industrial oxidation processes, since they allow the introduction of oxygen even into non-activated carbon atoms in a sterically and optically selective manner which is difficult or impossible to achieve by synthetic organic chemistry. Biooxidation using isolated enzymes is limited to oxidases and dehydrogenases. Surprisingly, cytochrome P450 monooxygenases (CYPs) have scarcely been studied for use in biooxidations, although they are one of the largest known superfamilies of enzyme proteins. Their gene sequences have been identified in various organisms such as humans, bacteria, algae, fungi and plants. The reactions catalyzed by P450s are quite diverse and range from biosynthetic pathways (e.g. those of animal hormones and secondary plant metabolites) to the activation or biodegradation of hydrophobic xenobiotic compounds (e. g. those of various drugs in the liver of higher animals). From a practical point of view, the great potential of P450s is limited by their functional complexity, low activity, and limited stability. In addition, P450-catalyzed reactions require a constant supply of NAD(P)H which makes continuous cell-free processes very expensive. Quite recently, several groups have started to investigate cost-efficient ways which could allow the continuous supply of electrons to the heme iron. These include, for example, the use of electron mediators, direct electron supply from electrodes and enzymatic approaches. In addition, methods of protein design and directed evolution have been applied in an attempt to enhance the activity of the enzymes and improve their selectivity. The promising application of bacterial P450s as catalyzing agents in biocatalytic reactions and recent progress made in this field are covered in this review.
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    Naming the pain in requirements engineering: a design for a global family of surveys and first results from Germany
    (2015) Méndez Fernández, Daniel; Wagner, Stefan
    Context: For many years, we have observed industry struggling in defining a high quality requirements engineering (RE) and researchers trying to understand industrial expectations and problems. Although we are investigating the discipline with a plethora of empirical studies, they still do not allow for empirical generalisations. Objective: To lay an empirical and externally valid foundation about the state of the practice in RE, we aim at a series of open and reproducible surveys that allow us to steer future research in a problem-driven manner. Method: We designed a globally distributed family of surveys in joint collaborations with different researchers and completed the first run in Germany. The instrument is based on a theory in the form of a set of hypotheses inferred from our experiences and available studies. We test each hypothesis in our theory and identify further candidates to extend the theory by correlation and Grounded Theory analysis. Results: In this article, we report on the design of the family of surveys, its underlying theory, and the full results obtained from Germany with participants from 58 companies. The results reveal, for example, a tendency to improve RE via internally defined qualitative methods rather than relying on normative approaches like CMMI. We also discovered various RE problems that are statistically significant in practice. For instance, we could corroborate communication flaws or moving targets as problems in practice. Our results are not yet fully representative but already give first insights into current practices and problems in RE, and they allow us to draw lessons learnt for future replications. Conclusion: Our results obtained from this first run in Germany make us confident that the survey design and instrument are well-suited to be replicated and, thereby, to create a generalisable empirical basis of RE in practice.
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    Introduction of static quality analysis in small- and medium-sized software enterprises: experiences from technology transfer
    (2014) Gleirscher, Mario; Golubitskiy, Dmitriy; Irlbeck, Maximilian; Wagner, Stefan
    Today, small- and medium-sized enterprises (SMEs) in the software industry face major challenges. Their resource constraints require high efficiency in development. Furthermore, quality assurance (QA) measures need to be taken to mitigate the risk of additional, expensive effort for bug fixes or compensations. Automated static analysis (ASA) can reduce this risk because it promises low application effort. SMEs seem to take little advantage of this opportunity. Instead, they still mainly rely on the dynamic analysis approach of software testing. In this article, we report on our experiences from a technology transfer project. Our aim was to evaluate the results static analysis can provide for SMEs as well as the problems that occur when introducing and using static analysis in SMEs. We analysed five software projects from five collaborating SMEs using three different ASA techniques: code clone detection, bug pattern detection and architecture conformance analysis. Following the analysis, we applied a quality model to aggregate and evaluate the results. Our study shows that the effort required to introduce ASA techniques in SMEs is small (mostly below one person-hour each). Furthermore, we encountered only few technical problems. By means of the analyses, we could detect multiple defects in production code. The participating companies perceived the analysis results to be a helpful addition to their current QA and will include the analyses in their QA process. With the help of the Quamoco quality model, we could efficiently aggregate and rate static analysis results. However, we also encountered a partial mismatch with the opinions of the SMEs. We conclude that ASA and quality models can be a valuable and affordable addition to the QA process of SMEs.
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    "In gel patch electrophoresis" : a new method for environmental DNA purification
    (2005) Roh, Changhyun; Villatte, Francois; Kim, Byung-Gee; Schmid, Rolf D.
    Most of the microorganism species are largely untapped and could represent an interesting reservoir of genes useful for biotechnological applications. Unfortunately, a major difficulty associated with the methods used to isolate environmental DNA is related to the contamination of the extracted material with humic substances. These polyphenolic compounds inhibit the DNA processing reactions and severely impede cloning procedures. In this work, we describe a rapid, simple and efficient method for the purification of genomic DNA from environmental samples: we added a chromatography step directly embedded into an agarose gel electrophoresis. This strategy enabled the DNA extraction from various environmental samples and it appeared that the purity grade was compatible with digestion by restriction enzymes and PCR amplifications.
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    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.
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    Field testing of feedforward collective pitch control on the CART2 using a nacelle-based lidar scanner
    (2012) Schlipf, David; Fleming, Paul; Haizmann, Florian; Scholbrock, Andrew; Hofsäß, Martin; Wright, Alan; Cheng, Po Wen
    This work presents the first results from a field test to proof the concept of LIDAR assisted collective pitch control using a scanning LIDAR device installed on the nacelle of a research turbine. The purpose of the campaign was to show that a reduction of rotor speed variation is feasible with a feedforward update without changing the feedback controller. Although only a small amount of data could be collected, positive effects can be observed not only on the rotor speed but also on tower, blade and shaft loads in the case that the correlation of the wind preview and the turbine reaction is taken into account.
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    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.
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    Kapitel 8, Grundwasserschutz : Sanierung kontaminierter Standorte
    (1987) Grimm-Strele, Jost; Kobus, Helmut; Geldner, Peter
    In der Bundesrepublik befinden sich nach Schätzungen ca. 30.000 kontaminierte Standorte, von denen etwa ein Fünftel in nächster Zeit saniert werden muß. Die Aufgabe, solch einen kontaminierten Standort zu sanieren, besteht darin, die Gefährdungen, die von dem Standort ausgehen, zu erkennen, zu bewerten und zu beseitigen bzw. weitgehend einzuschränken. Das Ausmaß der Gefährdung bestimmt dabei die Intensität der Erkundung und den Umfang der Sanierungsmaßnahmen. In Abhängigkeit von den Zielen der Messung der Grundwasserbeschaffenheit müssen verschiedene Vorgehensweisen oder Maßstrategien verfolgt werden. Dazu müssen vor allem Festlegungen über die Meßverfahren, die Meßpunkte, die Meßhäufigkeit und die Meßparameter getroffen werden. Das häufigste Meßverfahren, das bei den folgenden Ausführungen im Vordergrund steht, ist die Entnahme von Wasserproben aus der gesättigten Zone an geeigneten Meßstellen wie Beobachtungsrohren, Förderbrunnen, Quellen oder ähnlichem und die anschließende Analyse im chemischen Labor.