Fecal Sterols in Estuarine Sediments as Markers of Sewage Contamination in the Cubatao Area, São Paulo, Brazil

Sterol biomarkers serve as an alternative method for detecting sewage pollution. Sterols were extracted from samples of surface sediment collected in Cubato (the Vila dos Pescadores and Vila Esperan double dagger a communities) and quantified using GC-MS after Soxhlet extraction, cleanup, and derivatization. Fecal contamination was evaluated based on the concentration of coprostanol and the ratio of the selected sterols. The most abundant sterol was cholestanol, followed by coprostanol. The concentrations of coprostanol in surface sediments ranged from a minimum of 4.21 mu g g(-1) dry sediment (Vila dos Pescadores station) to a maximum of 8.32 mu g g(-1) dry sediment (Vila Esperan double dagger a station). A coprostanol concentration of about 10 mu g g(-1) was found, indicating areas of high sewage contamination. Coprostanol levels at sewage stations were higher than in other Brazilian coastal areas, which may be attributed to the fraction of the population without sanitation services.

Sewage sludge coke estimation using thermal analysis

The imposition of more stringent legislation by CETESB in the State of So Paulo (Brazil) governing the disposal and utilization of sewage sludge, coupled with the growth in its generation has prompted a drive for alternative uses of sewage sludge. One option that is especially promising, due to its potential to valorize sludge, is its conversion into carbonaceous adsorbents or coke for industrial effluents treatment. Thus, a methodology is presented to estimate the coke produced from the sludge of a sewage treatment station using thermal analysis. The used sewage sludge, which comes from aerobic treatment, was collected in the wastewater treatment station of Barueri, one of the largest of the So Paulo metropolitan area. The sludge samples were collected, dried, ground, and milled until they passed an ABNT 200 sieve. The inert ambient used during its thermal treatment produces inorganic matter and coke as residual materials. Coke formation occurs in the 200-500 A degrees C range and, between 500 and 900 A degrees C, its thermal decomposition occurs. The highest formation of coke occurs at 500 A degrees C.

Expression, purification and spectroscopic analysis of an HdrC: An iron-sulfur cluster-containing protein from Acidithiobacillus ferrooxidans

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

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 B.V. All rights reserved.

Purification, Characterization, and Preliminary X-Ray Diffraction Analysis of a Lactose-Specific Lectin from Cymbosema roseum Seeds

The unique carbohydrate-binding property of lectins makes them invaluable tools in biomedical research. Here, we report the purification, partial primary structure, carbohydrate affinity characterization, crystallization, and preliminary X-ray diffraction analysis of a lactose-specific lectin from Cymbosema roseum seeds (CRLII). Isolation and purification of CRLII was performed by a single step using a Sepharose-4B-lactose affinity chromatography column. The carbohydrate affinity characterization was carried using assays for hemagglutination activity and inhibition. CRLII showed hemagglutinating activity toward rabbit erythrocytes. O-glycoproteins from mucine mucopolysaccharides showed the most potent inhibition capacity at a minimum concentration of 1.2 A mu g mL(-1). Protein sequencing by mass spectrometry was obtained by the digestion of CRLII with trypsin, Glu-C, and AspN. CRLII partial protein sequence exhibits 46% similarity with the ConA-like alpha chain precursor. Suitable protein crystals were obtained with the hanging-drop vapor-diffusion method with 8% ethylene glycol, 0.1 M Tris-HCl pH 8.5, and 11% PEG 8,000. The monoclinic crystals belong to space group P2(1) with unit cell parameters a = 49.4, b = 89.6, and c = 100.8 A....

Partial purification and characterization of lysine-ketoglutarate reductase in normal and opaque-2 maize endosperms

