Stefan Lange

Reversible switching between p- and n-type conduction in the semiconductor Ag10Te4Br3

Semiconductors are key materials in modern electronics and are widely used to build, for instance, transistors in integrated circuits as well as thermoelectric materials for energy conversion, and there is a tremendous interest in the development and improvement of novel materials and technologies to increase the performance of electronic devices and thermoelectrics. Tetramorphic Ag(10)Te(4)Br(3) is a semiconductor capable of switching its electrical properties by a simple change of temperature. The combination of high silver mobility, a small non-stoichiometry range and an internal redox process in the tellurium substructure causes a thermopower drop of 1,400 microV K(-1), in addition to a thermal diffusivity in the range of organic polymers. The capability to reversibly switch semiconducting properties from ionic to electronic conduction in one single compound simply by virtue of temperature enables novel electronic devices such as semiconductor switches.

Cloning, recombinant expression and biochemical characterisation of novel esterases from Bacillus sp. associated with the marine sponge Aplysina aerophoba

Two novel esterases (EstB1 and EstB2) were isolated from a genomic library of Bacillus sp. associated with the marine sponge Aplysina aerophoba. EstB1 shows low identity (26–44%) with the published hydrolases of the genus Bacillus, whereas EstB2 shows high identity (73–74%) with the carboxylesterases from B. cereus and B. anthracis. Both esterases were efficiently expressed in Escherichia coli under the control of T7 promoter using the vector pET-22b(+). Recombinant EstB1 was purified in a single step to electrophoretic homogeneity by IMAC. A method for the refolding of inclusion bodies formed by the recombinant EstB2 was established to obtain active enzyme. Substrate specificity of the two enzymes towards p-nitrophenyl and methyl esters and the respective kinetic parameters Km and Vmax were determined. The temperature optima of EstB1 and EstB2 were determined to be in the range of 30–50°C and 20–35°C, respectively. The pH optima were found to be in the range of 6.5–7.5 and 6.5–8.0, respectively. Both enzymes showed the highest stability in up to 50% (v/v) DMSO followed by methanol, ethanol and 2-propanol. The influence of high NaCl and KCl concentrations was tested. The inhibition effect of 10–50 mM Zn2+ and 50 mM Mg2+ and Ca2+ ions was observed for both esterases. One to five millimolar PMSF deactivated the enzymes, whereas β-mercaptoethanol, DTT and EDTA had no effect on the enzymes activity.

High-yield expression of the recombinant, atrazine-specific Fab fragment K411B by the methylotrophic yeast Pichia pastoris

In this report, we describe the high-yield secretory expression ( approximately 40 mg x l(-1)) of pure, atrazine-specific Fab fragments (K411B) from Pichia pastoris that was achieved by co-integration of the genes encoding the heavy and light chains (both under the control of the alcohol oxidase promoter) into the genome of the yeast cells. Antibody-expressing clones were selected by SDS-PAGE and ELISA and fed-batch fermentations were carried out in a 5-l scale. Both chains of the Fab were successfully expressed upon methanol induction and almost no other proteins were secreted into the media. Approximately 30% of the two chains formed the active Fab fragment containing the intermolecular disulphide bond, as determined by Western blot analysis under non-reducing conditions. Crude culture supernatant was used to study the binding properties of the Fab fragment toward different s-triazines by means of competitive ELISA: the IC50 value for the detection of atrazine was determined from the standard curve as 3 microg x l(-1), which is one magnitude higher than the value obtained with the parental mAb K4E7 but equals that obtained when the same Fab fragment was expressed in Escherichia coli cells. In addition, the cross-reactivity pattern of the Fab from Pichia is comparable to that of E. coli and to the parental mAb K4E7.

By Overexpression in the YeastPichia pastoris to Enhanced Enantioselectivity: New Aspects in the Application of Pig Liver Esterase

Stable product quality without the interfering influences of other isoenzymes and hydrolases is possible through the application of recombinant pig liver esterase (rPLE), for which functional expression has now been achieved for the first time. In the hydrolysis of 1-phenyl-2-butyl acetate rPLE leads to substantially higher enantioselectivity than commercial PLE preparations.

Cloning, Functional Expression, and Characterization of Recombinant Pig Liver Esterase

