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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.
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.
Bestimmung der metabolischen Aktivität von Mikroorganismen während des Biogasbildungsprozesses
(2009)
Microfabrication, characterization and analytical application of a new thin-film silver microsensor
(2009)
Layer-by-Layer Assembly of Carbon Nanotubes Incorporated in Light-Addressable Potentiometric Sensors
(2009)
Optoelectronic Properties of Nanostructured Ensembles Controlled by Biomolecular Logic Systems
(2008)
Sensing charged macromolecules with nanocrystalline diamond-based field-effect capacitive sensors
(2008)
Heavy metal detection with semiconductor devices based on PLD-prepared chalcogenide glass thin films
(2007)
Handheld measurement device for field-effect sensor structures: Practical evaluation and limitations
(2007)
Online-Messsysteme für die automatisierte Charakterisierung von feldeffektbasierten Biosensoren
(2007)
Markierungsfreie DNA-Detektion mit Silizium-Feldeffekt-Sensoren – Messeffekte oder Artefakte?
(2007)
Novel organic membrane-based thin-film microsensors for the determination of heavy metal cations
(2006)
A first step towards the fabrication and electrochemical evaluation of thin-film microsensors based on organic PVC membranes for the determination of Hg(II), Cd(II), Pb(II) and Cu(II) ions in solutions has been realised. The membrane-coating mixture used in the preparation of this new type of microsensors is incorporating PVC as supporting matrix, o-nitrophenyloctylether (o-NPOE) as solvent mediator and a recently synthesized Hg[dimethylglyoxime(phene)]2+ and Bis-(4-hydroxyacetophenone)-ethylenediamine as electroactive materials for Hg(II) and Cd(II), respectively. A set of three commercialised ionophores for Cd(II), Pb(II) and Cu(II) has been also used for comparison. Thin-film microsensors based on these membranes showed a Nernstian response of slope (26-30 mV/dec.) for the respective tested cations. The potentiometric response characteristics (linear range, pH range, detection limit and response time) are comparable with those obtained by conventional membranes as well as coated wire electrodes prepared from the same membrane. The realisation of the new organic membrane-based thin-film microsensors overcomes the problem of an insufficient selectivity of solid-state-based thinfilm sensors.
An H2O2 sensor for the application in industrial sterilisation processes has been developed. Therefore, automated sterilisation equipment at laboratory scale has been constructed using parts from industrial sterilisation facilities. In addition, a software tool has been developed for the control of the sterilisation equipment at laboratory scale. First measurements with the developed sensor set-up as part of the sterilisation equipment have been performed and the sensor has been physically characterised by optical microscopy and SEM.
GaAs-based Gunn diodes with graded AlGaAs hot electron injector heterostructures have been developed under the special needs in automotive applications. The fabrication of the Gunn diode chips was based on total substrate removal and processing of integrated Au heat sinks. Especially, the thermal and RF behavior of the diodes have been analyzed by DC, impedance and S-parameter measurements. The electrical investigations have revealed the functionality of the hot electron injector. An optimized layer structure could fulfill the requirements in adaptive cruise control (ACC) systems at 77 GHz with typical output power between 50 and 90 mW.
A new and simple method for nanostructuring using conventional photolithography and layer expansion or pattern-size reduction technique is presented, which can further be applied for the fabrication of different nanostructures and nano-devices. The method is based on the conversion of a photolithographically patterned metal layer to a metal-oxide mask with improved pattern-size resolution using thermal oxidation. With this technique, the pattern size can be scaled down to several nanometer dimensions. The proposed method is experimentally demonstrated by preparing nanostructures with different configurations and layouts, like circles, rectangles, trapezoids, “fluidic-channel”-, “cantilever”- and meander-type structures.
Functional testing and characterisation of ISFETs on wafer level by means of a micro-droplet cell
(2006)
A wafer-level functionality testing and characterisation system for ISFETs (ionsensitive field-effect transistor) is realised by means of integration of a specifically designed capillary electrochemical micro-droplet cell into a commercial wafer prober-station. The developed system allows the identification and selection of “good” ISFETs at the earliest stage and to avoid expensive bonding, encapsulation and packaging processes for nonfunctioning ISFETs and thus, to decrease costs, which are wasted for bad dies. The developed system is also feasible for wafer-level characterisation of ISFETs in terms of sensitivity, hysteresis and response time. Additionally, the system might be also utilised for wafer-level testing of further electrochemical sensors.