Lysine-ketoglutaratc reductase catalyzes the first step of lysine catabolism in maize (Zea mays L.) endosperm. The enzyme condenses L-lysine and α-ketoglutarate into saccharopine using NADPH as cofactor. It is endosperm-specific and has a temporal pattern of activity, increasing with the onset of kernel development, reaching a peak 20 to 25 days after pollination, and thereafter decreasing as the kernel approaches maturity. The enzyme was extracted from the developing maize endosperm and partially purified by ammonium-sulfate precipitation, anion-exchange chromatography on DEAE-cellulose, and affinity chromatography on Blue-Sepharose CL-6B. The preparation obtained from affinity chromatography was enriched 275-fold and had a specific activity of 411 nanomoles per minute per milligram protein. The native and denaturated enzyme is a 140 kilodalton protein as determined by polyacrylamide gel electrophoresis. The enzyme showed specificity for its substrates and was not inhibited by either aminoethyl-cysteine or glutamate. Steady-state product-inhibition studies revealed that saccharopine was a noncompetitive inhibitor with respect to α-ketoglutarate and a competitive inhibitor with respect to lysine. This is suggestive of a rapid equilibriumordered binding mechanism with a binding order of lysine, α-ketoglutarate, NADPH. The enzyme activity was investigated in two maize inbred lines with homozygous normal and opaque-2 endosperms. The pattern of lysine-ketoglutarate reductase activity is coordinated with the rate of zein accumulation during endosperm development. A coordinated regulation of enzyme activity and zein accumulation was observed in the opaque-2 endosperm as the activity and zein levels were two to three times lower than in the normal endosperm. Enzyme extracted from L1038 normal and opaque-2 20 days after pollination was partially purified by DEAE-cellulose chromatography. Both genotypes showed a similar elution pattern with a single activity peak eluted at approximately 0.2 molar KCL. The molecular weight and physical properties of the normal and opaque-2 enzymes were essentially the same. We suggest that the Opaque-2 gene, which is a transactivator of the 22 kilodalton zein genes, may be involved in the regulation of the lysine-ketoglutarate reductase gene in maize endosperm. In addition, the decreased reductase activity caused by the opaque-2 mutation may explain, at least in part, the elevated concentration of lysine found in the opaque-2 endosperm.

Wetland projects developed in Brazil

The first studies with constructed wetlands undertaken in Brazil were the result of observations made from the Amazon flood plains. The first attempt to use this capacity to change the quality of the water, in the sense of purification performed in Brazil using constructed wetland systems, was made by Salati et al. After that, new technologies were developed in a focused attempt to increase the efficiency of the system and reduce investments. Over these 18 years, persuading the Brazilian scientific community as well as the environmental control agencies to give due attention to this kind of research has required endless efforts. Only in recent years have major institutions responsible for sewage treatment and potable water supply been concerned with this type of technology for solving real problems. These institutions are as follows: SABESP (Basic Sanitation Company of Sao Paulo State), SANEPAR (Sanitation Company of Parana State) and CESP (Electric Company of Sao Paulo State). One of the private institutions that has systematically worked in the design and projects of constructed wetlands is the Institute of Applied Ecology. This institution has enhanced and developed a water depuration system based on the purifying capacity of the soil. The wetlands with filtering soils are systems formed by overlapping layers of crushed stone, gravel and soil planted with rice. This technology has been used in sewage treatment and also in water supply systems.

Purification and characterization of two β-glucosidases from the thermophilic fungus Thermoascus aurantiacus

β-Glucosidase from the fungus Thermoascus aurantiacus grown on semi-solid fermentation medium (using ground corncob as substrate) was partially purified in 5 steps-ultrafiltration, ethanol precipitation, gel filtration and 2 anion exchange chromatography runs, and characterized. After the first anion exchange chromatography, β-glucosidase activity was eluted in 3 peaks (Gl-1, Gl-2, Gl-3). Only the Gl-2 and Gl-3 fractions were adsorbed on the gel matrix. Gl-2 and Gl-3 exhibited optimum pH at 4.5 and 4.0, respectively. The temperature optimum of both glucosidases was at 75-80°C. The pH stability of Gl-2 (4.0-9.0) was higher than Gl-3 (5.5-8.5); both enzyme activities showed similar patterns of thermostability. Under conditions of denaturing gel chromatography the molar mass of Gl-2 and Gl-3 was 175 and 157 kDa, respectively. Using 4-nitrophenyl β-D-glucopyranoside as substrate, Km values of 1.17 ± 0.35 and 1.38 ± 0.86 mmol/L were determined for Gl-2 and Gl-3, respectively. Both enzymes were inhibited by Ag+ and stimulated by Ca2+.

Cloning, overexpression, and purification of functional human purine nucleoside phosphorylase

