Identification and characterization of a glucan-producing enzyme from Lactobacillus hilgardii TMW 1.828 involved in granule formation of water kefir

Water kefir is a home made fermented beverage based on a sucrose solution with fruit extracts. The inoculum of such fermentations consists of macroscopic granula containing lactic and acetic acid bacteria, and yeasts, which are embedded in an exopolysaccharide (EPS) matrix. In this work, a strain of Lactobacillus hilgardii producing large amounts of the granule-forming dextran could be isolated. The glycosyltransferans (Gtf) commonly called glucansucrase responsible for the production of this dextran was purified from L. hilgardii. Characteristic enzyme kinetic data were obtained. Optimum activity was observed between pH 4.3 and 4.6 and temperatures between 40 °C and 45 °C. A Michaelis–Menten kinetic could be fit to the experimental data and a K_M of 0.0385 M was calculated. The corresponding gtf gene was identified and characterized. It encodes a 1448 amino acid protein with higher homologies to Gtfs of Lactobacillus parabuchneri, Lactobacillus sakei and Lactobacillus fermentum followed by lower homologies to Lactobacillus reuteri Gtfs. By knockout experiments the role of this gene in granule dextran production was demonstrated.     

Basic investigations on debris cooling

An experimental investigation of boiling phenomena in inductively heated particle beds has been performed. The major aim of these experiments is to provide data for validating numerical codes used in reactor safety. The experiments can be divided in three parts: boiling experiments, dryout experiments and quenching experiments. In boiling experiments, the pressure gradients have been measured along the bed height for different flow modes, different heat inputs and different system pressures. In dryout experiments, the minimum heat input has been determined for which the particle bed starts to superheat significantly above the saturation temperature. The final test series deals with the cool down behaviour of strongly superheated particles by flooding them with cold water. The initial temperatures ranged from 200 up to 900 °C in top-quenching experiments and from 230 up to 450 °C in bottom-quenching experiments. All experiments were performed with pre-oxidised stainless steel balls of 6 and 3 mm diameter in a cylindrical crucible. The bed height was 640 mm and the bed diameter was 125 mm for boiling and dryout experiments, respectively 150 mm for quenching experiments. The experimental results are compared with various available dryout models.     

Prognose der Sorptionskinetik organischer Schadstoffe in heterogenem Aquifermaterial

A model for the simulation of kinetic sorption processes in heterogeneous aquifer material is presented. Sorption kinetics is responsible for the long persistence of many organic contaminants in the subsurface. Therefore, reliable model predictions of these processes are of major importance concerning, for instance, the design of efficient remediation strategies. The modeling approach presented here recognizes sorption kinetics as retarded diffusion within the intra-particle pore space and, in particular, takes into account the sedimentological and petrographical composition of the aquifer material. This is in accordance with results from laboratory experiments quantifying sorption/desorption processes. For solving the model equations a finite-difference scheme is applied which incorporates several features proven to be relevant in practical model applications (mass balance, flexible choice of boundary conditions, easy handling). The simulation results shown here focus on the impact of aquifer heterogeneity (lithological composition, grain size distribution) on sorption and desorption kinetics of organic contaminants. Furthermore, this approach can be straightforwardly coupled to existing software for simulating multi-dimensional solute transport.     

Current in-service inspections of austenitic stainless steel and dissimilar metal welds in light water nuclear power plants

A tendency towards growing requirements for the inspection of austenitic piping can be observed in several countries. In Germany the revised KTA rule demands the UT inspection of austenitic and dissimilar metal welds in piping with diameters of 200 mm or more.On the basis of experience gained from austenitic piping with integranular stress corrosion cracking (IGSCC), longitudinal waves and mode conversion techniques are used. Depending on the geometry, material and grain orientation, spurious signals can be observed which require additional evaluation or analysis measurements.A promising new technique is based on horizontally polarized shear (SH) waves generated by electromagnetic acoustic transducers (EMATs). Investigations in the laboratory and field inspections showed that SH waves are well suited for the detection of longitudinal flaws, especially where the weld can be examined from one side only.For the complete solution of a given inspection problem SH waves can be combined with well-known standard techniques in order to provide redundant information for the characterization and sizing of indications.The investigation of possibilities of SH waves showed that the problem of cast austenitic steel inspection might not be solved using this technique. However, measurements using low frequency UT transducers showed promising results.     

Some nonrealizable line diagrams

We superimpose weaving patterns on planar line arrangements, and we face the question when they can be realized by lines in 3-space. Using the combinatorial type of the given lines in the plane we derive a class of nonrealizable weavings.     

