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Keywords
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.
Die Anforderungen an das energiesparende Bauen sind mit der Einführung der Energieeinsparverordnung (EnEV) 2009 auch im Industrie- und Gewerbebau deutlich verschärft worden. Einen wesentlichen Beitrag zur Energieeinsparung liefert die Minimierung des Transmissionswärmetransfers. Analysiert man Gebäudehüllen in Metallleichtbauweise stellt man fest, dass eine Erhöhung der Wärmedämmstärke allein noch nicht zielführend ist, zusätzlich sind Wärmebrückeneffekte zu berücksichtigen und deren Einflüsse auf die Wärmetransmission zu reduzieren. Neben der Bedeutung für die Energieeinsparung ist eine wärmetechnisch optimierte Detailausbildung auch erforderlich, um einen ausreichenden Feuchteschutz (Vermeidung von Tauwasser und Schimmelpilz) zu realisieren und so Schäden zu vermeiden. Ein wichtiges Hilfsmittel stellt hierzu der vom Industrieverband für Bausysteme im Metallleichtbau (IFBS) herausgegebene Wärmebrückenatlas der Metall-Sandwichbauweise dar.
Im Herbst 2009 wurde das “Deutsche Gütesiegel Nachhaltiges Bauen” für die Gebäudetypen Industriebauten und Handelsbauten offiziell vorgestellt. Die Ökobilanz als wichtiger Baustein der Nachhaltigkeitsbewertung wird somit auch im Industrie- und Gewerbebau zukünftig stark an Bedeutung gewinnen.
Der vorliegende Beitrag beinhaltet einen Vergleich der Umweltleistung verschiedener Bauweisen des Industrie- und Gewerbebaus, die durch die Ökobilanzierung quantifizierbar wird, anhand von momentan verfügbaren Daten und Methoden. Es werden ausgewählte Ergebnisse sowie vorhandene Unsicherheiten und daraus resultierender Forschungsbedarf aufgezeigt.
Bacillus subtilis and Bacillus licheniformis are widely used for the large-scale industrial production of proteins. These strains can efficiently secrete proteins into the culture medium using the general secretion (Sec) pathway. A characteristic feature of all secreted proteins is their N-terminal signal peptides, which are recognized by the secretion machinery. Here, we have studied the production of an industrially important secreted protease, namely, subtilisin BPN′ from Bacillus amyloliquefaciens. One hundred seventy-three signal peptides originating from B. subtilis and 220 signal peptides from the B. licheniformis type strain were fused to this secretion target and expressed in B. subtilis, and the resulting library was analyzed by high-throughput screening for extracellular proteolytic activity. We have identified a number of signal peptides originating from both organisms which produced significantly increased yield of the secreted protease. Interestingly, we observed that levels of extracellular protease were improved not only in B. subtilis, which was used as the screening host, but also in two different B. licheniformis strains. To date, it is impossible to predict which signal peptide will result in better secretion and thus an improved yield of a given extracellular target protein. Our data show that screening a library consisting of homologous and heterologous signal peptides fused to a target protein can identify more-effective signal peptides, resulting in improved protein export not only in the original screening host but also in different production strains.
The objectives of the present work are to characterize the Gas Metal Arc Welding process of DP 600 sheet steel and to summarize the modelling techniques. The time-temperature evolution during the welding cycle was measured experimentally and modelled with the softwaretool SimWeld. To model the phase transformations during the welding cycle dilatometer tests were done to quantify the parameters for phase field modelling by MICRESS®. The important input parameters are interface mobility, nucleation density, etc. A contribution was made to include austenite to bainite transformation in MICRESS®. This is useful to predict the microstructure in the fast cooling segments. The phase transformation model is capable to predict the microstructure along the heating and cooling cycles of welding. Tensile tests have shown the evidence of failure at the heat affected zone, which has the ferrite-tempered martensite microstructure.
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.
This paper aims to evaluate the formability of tailor welded blanks of dual phase (DP600)/transformation induced plasticity (TRIP700) steel sheets. In this work, bead on plate butt joints of 2·5 mm DP600 and 1·2 mm TRIP700 steel sheets were performed using CO2 laser beam welding. Microhardness measurements and transverse tensile testing were carried out to characterise the welds. The formability of base metals and welds were investigated by standard Erichsen test. In a perpendicular tensile test to the weld line, all specimens were fractured at the TRIP base metal, and the strengths were somewhat higher than those of base metal. There was a significant reduction in formability caused by welding of the DP600/TRIP700 steel sheets, and the formability increased with increasing welding speed.
In den letzten Jahrzehnten hat das Elektronenstrahlschweißen, das bereits im größeren Maßstab verwendet wird, seine Fähigkeit als qualitatives Werkzeug für die Verbindung verschiedener Materialen nachgewiesen. Das Non Vacuum Electron Beam Welding (NV-EBW) hat zahlreiche Vorteile im Vergleich zum Elektronenstrahlschweißen im Vakuum, da man unter normalem Atmosphärendruck arbeiten kann. Im Hinblick auf die reproduzierbare Qualität, insbesondere im Bereich der Massen-Fertigung, ist die Kontrolle der Strahlparameter sowie deren Einfluss auf das Schweißergebnis von großer Bedeutung. Durch eine genaue Kenntnis der Strahlkenngrößen wie des Strahldurchmessers und der Leistungsdichteverteilung kann eine Aussage über die sich ausbildende Schweißnaht sowie die Schweißbaddynamik getroffen werden. Messungen der Strahlkenngrößen im Prozess erlauben insbesondere die Untersuchung von Humping-Effekten. In diesem Beitrag wird der Prozess der Elektronenstrahlvermessung unter atmosphärischen Bedingungen beschrieben. Es wird zudem die Abhängigkeit der Elektronenstrahlcharakteristika von den verschiedenen Prozessparametern dargestellt.