Purine nucleoside phosphorylase (PNP) catalyzes the phosphorolysis of the N-ribosidic bonds of purine nucleosides and deoxynucleosides. A genetic deficiency due to mutations in the gene encoding for human PNP causes T-cell deficiency as the major physiological defect. Inappropriate activation of T-cells has been implicated in several clinically relevant human conditions such as transplant tissue rejection, psoriasis, rheumatoid arthritis, lupus, and T-cell lymphomas. Human PNP is therefore a target for inhibitor development aiming at T-cell immune response modulation. In addition, bacterial PNP has been used as reactant in a fast and sensitive spectrophotometric method that allows both quantitation of inorganic phosphate (Pi) and continuous assay of reactions that generate P i such as those catalyzed by ATPases and GTPases. Human PNP may therefore be an important biotechnological tool for P i detection. However, low expression of human PNP in bacterial hosts, protein purification protocols involving many steps, and low protein yields represent technical obstacles to be overcome if human PNP is to be used in either high-throughput drug screening or as a reagent in an affordable P i detection method. Here, we describe PCR amplification of human PNP from a liver cDNA library, cloning, expression in Escherichia coli host, purification, and activity measurement of homogeneous enzyme. Human PNP represented approximately 42% of total soluble cell proteins with no induction being necessary to express the target protein. Enzyme activity measurements demonstrated a 707-fold increase in specific activity of cloned human PNP as compared to control. Purification of cloned human PNP was achieved by a two-step purification protocol, yielding 48 mg homogeneous enzyme from 1 L cell culture, with a specific activity value of 80 U mg -1. © 2002 Elsevier Science (USA). All rights reserved.

One-step purification of 5-enolpyruvylshikimate-3-phosphate synthase enzyme from Mycobacterium tuberculosis

Currently, there are 8 million new cases and 2 million deaths annually from tuberculosis, and it is expected that a total of 225 million new cases and 79 million deaths will occur between 1998 and 2030. The reemergence of tuberculosis as a public health threat, the high susceptibility of HIV-infected persons, and the proliferation of multi-drug-resistant strains have created a need to develop new antimycobacterial agents. The existence of homologues to the shikimate pathway enzymes has been predicted by the determination of the genome sequence of Mycobacterium tuberculosis. We have previously reported the cloning and overexpression of M. tuberculosis aro A-encoded EPSP synthase in both soluble and active forms, without IPTG induction. Here, we describe the purification of M. tuberculosis EPSP synthase (mtEPSPS) expressed in Escherichia coli BL21(DE3) host cells. Purification of mtEPSPS was achieved by a one-step purification protocol using an anion exchange column. The activity of the homogeneous enzyme was measured by a coupled assay using purified shikimate kinase and purine nucleoside phosphorylase proteins. A total of 53 mg of homogeneous enzyme could be obtained from 1 L of LB cell culture, with a specific activity value of approximately 18 U mg-1. The results presented here provide protein in quantities necessary for structural and kinetic studies, which are currently underway in our laboratory. © 2002 Elsevier Science (USA). All rights reserved.

Assessment of anaerobic sewage sludge quality for agricultural application after metal bioleaching

The effects of metal bioleaching on nutrient solubilization, especially nitrogen and phosphorous, from anaerobically-digested sewage sludge were investigated in this work. The assessment of the sanitary quality of the anaerobic sludge after bioleaching was also carried out by enumerating indicator (total coliforms, fecal coliforms, and fecal streptococci) and total heterotrophic bacteria. The experiments of bioleaching were performed using indigenous sulphur-oxidizing bacteria (Thiobacillus spp.) as inoculum and samples of anaerobically-digested sludge. Nitrogen and phosphorous solubilization from sewage sludge was assessed by measuring, respectively, the concentration of Total Kjeldahl Nitrogen, ammonia, nitrate/nitrite, and soluble and total phosphorous before and after the bioleaching assays. At the end of the experiment, after 4 days of incubation (final pH of 1.4), the following metal solubilization yields were obtained: zinc, 91%; nickel, 87%; copper, 79%; lead, 52%; and chromium, 42%. As a result of sludge acidification, the viable counts of selected indicator bacteria were decreased to below the detection limit (4 × 103 cfu 100 ml-1), followed by an increase in the mineral fraction of nitrogen (from 6 to 10%) and in the soluble fraction of phosphorous (from 15 to 30%). Although some loss of sludge nutrients can occur during solid-liquid separation following bioleaching, its beneficial effects as metal removal and reduction of pathogenic bacteria are sufficient to consider the potential of this treatment before sludge disposal onto agricultural fields.

Purification and characterization of xylanases from Aspergillus giganteus

A strain of Aspergillus giganteus cultivated in a medium with xylan produced two xylanases (xylanase I and II) which were purified to homogeneity. Their molar mass, estimated by SDS-PAGE, were 21 and 24 kDa, respectively. Both enzymes are glycoproteins with 50°C temperature optimum; optimum pH was 6.0-6.5 for xylanase I and 6.0 for xylanase II. At 50°C xylanase I exhibited higher thermostability than xylanase II. Hg2+, Cu 2+ and SDS were strong inhibitors, 1,4-dithiothreitol stimulated the reaction of both enzymes. Both xylanases are xylan-specific; kinetic parameters indicated higher efficiency in the hydrolysis of oat spelts xylan. In hydrolysis of this substrate, xylotriose, xylotetraose and larger xylooligosaccharides were released and hence the enzymes were classified as endoxylanases.

