Purification, crystallization and preliminary crystallographic analysis of the catalytic domain of the extracellular cellulase CBHI from Trichoderma harzianum

The filamentous fungus Trichoderma harzianum has a considerable cellulolytic activity that is mediated by a complex of enzymes which are essential for the hydrolysis of microcrystalline cellulose. These enzymes were produced by the induction of T. harzianum with microcrystalline cellulose (Avicel) under submerged fermentation in a bioreactor. The catalytic core domain (CCD) of cellobiohydrolase I (CBHI) was purified from the extracellular extracts and submitted to robotic crystallization. Diffraction-quality CBHI CCD crystals were grown and an X-ray diffraction data set was collected under cryogenic conditions using a synchrotron-radiation source.

Protein preparation, crystallization and preliminary X-ray analysis of Trypanosoma cruzi nucleoside diphosphate kinase 1

The flagellated protozoan parasite Trypanosoma cruzi is the aetiological agent of Chagas disease. Nucleoside diphosphate kinases (NDPKs) are enzymes that are involved in energy management and nucleoside balance in the cell. T. cruzi TcNDPK1, a canonical isoform, was overexpressed in Escherichia coli as an N-terminally poly-His-tagged fusion protein and crystallized. Crystals grew after 72 h in 0.2 M MgCl(2), 20% PEG 3350. Data were collected to 3.5 angstrom resolution using synchrotron X-ray radiation at the National Synchrotron Light Laboratory (Campinas, Brazil). The crystals belonged to the trigonal space group P3, with unit-cell parameters a = b = 127.84, c = 275.49 angstrom. Structure determination is under way and will provide relevant information that may lead to the first step in rational drug design for the treatment of Chagas disease.

Sublattice magnetism in sigma-phase Fe(100-x)Vx (x=34.4, 39.9, and 47.9) studied via zero-field (51)V NMR

The successful measurements of a sublattice magnetism with (51)V NMR techniques in the sigma-phase Fe(100-x)V(x) alloys with x=34.4, 39.9, and 47.9 are reported. Vanadium atoms, which were revealed to be present on all five crystallographic sites, are found to be under the action of the hyperfine magnetic fields produced by the neighboring Fe atoms, which allow the observation of (51)V NMR signals. Their nuclear magnetic properties are characteristic of a given site, which strongly depend on the composition. Site A exhibits the strongest magnetism while site D is the weakest. The estimated average magnetic moment per V atom decreases from 0.36 mu(B) for x=34.4 to 0.20 mu(B) for x=47.9. The magnetism revealed at V atoms is linearly correlated with the magnetic moment of Fe atoms, which implies that the former is induced by the latter.

Crystallization and preliminary X-ray diffraction analysis of human IL-22 bound to its soluble decoy receptor IL-22BP

Interleukin-22 (IL-22) is a pleiotropic cytokine that is involved in inflammatory responses. Human IL-22 was incubated with its soluble decoy receptor IL-22BP (IL-22 binding protein) and the IL-22 -IL-22BP complex was crystallized in hanging drops using the vapour-diffusion method. Suitable crystals were obtained from polyethylene glycol solutions and diffraction data were collected to 2.75 angstrom resolution. The crystal belonged to the tetragonal space group P41, with unit-cell parameters a = b = 67.9, c = 172.5 angstrom, and contained two IL-22-IL- 22BP complexes per asymmetric unit.

Nonlinear refraction and absorption through phase transition in a Nd:SBN laser crystal

Time-resolved Z-scan measurements were performed in a Nd(3+)-doped Sr(0.61)Ba(0.39)Nb(2)O(6) laser crystal through ferroelectric phase transition. Both the differences in electronic polarizability (Delta alpha(p)) and cross section (Delta sigma) of the neodymium ions have been found to be strongly modified in the surroundings of the transition temperature. This observed unusual behavior is concluded to be caused by the remarkable influence that the structural changes associated to the ferro-to-paraelectric phase transition has on the 4f -> 5d transition probabilities. The maximum polarizability change value Delta alpha(p)=1.2x10(-25) cm(3) obtained at room temperature is the largest ever measured for a Nd(3+)-doped transparent material.

