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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.
A large strain collection comprising antagonistic bacteria was screened for novel detergent proteases. Several strains displayed protease activity on agar plates containing skim milk but were inactive in liquid media. Encapsulation of cells in alginate beads induced protease production. Stenotrophomonas maltophilia emerged as best performer under washing conditions. For identification of wash-active proteases, four extracellular serine proteases called StmPr1, StmPr2, StmPr3 and StmPr4 were cloned. StmPr2 and StmPr4 were sufficiently overexpressed in E. coli. Expression of StmPr1 and StmPr3 resulted in unprocessed, insoluble protein. Truncation of most of the C-terminal domain which has been identified by enzyme modeling succeeded in expression of soluble, active StmPr1 but failed in case of StmPr3.
From laundry application tests StmPr2 turned out to be a highly wash-active protease at 45 °C. Specific activity of StmPr2 determined with suc-l-Ala-l-Ala-l-Pro-l-Phe-p-nitroanilide as the substrate was 17 ± 2 U/mg. In addition we determined the kinetic parameters and cleavage preferences of protease StmPr2.
Germany is a frontrunner in setting frameworks for the transition to a low-carbon system. The mobility sector plays a significant role in this shift, affecting different people and groups on multiple levels. Without acceptance from these stakeholders, emission targets are out of reach. This research analyzes how the heterogeneous preferences of various stakeholders align with the transformation of the mobility sector, looking at the extent to which the German transformation paths are supported and where stakeholders are located.
Under the research objective of comparing stakeholders' preferences to identify which car segments require additional support for a successful climate transition, a status quo of stakeholders and car performance criteria is the foundation for the analysis. Stakeholders' hidden preferences hinder the derivation of criteria weightings from stakeholders; therefore, a ranking from observed preferences is used. This study's inverse multi-criteria decision analysis means that weightings can be predicted and used together with a recalibrated performance matrix to explore future preferences toward car segments.
Results show that stakeholders prefer medium-sized cars, with the trend pointing towards the increased potential for alternative propulsion technologies and electrified vehicles. These insights can guide the improved targeting of policy supporting the energy and mobility transformation. Additionally, the method proposed in this work can fully handle subjective approaches while incorporating a priori information. A software implementation of the proposed method completes this work and is made publicly available.
Modeling and upscaling of a pilot bayonettube reactor for indirect solar mixed methane reforming
(2020)
A 16.77 kW thermal power bayonet-tube reactor for the mixed reforming of methane using solar energy has been designed and modeled. A test bench for the experimental tests has been installed at the Synlight facility in Juelich, Germany and has just been commissioned. This paper presents the solar-heated reactor design for a combined steam and dry reforming as well as a scaled-up process simulation of a solar reforming plant for methanol production. Solar power towers are capable of providing large amounts of heat to drive high-endothermic reactions, and their integration with thermochemical processes shows a promising future. In the designed bayonet-tube reactor, the conventional burner arrangement for the combustion of natural gas has been substituted by a continuous 930 °C hot air stream, provided by means of a solar heated air receiver, a ceramic thermal storage and an auxiliary firing system. Inside the solar-heated reactor, the heat is transferred by means of convective mechanism mainly; instead of radiation mechanism as typically prevailing in fossil-based industrial reforming processes. A scaled-up solar reforming plant of 50.5 MWth was designed and simulated in Dymola® and AspenPlus®. In comparison to a fossil-based industrial reforming process of the same thermal capacity, a solar reforming plant with thermal storage promises a reduction up to 57 % of annual natural gas consumption in regions with annual DNI-value of 2349 kWh/m2. The benchmark solar reforming plant contributes to a CO2 avoidance of approx. 79 kilotons per year. This facility can produce a nominal output of 734.4 t of synthesis gas and out of this 530 t of methanol a day.