DAHP synthase from Mycobacterium tuberculosis H37Rv: Cloning, expression, and purification of functional enzyme

Tuberculosis (TB), caused by Mycobacterium tuberculosis, remains the leading cause of mortality due to a bacterial pathogen. According to the 2004 Global TB Control Report of the World Health Organization, there are 300,000 new cases per year of multi-drug resistant strains (MDR-TB), defined as resistant to isoniazid and rifampicin, and 79% of MDR-TB cases are now super strains, resistant to at least three of the four main drugs used to treat TB. Thus there is a need for the development of effective new agents to treat TB. The shikimate pathway is an attractive target for the development of antimycobacterial agents because it has been shown to be essential for the viability of M. tuberculosis, but absent from mammals. The M. tuberculosis aroG-encoded 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (mtDAHPS) catalyzes the first committed step in this pathway. Here we describe the PCR amplification, cloning, and sequencing of aroG structural gene from M. tuberculosis H37Rv. The expression of recombinant mtDAHPS protein in the soluble form was obtained in Escherichia coli Rosetta-gami (DE3) host cells without IPTG induction. An approximately threefold purification protocol yielded homogeneous enzyme with a specific activity value of 0.47 U mg-1 under the experimental conditions used. Gel filtration chromatography results demonstrate that recombinant mtDAHPS is a pentamer in solution. The availability of homogeneous mtDAHPS will allow structural and kinetics studies to be performed aiming at antitubercular agents development. © 2004 Elsevier Inc. All rights reserved.

Purification and biochemical characterization of two xylanases produced by Aspergillus caespitosus and their potential for kraft pulp bleaching

Two extracellular xylanases produced by the thermotolerant fungus Aspergillus caespitosus grown in sugar cane bagasse were purified and characterized. Estimated molecular masses were 26.3 and 27 kDa (xyl I); 7.7 and 17.7 kDa (xyl II) for gel filtration and SDS-PAGE, respectively. Optimal temperature for both xylanases was 50-55°C. Optimal pH was 6.5-7.0 for xyl I, and 5.5-6.5 for xyl II. The thermostability (T half) at 55°C was 27.3 min (xyl I) and >90 min (xyl II). Xylanase activity was inhibited by several ions. β-mercaptoethanol activated 59 and 102% xyl I and xyl II activities, respectively. These enzymes preferentially hydrolyzed birchwood xylan, and the K m and V max values were 2.5 mg/ml and 1679 U/mg protein (xyl I), and 3.9 mg/ml and 113 U/mg protein (xyl II). The action of both xylanases mainly that of xyl II, on kraft pulp reduced kappa number and increased pulp viscosity. © 2004 Elsevier Ltd. All rights reserved.

Nickel in a tropical soil treated with sewage sludge and cropped with maize in a long-term field study

Sewage sludge produced by the SABESP wastewater treatment plant (Companhia de Saneamento Básico do Estado de São Paulo), located in Barueri, SP, Brazil, may contain high contents of nickel (Ni), increasing the risk of application to agricultural soils. An experiment was carried out under field conditions in Jaboticabal, SP, Brazil, with the objective of evaluating the effects on soil properties and on maize plants of increasing rates of a sewage sludge rich in Ni that had been applied for 6 consecutive years. The experiment was located on a Typic Haplorthox soil, using an experimental design of randomized blocks with four treatments (rates of sewage sludge) and five replications. At the end of the experiment the accumulated amounts of sewage sludge applied were 0.0, 30.0, 60.0 and 67.5 t ha-1. Maize (Zea mays L.) was the test plant. Soil samples were collected 60 d after sowing at depths of 0-20 cm for Ni studies and from 0 to 10 cm and from 10 to 20 cm for urease studies. Sewage sludge did not cause toxicity or micronutrient deficiencies to maize plants and increased grain production. Soil Ni appeared to be associated with the most stable fractions of the soil organic matter and was protected against strong extracting solutions such as concentrated and hot HNO3 and HCl. Ni added to the soil by sewage sludge increased the metal concentration in the shoots, but not in the grain. The Mehlich 3 extractor was not efficient to evaluate Ni phytoavailability to maize plants. Soil urease activity was increased by sewage sludge only in the layer where the residue was applied. © 2006 Elsevier Ltd. All rights reserved.