Culture medium for isolating chitinolytic bacteria from seawater and plankton

Chitin degradation is a key step in the cycling of nutrients in marine ecosystems and chitinolytic bacteria are the primary agents of this process. Chitinases, produced by bacteria, have potential applications in agriculture, medicine and in a wide range of biotechnological processes. We utilized a simple, rapid and cost-effective method of colloidal chitin preparation and a culture medium, in which colloidal chitin is the sole carbon source for the purpose of counting and isolating chitinolytic bacteria from seawater and plankton. This culture medium could be useful to isolate bacteria with the ability to produce extracellular chitinases for biotechnological applications.

Gene optimization leads to robust expression of human respiratory syncytial virus nucleoprotein and phosphoprotein in human cells and induction of humoral immunity in mice

Human respiratory syncytial virus (HRSV) is the major pathogen leading to respiratory disease in infants and neonates worldwide. An effective vaccine has not yet been developed against this virus, despite considerable efforts in basic and clinical research. HRSV replication is independent of the nuclear RNA processing constraints, since the virus genes are adapted to the cytoplasmic transcription, a process performed by the viral RNA-dependent RNA polymerase. This study shows that meaningful nuclear RNA polymerase II dependent expression of the HRSV nucleoprotein (N) and phosphoprotein (F) proteins can only be achieved with the optimization of their genes, and that the intracellular localization of N and P proteins changes when they are expressed out of the virus replication context. Immunization tests performed in mice resulted in the induction of humoral immunity using the optimized genes. This result was not observed for the non-optimized genes. In conclusion, optimization is a valuable tool for improving expression of HRSV genes in DNA vaccines. (c) 2009 Elsevier B.V. All rights reserved.

The potential for first-generation ethanol production from sugarcane

Ethanol production from sugarcane, mainly in Brazil, on the basis of first-generation technology (22.5 billion liters, in 2007/2008 season, in 3.4 million hectares) replaces 1% of the gasoline used in the world today and is highly competitive in economic terms with ethanol produced from other crops in the USA and Europe. In this paper we discuss the potential for sugarcane ethanol expansion from two angles: (1) productivity gains which would allow greater production in the same area and (2) geographical expansion to larger areas. The potential of first-generation technology for the production of ethanol from sugarcane is far from being exhausted. There are gains in productivity of approximately a factor of two from genetically modified varieties and a geographical expansion by a factor of ten of the present level of production in many sugar-producing countries. The replacement of 10% of the gasoline used in the world by ethanol from sugarcane seems possible before second-generation technology reaches technological maturity and possibly economic competitiveness. (C) 2009 Society of Chemical Industry and John Wiley & Sons, Ltd

Chromium ions phytoaccumulation by three floating aquatic macrophytes from a nutrient medium

In the present work, the trivalent and hexavalent chromium phytoaccumulation by three living free floating aquatic macrophytes Salvinia auriculata, Pistia stratiotes, and Eicchornia crassipes was investigated in greenhouse. These plants were grown in hydroponic solutions supplied with non-toxic Cr3+ and Cr6+ chromium concentrations, performing six collections of nutrient media and plants in time from a batch system. The total chromium concentrations into Cr-doped hydroponic media and dry roots and aerial parts were assayed, by using the Synchrotron radiation X-ray fluorescence technique. The aquatic plant-based chromium removal data were described by using a nonstructural kinetic model, obtaining different bioaccumulation rate, ranging from 0.015 to 0.837 1 mg(-1) d(-1). The Cr3+ removal efficiency was about 90%, 50%, and 90% for the E. crassipes, P. stratiotes, and S. auriculata, respectively; while it was rather different for Cr6+ one, with values about 50%, 70%, and 90% for the E. crassipes, P. stratiotes, and S. auriculata.

Enzyme immobilization on Ag nanoparticles/polyaniline nanocomposites

We show a simple strategy to obtain all efficient enzymatic broelectrochemical device, in which urease was immobilized oil electroactive nanostructured membranes (ENMs) made with polyaniline and silver nanoparticles (AgNP) stabilized in polyvinyl alcohol (PAni/PVA-AgNP). Fabrication of the modified electrodes comprised the chemical deposition of polyaniline followed by drop-coating of PVA-AgNP and urease, resulting in a final ITO/PAni/PVA-AgNP/urease electrode Configuration. For comparison. the electrochemical performance of ITO/PAni/urease electrodes (without Ag nanoparticles) was also studied. The performance of the modified electrodes toward Urea hydrolysis was investigated via amperometric measurements, revealing a fast increase in cathodic current with a well-defined peak upon addition of urea to the electrolytic solution. The cathodic currents for the ITO/PAni/PVA-AgNP urease electrodes were significantly higher than for the ITO/PAni/urease electrodes. The friendly environment provided by the ITO/PAni/PVA-AgNP electrode to the immobilized enzyme promoted efficient catalytic conversion of urea into ammonium and bicarbonate tons. Using the Michaelis-Menten kinetics equation, a K(M)(aPP) of 2.7 mmol L(-1) was obtained. indicating that the electrode architecture employed may be advantageous for fabrication of enzymatic devices with improved biocatalytic properties. Crown Copyright (C) 2009 Published by Elsevier B.V. All rights reserved.