Retrofitting of existing parabolic trough collector power plants with molten salt tower systems
(2018)
In this paper we report on an architecture for a self-driving car that is based on ROS2. Self-driving cars have to take decisions based on their sensory input in real-time, providing high reliability with a strong demand in functional safety. In principle, self-driving cars are robots. However, typical robot software, in general, and the previous version of the Robot Operating System (ROS), in particular, does not always meet these requirements. With the successor ROS2 the situation has changed and it might be considered as a solution for automated and autonomous driving. Existing robotic software based on ROS was not ready for safety critical applications like self-driving cars. We propose an architecture for using ROS2 for a self-driving car that enables safe and reliable real-time behaviour, but keeping the advantages of ROS such as a distributed architecture and standardised message types. First experiments with an automated real passenger car at lower and higher speed-levels show that our approach seems feasible for autonomous driving under the necessary real-time conditions.
On the Vanishing Displacement Current Limit for Eddy-Current Problems / Quell, P. ; Reißel, M.
(1996)
Magnetotomography and Electric Currents in a Fuel Cell / Lustfeld, H. ; Reißel, M. ; Steffen, B.
(2009)
Optical Instruments require an extremely stable thermal surrounding to prevent loss of data quality by misalignments of the instrument components resulting from material deformation due to temperature f luctuations (e.g. from solar intrusion). Phase Change Material (PCM) can be applied as a thermal damper to achieve a more uniform temperature distribution. The challenge of this method is, among others, the integration of PCM into affected areas. If correctly designed, incoming heat is latently absorbed during phase change of the PCM, i.e. the temperature of a structure remains almost constant. In a cold phase, the heat during phase change is released again latently until the PCM returns to its original state of aggregation. Thus, the structure is thermally stabilized. At FH Aachen– University of Applied Sciences research is conducted to apply PCM directly into the structures of affected components (baffles, optical benches, electronic boxes, etc.). Through the application of Additive Manufacturing, the necessary voids are directly printed into these structures and filled later with PCM. Additive Manufacturing enables complex structures that would not have been possible with conservative manufacturing methods. A corresponding Breadboard was developed and manufactured by Selective Laser Melting (SLM). The current state of research includes the handling and analysis of the Breadboard, tests and a correlation of the thermal model. The results have shown analytically and practically that it is possible to use PCM as an integral part of the structure as a thermal damper. The results serve as a basis for the further development of the technology, which should maximize performance and enable the integration of PCM into much more complex structures.
Bead-on-plate butt joints of 2.5 mm hot rolled DP600/DP600 and 1.2 mm cold rolled TRIP700/TRIP700 steel sheets were performed using 6 kW CO2 laser beam welding. The welding speed ranged from 1.5 to 3.0 and from 2.1 to 3.9 m/min in DP/DP and TRIP/TRIP steel weldments respectively. A top surface helium gas was used as a shielding gas at a flow rate of 20 l/min. Metallographic examinations and transverse tensile testing (DIN EN 895: 1995) were carried out to characterize the weldments. The formability of base metals and weldments were investigated by standard Erichsen test (DIN EN ISO 20482). It was found that the uniaxial plastic behavior of both DP600 and TRIP700 base metals was in agreement with Swift and modified Mecking–Kocks models respectively. In a perpendicular tensile test to the weld line, all specimens were fractured at the base metal however the strengths were somewhat higher than those of base metal. There was a significant reduction in formability caused by welding of both DP/DP and TRIP/TRIP steel weldments and the formability has been improved with the increase of the welding speed.
Shielding gas influences on laser weldability of tailored blanks of advanced automotive steels
(2010)
The effects of shielding gas types and flow rates on CO2 laser weldability of DP600/TRIP700 steel sheets were studied in this work. The evaluated shielding gases were helium (He), argon (Ar) and different mixtures of He and Ar. Weld penetration, tensile strength and formability (Erichsen test) of laser welds were found to be strongly dependent upon the shielding gas types. The ability of shielding gas in removing plasma plume and thus increasing weld penetration is believed to be closely related to ionization potential and atomic weight which determine the period of plasma formation and disappearance. It was found that the higher helium shielding gas flow rate, the deeper weld penetration and the lower weld width.
Numerical and experimental investigation of tensile behavior of laser beam welded TRIP700 steel
(2011)
In this research work, a statistical analysis of the CO2 laser beam welding of dual phase (DP600)/transformation induced plasticity (TRIP700) steel sheets was done using response surface methodology. The analysis considered the effect of laser power (2–2.2 kW), welding speed (40–50 mm/s) and focus position (−1 to 0 mm) on the heat input, the weld bead geometry, uniaxial tensile strength, formability limited dome height and welding operation cost. The experimental design was based on Box–Behnken design using linear and quadratic polynomial equations for predicting the mathematical models. The results indicate that the proposed models predict the responses adequately within the limits of welding parameters being used and the welding speed is the most significant parameter during the welding process.
