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C-terminal truncation of a metagenome-derived detergent protease for effective expression in E. coli
(2010)
Recently, a new alkaline protease named HP70 showing highest homology to extracellular serine proteases of Stenotrophomonas maltophilia and Xanthomonas campestris was found in the course of a metagenome screening for detergent proteases (Niehaus et al., submitted for publication). Attempts to efficiently express the enzyme in common expression hosts had failed. This study reports on the realization of overexpression in Escherichia coli after structural modification of HP70. Modelling of HP70 resulted in a two-domain structure, comprising the catalytic domain and a C-terminal domain which includes about 100 amino acids. On the basis of the modelled structure the enzyme was truncated by deletion of most of the C-terminal domain yielding HP70-C477.
This structural modification allowed effective expression of active enzyme using E. coli BL21-Gold as the host. Specific activity of HP70-C477 determined with suc-l-Ala-l-Ala-l-Pro-l-Phe-p-nitroanilide as the substrate was 30 ± 5 U/mg compared to 8 ± 1 U/mg of the native enzyme. HP70-C477 was most active at 40 °C and pH 7–11; these conditions are prerequisite for a potential application as detergent enzyme. Determination of kinetic parameters at 40 °C and pH = 9.5 resulted in KM = 0.23 ± 0.01 mM and kcat = 167.5 ± 3.6 s⁻¹. MS-analysis of peptide fragments obtained from incubation of HP70 and HP70-C477 with insulin B indicated that the C-terminal domain influences the cleavage preferences of the enzyme. Washing experiments confirmed the high potential of HP70-C477 as detergent protease.
Eine SPS im Kraftfahrzeug? Das geht ja gar nicht – da gibt es doch all die Hersteller für Kfz-Steuergeräte, die konfigurierbare Hardware anbieten. Im Prinzip ja, aber leider sind gerade die Sensoren oder Aktoren nicht ansteuerbar, die der Entwickler genau für sein aktuelles Projekt benötigt. Dieser Beitrag gibt einen kleinen Einblick in neue Möglichkeiten des Rapid Prototyping für mechatronische Systeme auf der Basis von Speicherprogrammierbaren Steuerungen (SPS).
In den letzten drei bis vier Jahren unterlag der Bereich der Bilanzierung und Offenlegung von unsicheren Steuerpositionen im IFRS- und US-GAAP-Abschluss einem stetigen Wandel. Sowohl nach US-GAAP als auch nach IFRS nehmen die Anforderungen an die Bilanzierung und Offenlegung beständig zu. Schon allein dies zeigt die Bedeutung, die dem Themenbereich der Steuerrisiken beigemessen wird. Aber nicht nur im Rahmen der Berichterstattung im Jahresabschluss stehen Steuerrisiken oben auf der Agenda, auch der amerikanische Internal Revenue Service (IRS) verfolgt das Thema sehr konsequent und plant, bestimmte Unternehmen zu Angaben von Steuerrisiken im Rahmen der Steuererklärungen zu verpflichten.
Der Aufsatz befasst sich mit den neueren Entwicklungen im Bereich der Bilanzierung und Offenlegung von unsicheren Steuerpositionen in der Rechnungslegung nach IFRS, US-GAAP und HGB und der Offenlegung von Steuerrisiken im Rahmen der Steuererklärung.
Latente Steuern stellen eine Schnittstelle zwischen handelsrechtlicher Rechnungslegung und Steuerrecht dar. Das Verhältnis von Handels- und Steuerrecht ist dabei keinesfalls stetig; vielmehr fordert die Dynamik des deutschen Steuerrechts in vielen Bereichen regelmäßige Anpassungen in der Bilanzierung in Handels- und Steuerbilanz. Dies gilt insbesondere für die Bilanzierung latenter Steuern im handelsrechtlichen Jahresabschluss, welche durch das BilMoG deutlich an Bedeutung gewonnen hat. Neben latenten Steuern auf temporäre Differenzen ist es nunmehr erforderlich, Verlustvorträge und die Folgen der Zinsschranke bei der Berechnung latenter Steuern zu berücksichtigen. Der nachfolgende Beitrag geht auf die Berücksichtigung von Verlust- und Zinsvorträgen und die Berücksichtigungsfähigkeit von EBITDA-Vorträgen bei der Berechnung latenter Steuern ein und beleuchtet dabei insbesondere auch praktische Fragestellungen.
Quantifying and minimizing uncertainty is vital for simulating technically and economically successful geothermal reservoirs. To this end, we apply a stochastic modelling sequence, a Monte Carlo study, based on (i) creating an ensemble of possible realizations of a reservoir model, (ii) forward simulation of fluid flow and heat transport, and (iii) constraining post-processing using observed state variables. To generate the ensemble, we use the stochastic algorithm of Sequential Gaussian Simulation and test its potential fitting rock properties, such as thermal conductivity and permeability, of a synthetic reference model and—performing a corresponding forward simulation—state variables such as temperature. The ensemble yields probability distributions of rock properties and state variables at any location inside the reservoir. In addition, we perform a constraining post-processing in order to minimize the uncertainty of the obtained distributions by conditioning the ensemble to observed state variables, in this case temperature. This constraining post-processing works particularly well on systems dominated by fluid flow. The stochastic modelling sequence is applied to a large, steady-state 3-D heat flow model of a reservoir in The Hague, Netherlands. The spatial thermal conductivity distribution is simulated stochastically based on available logging data. Errors of bottom-hole temperatures provide thresholds for the constraining technique performed afterwards. This reduce the temperature uncertainty for the proposed target location significantly from 25 to 12 K (full distribution width) in a depth of 2300 m. Assuming a Gaussian shape of the temperature distribution, the standard deviation is 1.8 K. To allow a more comprehensive approach to quantify uncertainty, we also implement the stochastic simulation of boundary conditions and demonstrate this for the basal specific heat flow in the reservoir of The Hague. As expected, this results in a larger distribution width and hence, a larger, but more realistic uncertainty estimate. However, applying the constraining post-processing the uncertainty is again reduced to the level of the post-processing without stochastic boundary simulation. Thus, constraining post-processing is a suitable tool for reducing uncertainty estimates by observed state variables.
This paper proposes an approach to the choice and evaluation of engineering models with the aid of a typical application in geotechnics. An important issue in the construction of shallow tunnels, especially in weak ground conditions, is the tunnel face stability. Various theoretical and numerical models for predicting the necessary support pressure have been put forth in the literature. In this paper, we combine laboratory experiments performed at the University of Innsbruck with current methods of uncertainty and sensitivity analysis for assessing adequacy, predictive power and robustness of the models. The major issues are the handling of the twofold uncertainty of test results and of model predictions as well as the decision about what are the influential input parameters.
Various models have been proposed for the prediction of the necessary support pressure at the face of a shallow tunnel. To assess their quality, the collapse of a tunnel face was modelled with small-scale model tests at single gravity. The development of the failure mechanism and the support force at the face in dry sand were investigated. The observed displacement patterns show a negligible influence of overburden on the extent and evolution of the failure zone. The latter is significantly influenced, though, by the initial density of the sand: in dense sand a chimney-wedge-type collapse mechanism developed, which propagated towards the soil surface. Initially, loose sand did not show any discrete collapse mechanism. The necessary support force was neither influenced by the overburden nor the initial density. A comparison with quantitative predictions by several theoretical models showed that the measured necessary support pressure is overestimated by most of the models. Those by Vermeer/Ruse and Léca/Dormieux showed the best agreement to the measurements.