Detection of phenolic compounds using impedance spectroscopy measurements

Langmuir-Blodgett (LB) and layer-by-layer films (LbL) of a PPV (p-phenylenevinylene) derivative, an azo compound and tetrasulfonated phthalocyanines were successfully employed as transducers in an ""electronic tongue"" system for detecting trace levels of phenolic compounds in water. The choice of the materials was based on their distinct electrical natures, which enabled the array to establish a fingerprint of very similar liquids. Impedance spectroscopy measurements were taken in the frequency range from 10 Hz to 1 MHz, with the data analysed with principal component analysis (PCA). The sensing units were obtained from five-layer LB films of (poly[(2-methoxy-5-n-hexyloxy)-p-phenylenevinylene]), OC(1)OC(18)-PPV (poly(2-methoxy,5-(n-octadecyl)-p-phenylenevinylene)), DR (HEMA-co-DR13MA (poly-(hydroxyethylmethacrylate-co-[4`-[[2-(methacryloyloxy)-ethyl]ethylamino]-2-chloro-4-nitroazobenzene]))) and five-bilayer LbL films of tetrasulfonated metallic phthalocyanines deposited onto gold interdigitated electrodes. The sensors were immersed into phenol, 2-chloro-4-methoxyphenol, 2-chlorophenol and 3-chlorophenol (isomers) solutions at 1 x 10(-9) mol L(-1), with control experiments carried out in ultra pure water. Samples could be distinguished if the principal component analysis (PCA) plots were made with capacitance values taken at 10(3) Hz, which is promising for detection of trace amounts of phenolic pollutants in natural water.

Biomimetic biosensor based on lipidic layers containing tyrosinase and lutetium bisphthalocyanine for the detection of antioxidants

This paper describes the preparation of a biomimetic Langmuir-Blodgett film of tyrosinase incorporated in a lipidic layer and the use of lutetium bisphthalocyanine as an electron mediator for the voltammetric detection of phenol derivatives, which include one monophenol (vanillic acid), two diphenols (catechol and caffeic acid) and two triphenols (gallic acid and pyrogallol). The first redox process of the voltammetric responses is associated with the reduction of the enzymatically formed o-quinone and is favoured by the lutetium bisphthalocyanine because significant signal amplification is observed, while the second is associated with the electrochemical oxidation of the antioxidant and occurs at lower potentials in the presence of an electron mediator. The biosensor shows low detection limit (1.98 x 10(-6)-27.49 x 10(-6) M), good reproducibility, and high affinity to antioxidants (Km in the range of 62.31-144.87 mu M). The excellent functionality of the enzyme obtained using a biomimetic immobilisation method, the selectivity afforded by enzyme catalysis, the signal enhancement caused by the lutetium bisphthalocyanine mediator and the increased selectivity of the curves due to the occurrence of two redox processes make these sensors exceptionally suitable for the detection of phenolic compounds. (C) 2010 Elsevier B.V. All rights reserved.

PK/DB: database for pharmacokinetic properties and predictive in silico ADME models

The study of pharmacokinetic properties (PK) is of great importance in drug discovery and development. In the present work, PK/DB (a new freely available database for PK) was designed with the aim of creating robust databases for pharmacokinetic studies and in silico absorption, distribution, metabolism and excretion (ADME) prediction. Comprehensive, web-based and easy to access, PK/DB manages 1203 compounds which represent 2973 pharmacokinetic measurements, including five models for in silico ADME prediction (human intestinal absorption, human oral bioavailability, plasma protein binding, bloodbrain barrier and water solubility).