Generally, the quality of a weld joint is directly influenced by the welding input parameter settings. Selection of proper process parameters is important to obtain the desired weld bead profile and quality. In this research work, numerical and graphical optimization techniques of the CO2 laser beam welding of dual phase (DP600)/transformation induced plasticity (TRIP700) steel sheets were carried out using response surface methodology (RSM) based on Box–Behnken design. The procedure was established to improve the weld quality, increase the productivity and minimize the total operation cost by considering the welding parameters range of laser power (2–2.2 kW), welding speed (40–50 mm/s) and focus position (−1 to 0 mm). It was found that, RSM can be considered as a powerful tool in experimental welding optimization, even when the experimenter does not have a model for the process. Strong, efficient and low cost weld joints could be achieved using the optimum welding conditions.
Electron beam plasma measurement was realised by means of DIABEAM system invented by ISF RWTH Aachen. The Langmuir probe method is used for measurement. The relative simplicity of the method and the possibility of dispersion of high power on the probe allow its application for the investigation of high-power electron beams. The key element of the method is a rotating thin tungsten wire, which intersects the beam transversely on its axis and collects part of the current by itself. The signals, which are registered in the DIABEAM as a voltage, were taken in the form of amplitude. The conversion of the probe current into the distribution along the beam radius was realised using the Abel’s method. A voltage-current characteristic was built for the beam current. The local electron density as well as the electron temperature, the floating potential and the plasma potential were measured and calculated by means of this characteristic.
The laser beam-submerged arc hybrid welding method originates from the knowledge that, with increasing penetration depth, the laser beam process has a tendency to pore formation in the lower weld regions. The coupling with the energy-efficient submerged-arc process improves degassing and reduces the tendency to pore formation. The high deposition rate of the SA process in combination with the laser beam process offers, providing the appropriate choice of weld preparation, the possibility of welding plates with a thickness larger than 20° mm in a single pass, and also of welding thicker plates with the double-sided single pass technique.
In this work, a multi-sensor chip for the investigation of the sensing properties of different types of metal oxides towards hydrogen peroxide in the ppm range is presented. The fabrication process and physical characterization of the multi-sensor chip are described. Pure SnO2 and WO3 as well as Pd- and Pt-doped SnO2 films are characterized in terms of their sensitivity to H2O2. The sensing films have been prepared by drop-coating of water-dispensed nano-powders. A physical characterization, including scanning electron microscopy and X-ray diffraction analysis of the deposited metal-oxide films, was done. From the measurements in hydrogen peroxide atmosphere, it could be shown, that all of the tested metal oxide films are suitable for the detection of H2O2 in the ppm range. The highest sensitivity and reproducibility was achieved using Pt-doped SnO2.
Calibration plot of a SnO2, WO3, Pt-, and Pd-doped SnO2 gas sensor for H2O2 concentrations in the ppm range.
The present work describes a novel multiple sensor-type system for the real-time analysis of aseptic sterilisation processes employing gaseous hydrogen peroxide (H2O2) as a sterilant. The inactivation kinetics of Bacillus atrophaeus by gaseous H2O2 have been investigated by means of a methodical calibration experiment, taking into account the process variables H2O2 concentration, humidity and gas temperature. It has been found that the microbicidal effectiveness at H2O2 concentrations above 2% v/v is largely determined by the concentration itself, while at lower H2O2 concentrations, the gas temperature and humidity play a leading role. Furthermore, the responses of different types of gas sensors towards the influencing factors of the sterilisation process have been analysed within the same experiment. Based on a correlation established between the inactivation kinetics and the sensor responses, a calorimetric H2O2 sensor and a metal-oxide semiconductor (MOX) sensor have been identified as possible candidates for monitoring the microbicidal effectiveness of aseptic sterilisation processes employing gaseous H2O2. Therefore, two linear models that describe the relationship between sensor response and microbicidal effectiveness have been proposed.