The N‐terminal amino acid sequence of pig liver esterase (PLE) from a commercial sample was determined and shown to match closely to a published sequence encoding a proline‐β‐naphthylamidase from pig liver. Next, mRNA isolated from pig liver was transcribed into cDNA and primers deduced from the N‐terminal sequence were used to clone the 1698 base pairs of PLE cDNA. Initial attempts to express the cDNA in Escherichia coli and Pichia pastoris with different expression vectors and secretion signal sequences failed. Only after deletion of the putative C‐terminal sequence His‐Ala‐Glu‐Leu, usually considered as an endoplasmic reticulum retention signal, could heterologous expression of PLE be readily achieved in the methylotrophic yeast P. pastoris. Recombinant PLE (rPLE) was secreted into the medium and exhibited a specific activity of approximately 600 Uu2009mg−1 and a Vmax/Km value of 139 μmolu2009min−1u2009mM−1 with p‐nitrophenyl acetate as a substrate. Activity staining of renatured sodium dodecylsulfate–polyacrylamide gels gave a single band with esterolytic activity for rPLE, whereas several bands are visible in crude commercial PLE preparations. This was confirmed by native gels, which also show that rPLE is active as a trimer. Biochemical characterization of the recombinant enzyme and comparison with properties of commercial PLE preparations as well as with published data confirmed that we expressed a single PLE isoenzyme which showed a high preference for proline‐β‐naphthylamide. This is a substrate specificity for the so‐called γ subunit of PLE. The optimum pH value and temperature for the recombinant PLE were 8.0 and 60u2009°C, respectively. The determined molecular weight of the secreted enzyme was approximately 61–62 kDa, which closely matches the calculated value of 62.419 kDa. The active site residues are located at Ser203, His448, and Asp97, and the typical consensus sequence motif for hydrolases was found around the active site serine (Gly‐Glu‐Ser‐Ala‐Gly).

Mineralization Routes to Polyphosphides: Cu2P20 and Cu5InP16

The element chemistry of phosphorus is one the most complex but also one of the most exciting of all chemical elements. Both the element and the binary and ternary derivatives show a great diversity in terms of reactivity, structural chemistry, and physical properties, such as polymorphism, magnetism, and superconductivity, and are applied, for example, as thermoelectrics, catalysts, or in precipitation hardening. Four allotropic modifications of phosphorus are known to date at standard conditions, namely white, violet, fibrous, and black phosphorus, besides the amorphous red phosphorus. White phosphorus comprises molecular P4 units, and black phosphorus is a layered compound, whereas violet and fibrous phosphorus are characterized by polymeric phosphorus stands of tubular [P20] 2 units connected via P2 bridges. Structural fragments of these units have been identified in amorphous red phosphorus by vibrational spectroscopy and in KP15 by structural analysis. [7] Some theoretically predicted allotropes featuring polymeric phosphorus units were successfully isolated from a copper halide matrix. P15Se and P19Se are two examples of heteroatomic polymer chains that have similar but not identical structural motifs to [P20] 2 . In the past decades elemental phosphorus was used to prepare a plethora of binary and multinary phosphides and polyphosphides. Thermodynamically and also kinetically controlled reactions were developed to derive new compounds from elemental phosphorus. Surprisingly none of the developed methods led to a binary derivative of violet or fibrous phosphorus with retention of the polyphosphide substructure. Only one ternary compound featuring a [P20] 2 unit, the direct subunit of the element structure, embedded in a copper(I) halide matrix, was reported. Recently we prepared the novel polyphosphide AgSbP14, the first pure inorganic material containing a covalent Sb P interaction, and developed a low-pressure route to black phosphorus, making this phosphorus modification commercially available and accessible for applications. Both compounds have been synthesized by a kinetically controlled reaction route using main group metal halides such as SbI3 or SnI4 as reaction promoters (mineralizers). The general reaction principle is closely related to the well-known concept of mineralization reactions described by Sch6fer. Phosphides and polyphosphides such as Zn3P2, Cu3P, LiCu2P2, and Li7Cu5P8 are considered to be promising materials for electrodes in rechargeable batteries, and a carbon–phosphorus composite was successfully tested as an electrode material. The ongoing interest in new energy storage materials on the one hand and the still not completely solved material and engineering problems with present battery systems on the other hand are stimulating the development of new synthesis routes to new materials. Polyphosphides with anisotropic subunits (2D layers) are potential candidates for intercalation reactions, as shown in case of black phosphorus. Herein we report on the CuImediated synthesis of Cu2P20 and Cu5InP16 as well as on their structures and physical properties. X-ray powder diffraction and EDX analyses for both polyphosphides substantiated the phase purity of the bulk phases and the composition of the single crystals selected for the structure determinations. The crystal structure of Cu2P20 was solved from single-crystal X-ray data at room temperature (Figure 1, top). Tubular [P20] 2 units are stacked parallel to each other and connected through tetrahedrally coordinated copper(I) ions; the Cu P bond lengths range between 2.271(3) and 2.317(3) ?. The P P bond lengths (2.154(4)–2.322(3) ?) within the tubular [P20] 2

Fluorophor-linked immunosorbent assay: a time- and cost-saving method for the characterization of antibody fragments using a fusion protein of a single-chain antibody fragment and enhanced green fluorescent protein