Enzymatic hydrolysis of pretreated sugar cane bagasse using Penicillium funiculosum and Trichoderma harzianum cellulases

In this study, we investigated the enzymatic hydrolysis of pretreated sugarcane bagasse using eight different enzymatic blends obtained from concentrated crude enzyme extracts produced by Penicillium funiculosum and Trichoderma harzianum as well as from the extracts in combination with a commercial enzymatic cocktail. The influence of different levels of biomass delignification, degree of crystallinity of lignicellulose, composition of enzymatic activities and BSA on enzymatic hydrolysis yields (HYs) was evaluated. Our X-ray diffraction studies showed that crystallinity of lignocellulose is not a key determinant of its recalcitrance toward enzymatic hydrolysis. In fact, under the experimental conditions of our study, an increase in crystallinity of lignocellulosic samples resulted in increased glucose release by enzymatic hydrolysis. Furthermore, under the same conditions, the addition of BSA had no significant effect on enzymatic hydrolysis. The most efficient enzyme blends were obtained by mixing a commercial enzymatic cocktail with P. funiculosum or T. harzianum cellulase preparations (HYs above 97%) followed by the concentrated extract of P. funiculosum alone (HY= 88.5%). Increased hydrolytic efficiencies appeared to correlate with having an adequate level of both beta-glucosidase and xylanase activities in the blends. (C) 2011 Elsevier Ltd. All rights reserved.

Culture and exopolysaccharide production from sugarcane molasses by Gordonia polyisoprenivorans CCT 7137, isolated from contaminated groundwater in Brazil

Gordonia polyisoprenivorans CCT 7137 was isolated from groundwater contaminated with leachate in an old controlled landfill (Sauo Paulo, Brazil), and cultured in GYM medium at different concentrations of sugarcane molasses (2%, 6%, and 10%). The strain growth was analyzed by monitoring the viable cell counts (c.f.u. mL(-1)) and optical density and EPS production was evaluated at the end of the exponential phase and 24 h after it. The analysis of the viable cell counts showed that the medium that most favored bacterial growth was not the one that favored EPS production. The control medium (GYM) was the one that most favored the strain growth, at the maximum specific growth rate of 0.232 h(-1). Differences in bacterial growth when cultured at three different concentrations of molasses were not observed. Production of EPS, in all culture media used, began during the exponential phase and continued during the growth stationary phase. The highest total EPS production, after 24 h of stationary phase, was observed in 6% molasses medium (172.86 g L(-1)) and 10% (139.47 g L(-1)) and the specific total EPS production was higher in 10% molasses medium (39.03 x10(-11)g c.f.u.(-1)). After the exponential phase, in 2%, 6%, and 10% molasses media, a higher percentage of free exopolysaccharides (EPS) was observed, representing 88.4%, 62.4%, and 64.2% of the total, respectively. A different result was observed in pattern medium, which presented EPS made up of higher percentage of capsular EPS (66.4% of the total). This work is the first study on EPS production by G. polyisoprenivorans strain in GYM medium and in medium utilizing sugarcane molasses as the sole nutrient source and suggests its potential use for EPS production by G. polyisoprenivorans CCT 7137 aiming at application in biotechnological processes.

Cloning, expression, purification and characterization of recombinant glutathione-S-transferase from Xylella fastidiosa

Xylella fastidiosa is an important pathogen bacterium transmitted by xylem-feedings leafhoppers that colonizes the xylem of plants and causes diseases on several important crops including citrus variegated chlorosis (CVC) in orange and lime trees. Glutathione-S-transferases (GST) form a group of multifunctional isoenzymes that catalyzes both glutathione (GSH)-dependent conjugation and reduction reactions involved in the cellular detoxification of xenobiotic and endobiotic compounds. GSTs are the major detoxification enzymes found in the intracellular space and mainly in the cytosol from prokaryotes to mammals, and may be involved in the regulation of stress-activated signals by suppressing apoptosis signal-regulating kinase 1. In this study, we describe the cloning of the glutathione-S-transferase from X. fastidiosa into pET-28a(+) vector, its expression in Escherichia coli, purification and initial structural characterization. The purification of recombinant xfGST (rxfGST) to near homogeneity was achieved using affinity chromatography and size-exclusion chromatography (SEC). SEC demonstrated that rxfGST is a homodimer in solution. The secondary and tertiary structures of recombinant protein were analyzed by circular dichroism and fluorescence spectroscopy, respectively. The enzyme was assayed for activity and the results taken together indicated that rxfGST is a stable molecule, correctly folded, and highly active. Several members of the GST family have been extensively studied. However, xfGST is part of a less-studied subfamily which yet has not been structurally and biochemically characterized. In addition, these studies should provide a useful basis for future studies and biotechnological approaches of rxfGST. (C) 2008 Elsevier Inc. All rights reserved.