Crystallization and preliminary X-ray diffraction analysis of recombinant chlorocatechol 1,2-dioxygenase from Pseudomonas putida

Chlorocatechol 1,2-dioxygenase from the Gram-negative bacterium Pseudomonas putida (Pp 1,2-CCD) is considered to be an important biotechnological tool owing to its ability to process a broad spectrum of organic pollutants. In the current work, the crystallization, crystallographic characterization and phasing of the recombinant Pp 1,2-CCD enzyme are described. Reddish-brown crystals were obtained in the presence of polyethylene glycol and magnesium acetate by utilizing the vapour-diffusion technique in sitting drops. Crystal dehydration was the key step in obtaining data sets, which were collected on the D03B-MX2 beamline at the CNPEM/MCT - LNLS using a MAR CCD detector. Pp 1,2-CCD crystals belonged to space group P6(1)22 and the crystallographic structure of Pp 1,2-CCD has been solved by the MR-SAD technique using Fe atoms as scattering centres and the coordinates of 3-chlorocatechol 1,2-dioxygenase from Rhodococcus opacus (PDB entry 2boy) as the search model. The initial model, which contains three molecules in the asymmetric unit, has been refined to 3.4 A resolution.

Influence of film thickness on the crystallization of Ni-doped amorphous silicon samples

This work reports on the crystallization of amorphous silicon (a-Si) films doped with 1 at. % of nickel. The films, with thicknesses ranging from 10 to 3000 nm, were deposited using the cosputtering method onto crystalline quartz substrates. In order to investigate the crystallization mechanism in detail, a series of undoped a-Si films prepared under the same deposition conditions were also studied. After deposition, all a-Si films were submitted to isochronal thermal annealing treatments up to 1000 degrees C and analyzed by Raman scattering spectroscopy. Based on the present experimental results, it is possible to state that (a) when compared to the undoped a-Si films, those containing 1 at. % of Ni crystallize at temperatures similar to 100 degrees C lower, and that (b) the film thickness influences the temperature of crystallization that, in principle, tends to be lower in films thinner than 1000 nm. The possible reasons associated to these experimental observations are presented and discussed in view of some experimental and thermodynamic aspects involved in the formation of ordered Si-Si bonds and in the development of Ni-silicide phases. (c) 2008 American Institute of Physics.

Effective coordination concept applied for phase change (GeTe)(m)(Sb(2)Te(3))(n) compounds

In this work, we employed the effective coordination concept to study the local environments of the Ge, Sb, and Te atoms in the Ge(m)Sb(2n)Te(m+3n) compounds. From our calculations and analysis, we found an average effective coordination number (ECN) reduction of 1.59, 1.42, and 1.37, for the Ge, Sb, Te atoms in the phase transition from crystalline, ECN=5.55 (Ge), 5.73 (Sb), 4.37 (Te), to the amorphous phase, ECN=3.96 (Ge), 4.31 (Sb), 3.09 (Te), for the Ge(2)Sb(2)Te(5) composition. Similar changes are observed for other compositions. Thus, our results indicate that the coordination changes from the crystalline to amorphous phase are not large as previously assumed in the literature, i.e., from sixfold to fourfold for Ge, which can contribute to obtain a better understanding of the crystalline to amorphous phase transition. (C) 2011 American Institute of Physics. [doi:10.1063/1.3533422]

A PHASE TRANSITION FOR COMPETITION INTERFACES

We study the competition interface between two growing clusters in a growth model associated to last-passage percolation. When the initial unoccupied set is approximately a cone, we show that this interface has an asymptotic direction with probability 1. The behavior of this direction depends on the angle theta of the cone: for theta >= 180 degrees, the direction is deterministic, while for theta < 180 degrees, it is random, and its distribution can be given explicitly in certain cases. We also obtain partial results on the fluctuations of the interface around its asymptotic direction. The evolution of the competition interface in the growth model can be mapped onto the path of a second-class particle in the totally asymmetric simple exclusion process; from the existence of the limiting direction for the interface, we obtain a new and rather natural proof of the strong law of large numbers (with perhaps a random limit) for the position of the second-class particle at large times.

Metastable BrO(2+) and NBr(2+) molecules in the gas phase

The doubly positively charged gas-phase molecules BrO(2+) and NBr(2+) have been produced by prolonged high-current energetic oxygen (17 keV (16)O(-)) ion surface bombardment (ion beam sputtering) of rubidium bromide (RbBr) and of ammonium bromide (NH(4)Br) powdered ionic salt samples, respectively, pressed into indium foil. These novel species were observed at half-integer m/z values in positive ion mass spectra for ion flight times of roughly similar to 12 mu s through a magnetic-sector secondary ion mass spectrometer. Here we present these experimental results and combine them with a detailed theoretical investigation using high level ab initio calculations of the ground states of BrO(2+) and NBr(2+), and a manifold of excited electronic states. NBr(2+) and BrO(2+), in their ground states, are long-lived metastable gas-phase molecules with well depths of 2.73 x 10(4) cm(-1) (3.38 eV) and 1.62 x 10(4) cm(-1) (2.01 eV); their fragmentation channels into two monocations lie 2.31 x 10(3) cm(-1) (0.29 eV) and 2.14 x 10(4) cm(-1) (2.65 eV) below the ground state minimum. The calculated lifetimes for NBr(2+) (v '' < 35) and BrO(2+) (v '' < 18) are large enough to be considered stable against tunneling. For NBr(2+), we predicted R(e) = 3.051 a(0) and omega(e) = 984 cm(-1); for BrO(2+), we obtained 3.033 a(0) and 916 cm(-1), respectively. The adiabatic double ionization energies of BrO and NBr to form metastable BrO(2+) and NBr(2+) are calculated to be 30.73 and 29.08 eV, respectively. The effect of spin-orbit interactions on the low-lying (Lambda + S) states is also discussed. (C) 2011 American Institute of Physics. [doi:10.1063/1.3562121]