Comparison of pressure and heat resistance of Clostridium botulinum and other endospores in mashed carrots

Inactivation of bacterial endospores in food requires a combination of pressure and moderate heat. Endospore resistance of seven Clostridium botulinum strains was compared with those of Bacillus spp. (B. cereus, B. subtilis, B. licheniformis, B. smithii, B. amyloliquefaciens) and Thermoanaerobacterium thermosaccharolyticum with respect to pressure (600 to 800 MPa) and temperature (80 to 116 degrees C) treatments in mashed carrots. A large variation was observed in the pressure resistance of C. botulinum spores. Their reduction after treatments with 600 MPa at 80 degrees C for 1 s ranged from more than 5.5 log units to no reduction. Spores of the proteolytic C. botulinum TMW 2.357 exhibited a greater resistance to pressure than spores from all other bacteria examined, with the exception of B. amyloliquefaciens. Heat resistance of spores did not correlate with pressure resistance, either within strains of C. botulinum or when C. botulinum spores were compared with spores of T. thermosaccharolyticum. A quantitative release of dipicolinic acid was observed from C. botulinum spores on combined pressure and temperature treatments only after inactivation of more than 99.999% of the spores. Thus, dipicolinic acid is released by a physicochemical rather than a physiological process. The resistance of spores to combined pressure and temperature treatments correlated with their ability to retain dipicolinic acid. B. amyloliquefaciens, a mesophilic organism that forms highly pressure-resistant spores is proposed as a nonpathogenic target organism for high-pressure process development.     

Encoding microcarriers by spatial selective photobleaching

Bead-based assays on very large numbers of molecules in gene expression studies, drug screening and clinical diagnostics, require the encoding of each of the microspheres according to the particular ligand bound to its surface. This allows mixing the uniquely encoded microspheres and subjecting them to an assay simultaneously. When a particular microsphere gives a positive reaction, the substance on its surface can be identified by reading the code. Previously reported techniques for colour encoding polymer microspheres only allow for a limited number of unique codes. Graphical encoding methods use metallic particles, which are rather uncommon in screening applications. Here, we demonstrate a new approach to encode polymer microspheres that are commonly used in screening applications, such as polystyrene microspheres, with a method that provides a virtually unlimited number of unique codes. Patterns can be written in fluorescently dyed microspheres by 'spatial selective photobleaching' and can be identified by confocal microscopy. Such encoded microparticles can find broad application in the collection and analysis of genetic information, high-throughput screening, medical diagnostics and combinatorial chemistry, and can also be used for labelling of consumer goods or as security labels to prevent counterfeiting.     

Macrocyclization of Organo‐Peptide Hybrids through a Dual Bio‐orthogonal Ligation: Insights from Structure–Reactivity Studies

Macrocycles constitute an attractive structural class of molecules for targeting biomolecular interfaces with high affinity and specificity. Here, we report systematic studies aimed at exploring the scope and mechanism of a novel chemo‐biosynthetic strategy for generating macrocyclic organo‐peptide hybrids (MOrPHs) through a dual oxime‐/intein‐mediated ligation reaction between a recombinant precursor protein and bifunctional, oxyamino/1,3‐amino‐thiol compounds. An efficient synthetic route was developed to access structurally different synthetic precursors incorporating a 2‐amino‐ mercaptomethyl‐aryl (AMA) moiety previously found to be important for macrocyclization. With these compounds, the impact of the synthetic precursor scaffold and of designed mutations within the genetically encoded precursor peptide sequence on macrocyclization efficiency was investigated. Importantly, the desired MOrPHs were obtained as the only product from all the different synthetic precursors probed in this study and across peptide sequences comprising four to 15 amino acids. Systematic mutagenesis of the “i?1” site at the junction between the target peptide sequence and the intein moiety revealed that the majority of the 20 amino acids are compatible with MOrPH formation; this enables the identification of the most and the least favorable residues for this critical position. Furthermore, interesting trends with respect to the positional effect of conformationally constrained (Pro) and flexible (Gly) residues on the reactivity of randomized hexamer peptide sequences were observed. Finally, mechanistic investigations enabled the relative contributions of the two distinct pathways (side‐chain→C‐end ligation versus C‐end→side‐chain ligation) to the macrocyclization process to be dissected. Altogether, these studies demonstrate the versatility and robustness of the methodology to enable the synthesis and diversification of a new class of organo‐peptide macrocycles and provide valuable structure–reactivity insights to inform the construction of macrocycle libraries through this chemo‐biosynthetic strategy.     

Mobile Camera Localization Using Apollonius Circles and Virtual Landmarks

We investigate the localization of a camera subject to a planar motion with horizontal optical axis in the presence of known vertical landmarks. Under these assumptions, a calibrated camera can measure the distance to the viewed landmarks. We propose to replace the trilateration method by intersecting a pair of Chasles-Apollonius circles. In the case of square pixels but unknown focal length we introduce a new method to recover the camera position from one image with three vertical landmarks. To this end we consider virtual landmarks and Apollonius-like circles. We extend this method in order to deal with an unknown principal point by using four landmarks.