Purification, and Biochemical and Biophysical Characterization of Cellobiohydrolase I from Trichoderma harzianum IOC 3844

Because of its elevated cellulolytic activity, the filamentous fungus Trichoderma harzianum has a considerable potential in biomass hydrolysis applications. Trichoderma harzianum cellobiohydrolase I (ThCBHI), an exoglucanase, is an important enzyme in the process of cellulose degradation. Here, we report an easy single-step ion-exchange chromatographic method for purification of ThCBHI and its initial biophysical and biochemical characterization. The ThCBHI produced by induction with microcrystalline cellulose under submerged fermentation was purified on DEAE-Sephadex A-50 media and its identity was confirmed by mass spectrometry. The ThCBHI biochemical characterization showed that the protein has a molecular mass of 66 kDa and pi of 5.23. As confirmed by small-angle X-ray scattering (SAXS), both full-length ThCBHI and its catalytic core domain (CCD) obtained by digestion with papain are monomeric in solution. Secondary structure analysis of ThCBHI by circular dichroism revealed alpha-helices and beta-strands contents in the 28% and 38% range, respectively. The intrinsic fluorescence emission maximum of 337 nm was accounted for as different degrees of exposure of ThCBHI tryptophan residues to water. Moreover, ThCBHI displayed maximum activity at pH 5.0 and temperature of 50 degrees C with specific activities against Avicel and p-nitrophenyl-beta-D-cellobioside of 1.25 U/mg and 1.53 U/mg, respectively.

Immobilization of lutetium bisphthalocyanine in nanostructured biomimetic sensors using the LbL technique for phenol detection

This study describes the development of amperometric sensors based on poly(allylamine hydrochloride) (PAH) and lutetium bisphthalocyanine (LuPc(2)) films assembled using the Layer-by-Layer (LbL) technique. The films have been used as modified electrodes for catechol quantification. Electrochemical measurements have been employed to investigate the catalytic properties of the LuPc(2) immobilized in the LbL films. By chronoamperometry, the sensors present excellent sensitivity (20 nA mu M(-1)) in a wide linear range (R(2) = 0.994) up to 900 mu M and limit of detection (s/n = 3) of 37.5 x 10(-8) M for catechol. The sensors have good reproducibility and can be used at least for ten times. The work potential is +0.3 V vs. saturated calomel electrode (SCE). In voltammetry measurements, the calibration curve shows a good linearity (R(2) = 0.992) in the range of catechol up to 500 mu M with a sensitivity of 90 nA mu M(-1) and LD of 8 mu M. (C) 2011 Elsevier B.V. All rights reserved.

Establishment of the comet assay in the freshwater snail Biomphalaria glabrata (Say, 1818)

The single cell gel eletrophoresis or the comet assay was established in the freshwater snail Biomphalaria glabrata. For detecting DNA damage in circulating hemocytes, adult snails were irradiated with single doses of 2.5. 5, 10 and 20 Gy of Co-60 gamma radiation. Genotoxic effect of ionizing radiation was detected at all doses as a dose-related increase in DNA migration. Comet assay in B. glabrata demonstrated to be a simple, fast and reliable tool in the evaluation of genotoxic effects of environmental mutagens. (c) 2008 Elsevier B.V. All rights reserved.

Recovery of DNA for the detection and genotyping of human papillomavirus from clinical cervical specimens stored for up to 2 years in a universal collection medium with denaturing reagent

The recovery and stability of DNA for the detection and genotyping of HPV in UCM-containing specimens, after exposure to denaturing reagents and stored for up to 2 years were evaluated. Samples were collected from 60 women who had cervical cytology specimens harboring cervical intraepithelial neoplasia (CIN) 2 or 3. All samples were stored in UCM and had been frozen at -20 degrees C following the addition of the denaturing reagent (sodium hydroxide) and the removal of the aliquot required for Hybrid Capture 2 testing for the identification of HPV DNA. The samples had been stored for 6, 12 and 24 months (20 samples for each storage time). HPV DNA extraction was performed according to a protocol designed specifically and the presence and quality of DNA was confirmed by human P-globin detection using the consensus primers G73 and G74. HPV DNA was amplified using the consensus primers PGMY09 and PGMY11, and reverse line-blot hybridization was used to detect type-specific amplicons for 37 HPV types. The DNA extracted from the denatured specimen was recovered in 57/60 (95%) of the samples. HPV DNA was detected in 56/57 (98%) of the recovered samples. Twenty-six of the 56 samples recovered (48%) were genotyped successfully. (c) 2007 Elsevier B.V. All rights reserved.