A novel assay, referred to as fluorophor-linked immunosorbent assay (FLISA), for the characterization of single-chain antibody fragments (scFvs) is described. The principle of the method is the fusion of an scFv to enhanced green fluorescent protein (EGFP). The scFv domain, which binds to the immobilized hapten, can be detected by measuring the fluorescence of the EGFP domain. The time-consuming binding of secondary antibodies and enzyme reaction, necessary for enzyme-linked immunosorbent assays (ELISAs) are not required. Consequently, the assay time of 1.5 h needed to complete the FLISA is much shorter than that of comparable ELISAs, which require about 5 h. This renders the FLISA suitable for applications where a short assay time is essential, such as screening of mutant libraries of scFvs in directed evolution experiments or monitoring of the amount of functionally expressed recombinant protein during production processes. In contrast to a comparable ELISA, the FLISA showed no saturation when determining the relative amount of functional scFv. The amount of the soluble fraction of cell extracts from Escherichia coli expressing the fusion protein and the normalized fluorescence signal showed a linear correlation with R(2)>0.99. The usefulness of a competitive FLISA for the detection of analytes is shown exemplarily by the detection of s-triazines with the s-triazine-specific scFv K411B.

Recombinant production of human microsomal cytochrome P450 2D6 in the methylotrophic yeast Pichia pastoris

Microsomal cytochrome P450 monooxygenases of groups 1–3 are mainly expressed in the liver and play a crucial role in phase 1 reactions of xenobiotic metabolism. The cDNAs encoding human CYP2D6 and human NADPH‐P450 oxidoreductase (CPR) were transformed into the methylotrophic yeast Pichia pastoris and expressed with control of the methanol‐inducible AOX1 promoter. The determined molecular weights of the recombinant CYP2D6 and CPR closely matched the calculated values of 55.8 and 76.6 kDa. CPR activity was detected by conversion of cytochrome c by using isolated microsomes. Nearly all of the recombinant CYP was composed of the active holoenzyme, as confirmed by reduced CO difference spectra, which showed a single peak at 450 nm. Only by coexpression of human CPR and CYP was CYP2D6 activity obtained. Microsomes containing human CPR and CYP2D6 converted different substrates, such as 3‐cyano‐7‐ethoxycoumarin, parathion and dextrometorphan. The kinetic parameters of dextrometorphan conversion closely matched those of CYP2D6 from other recombinant expression systems and human microsomes. The endogenous NADPH‐P450 oxidoreductase of Pichia pastoris seems to be incompatible with human CYP2D6, as expression of CYP2D6 without human CPR did not result in any CYP activity. These recombinant strains provide a novel, easy‐to‐handle and cheap source for the biochemical characterisation of single microsomal cytochromes, as well as their allelic variants.

Crystal Structures and Thermal and Electrical Properties of the New Silver (poly)Chalcogenide Halides Ag23Te12Cl and Ag23Te12Br

Two new silver (poly)chalcogenide halides, Ag23Te12Cl and Ag23Te12Br, were characterized by powder X-ray phase analysis, energy dispersive X-ray analysis, and crystal structure determinations at various temperatures. Thermal analyses of both compounds and electrochemical measurements for the bromide completed the investigation. The compounds Ag23Te12X (X = Cl, Br) are isostructural and crystallize orthorhombically (space group Pnnm, Z = 4) as systematic twins. The lattice parameter values derived from X-ray powder data were a = 21.214(2) A, b = 21.218(2) A, c = 7.7086(7) A, and V = 3469.8(6) A (3) for Ag23Te12Cl at 293 K and a = 21.170(1) A, b = 21.170(1) A, c = 7.7458(5) A, and V = 3471.4(4) A (3) for Ag23Te12Br at 298 K. An enhanced silver ion mobility was revealed by impedance spectroscopy investigations. No phase transitions were observed in the temperature range 100-750 K. These two silver(I) (poly)chalcogenide halides are the second set of representatives of a new class of coinage-metal (poly)chalcogenide halides in which both covalently bonded [Te2](2-) dumbbells and ionically bonded Te(2-) anions appear.

Identification of factors impeding the production of a single-chain antibody fragment in Escherichia coli by comparing in vivo and in vitro expression

The atrazine-specific single-chain variable antibody fragments (scFv) K411B was produced by expression in either the cytoplasm or the periplasm of Escherichia coli BL21(DE3). For periplasmic production, the pelB leader was N-terminally fused to scFv, whereas the unfused variant resulted in cytoplasmic expression. The extent of protein accumulation differed significantly. Expression of scFv with leader was 2.3 times higher than that of the protein without leader. This was further investigated by generating the respective translation profiles using coupled in vitro transcription/translation assays, the results of which were in agreement. This comparative approach was also applied to functionality: Periplasmic expression and in vitro expression resulted in only 10% correctly folded scFv, indicating that the oxidizing environment of the periplasm did not increase proper folding. Thus, the data obtained in vitro confirmed the findings observed in vivo and suggested that the discrepancy in expression levels was due to different translation efficiencies. However, the in vivo production of scFv with enhanced green fluorescent protein (EGFP) fused C-terminally (scFv-EGFP) was only successful in the cytoplasm, although in vitro the expression with and without the leader rendered the same production profile as for scFv. This indicated that neither the translation efficiency nor the solubility but other factors impeded periplasmic expression of the fusion protein.