Characterization of dynamic solid phase DNA extraction from blood with magnetically controlled silica beads

A novel solid phase extraction technique is described where DNA is bound and eluted from magnetic silica beads in a manner where efficiency is dependent on the magnetic manipulation of the beads and not on the flow of solution through a packed bed. The utility of this technique in the isolation of reasonably pure, PCR-amplifiable DNA from complex samples is shown by isolating DNA from whole human blood, and subsequently amplifying a fragment of the beta-globin gene. By effectively controlling the movement of the solid phase in the presence of a static sample, the issues associated with reproducibly packing a solid phase in a microchannel and maintaining consistent flow rates are eliminated. The technique described here is rapid, simple, and efficient, allowing for recovery of more than 60% of DNA from 0.6 mu L of blood at a concentration which is suitable for PCR amplification. In addition, the technique presented here requires inexpensive, common laboratory equipment, making it easily adopted for both clinical point-of-care applications and on-site forensic sample analysis.

Direct Solid-Phase Optical Measurements in Flow Systems: A Review

Measurements based on absorption, reflectance, or luminescence of molecular species or complex ions can be carried out directly on a solid support simultaneously to the retention of the analyte. The use of this strategy in flow-based systems is advantageous in view of the reproducible handling of solutions in retention and elution steps of the analyte. This approach can be exploited to increase sensitivity, minimize reagent consumption as well as waste generation, improve selectivity or for simultaneous determination based on selective retention or differences in sorption rates of the analytes. This review focuses on the main characteristics of direct solid-phase measurements in flow systems, including the discussion of advantages and limitations and practical guidelines to the successful implementation of this approach. Selected applications in diverse fields, such as pharmaceutical, food, and environmental analysis are discussed.

Liquid-liquid microextraction without phase separation in a multicommuted flow system for diltiazem determination in pharmaceuticals

Liquid-liquid microextraction without phase segmentation was implemented in a multicommuted flow system for determination of the anti-hypertensive diltiazem. The procedure was based on ion pair formation between the drug and the dye bromothymol blue at pH 3.5. The detection was performed without phase separation in a glass tube coupled to a fiber-optics spectrophotometer. The total volume of chloroform was reduced to 50 mu L in comparison with 10 mL consumed in batch. A linear response was observed between 9 and 120 mu mol L(-1), with a detection limit of 0.9 mu mol L(-1) (99.7% confidence level). The coefficient of variation (n = 10), sampling rate and extraction efficiency were estimated as 0.6%, 78 determinations per hour and 61%, respectively. About 30 mu g of bromothymol blue was consumed and the waste volume was 380 mu L per determination. The results for pharmaceutical samples agreed with those obtained by the reference procedure at the 95% confidence level. (C) 2011 Elsevier B.V. All rights reserved.

Analytical calculation of the transition to complete phase synchronization in coupled oscillators

Here we present a system of coupled phase oscillators with nearest neighbors coupling, which we study for different boundary conditions. We concentrate at the transition to the total synchronization. We are able to develop exact solutions for the value of the coupling parameter when the system becomes completely synchronized, for the case of periodic boundary conditions as well as for a chain with fixed ends. We compare the results with those calculated numerically.

Batch cooling crystallization of xylitol produced by biotechnological route

BACKGROUND: This work deals with the xylitol production by biotechnological routes emphasizing the purification process using crystallization. RESULTS: Xylitol volumetric productivity of 0.665 g L(-1) h(-1) and yield of 0.7024 g g(-1) were obtained after 92 h fermentation. The fermented broth (61.3 g L(-1) xylitol) was centrifuged, treated and concentrated obtain a syrup (745.3 g L(-1) xylitol) which was crystallized twice, xylitol crystals with 98.5-99.2% purity being obtained. CONCLUSION: The hypothetical distribution obtained permits the determination of modeling parameters, which make possible the estimation of crystal dominant size from different initial experimental conditions. (C) 2008 Society of Chemical Industry