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.

Purification of an exopolygalacturonase from Penicillium viridicatum RFC3 produced in submerged fermentation

An exo-PG obtained from Penicillium viridicatum in submerged fermentation was purified to homogeneity. The apparent molecular weight of the enzyme was 92 kDa, optimum pH and temperature for activity were pH 5 and 50-55°C. The exo-PG showed a profile of an exo-polygalacturonase, releasing galacturonic acid by hydrolysis of pectin with a high degree of esterification (D.E.). Ions Ca 2+ enhanced the stability of enzyme and its activity by 30%. The K m was 1.30 in absence of Ca 2+ and 1.16mg mL -1 in presence of this ion. In relation to the Vmax the presence of this ion increased from 1.76 to 2.07 μmol min -1mg -1. Copyright © 2009 Eleni Gomes et al.

Human interferon B1 ser17: Coding DNA synthesis, expression, purification and characterization of bioactive recombinant protein

A protocol to produce large amounts of bioactive homogeneous human interferon β1 expressed in Escherichia coli was developed. Human interferon β1 ser17 gene was constructed, cloned and subcloned, and the recombinant protein expressed in E. coli cells. Solubilization of recombinant human interferon β1 ser17 (rhIFN-β1 ser17) was accomplished by employing a brief shift to high alkaline pH in the presence of non-ionic detergent. The recombinant protein was purifi ed by three chromatographic steps. N-terminal amino acid sequencing and mass spectrometry analysis provided experimental evidence for the identity of the recombinant protein. Reverse phase liquid chromatography demonstrated that the content of deamidates and sulphoxides was similar to a commercial standard. Size exclusion chromatography demonstrated the absence of high molecular mass aggregates and dimers. The protocol represents an effi cient and high-yield method to obtain bioactive homogeneous monomeric rhIFN-β1 ser17 protein. It may thus represent an important step towards scaling up for rhIFN-β1 ser17 large-scale production. © 2010 Villela AD, et al.

Alterations in the fat body cells of rhinocricus padbergi (Diplopoda) resulting from exposure to substrate containing sewage sludge

Abstract The final disposal of residues generated at sewage treatment plants (STPs) has become a major problem for cities, due to the increase in the amount of treated sewage. One of the alternatives for the residue, labeled sewage sludge, is its reuse in agriculture and in degraded soil. However, not all pathogens and metals present in it are eliminated during treatment. Diplopods have been used as bioindicators in ecotoxicological tests as they are constantly in close contact with the soil. Owing to this fact, the purpose of this study was to expose specimens of the diplopod Rhinocricus padbergi to substrate containing sewage sludge collected at STPs to analyze morphological alterations in their parietal and perivisceral fat body, where substances are stored. The exposures were held for 7, 15, or 90 days at different concentrations of sewage sludge (control, 1%, 10%, and 50%). The parietal fat body showed no alterations in any of the three exposure periods or concentrations. Alterations in the perivisceral fat body were observed for all exposure periods. According to the results, we suggest that the sludge used has toxic agents responsible for changing the animal's perivisceral fat body. © 2012 Microscopy Society of America.

Wireless sensor networks Zigbee applied on sewage treatment station

The good efficiency in a sewage treatment plant (WWTP) is a great importance to the environment. The management of electromechanical equipment installed in these stations is a major challenge due to the fact that they are installed on areas of difficult access and maintenance unhealthy and making the time for the correction of any faults is extended. This paper proposes the development of a Wireless Sensor Network (WSN), in order to monitor electromechanical equipment, allowing the Concessionaire a predictive control in real time. The design of a wireless sensors network for monitoring equipment requires not only the development and assembly of the sensor modules, but must also include the development of software for managing the data collected. Thus, this work includes a Zigbee WSN, small, adapted for monitoring of electromechanical equipment and environmental conditions of a WWTP, type stabilization pond, installed in an area of approximately 0.15 km 2 and the average flow of 320 liters of treatment per second. The experimental results show that this monitoring system can perform with the collection of parameters of performance and quality assessment at the station.

Biomass and yield of peanut grown on tropical soil amended with sewage sludge contaminated with lead

Application of sewage sludge with high lead (Pb) contents may pollute soils and contaminate crops. The objective of this work was to evaluate peanut responses to application of sewage sludge with varying Pb contents in order to supply phosphorus (P) to the plant. A greenhouse experiment was carried out with peanut grown on soil sample from a medium-textured Haplustox. Treatments were arranged in 3 × 2 + 2 factorial scheme, replicated three times, distributed in randomized block design, and consisted of: three Pb rates applied to soil with sewage sludge (3, 21, and 42 mg kg-1) × two times of sewage sludge application (30 days before peanut sowing and at the day of the sowing) + mineral fertilization + control (without sewage sludge and mineral fertilization). Sewage sludge was efficient to supply P to peanut. Sewage sludge containing high rates of Pb, when applied, did not harm biomass and yield of the plant, but increased HCl-extractable Pb in soil and Pb content in shoot, roots, and pod husks. Increase of Pb content in pod husks may represent contamination risk of kernels and their products with fragments from husks detached during manipulation or industrial processing of peanuts. © 2012 Fábio Camilotti et al.

Purification, characterization, and specificity determination of a new serine protease secreted by penicillium waksmanii

The purpose of this work was to purify a protease from Penicillium waksmanii and to determine its biochemical characteristics and specificity. The extracellular protease isolated that was produced by P. waksmanii is a serine protease that is essential for the reproduction and growth of the fungus. The protease isolated showed 32 kDa, and has optimal activity at pH 8.0 and 35 C towards the substrate Abz-KLRSSKQ-EDDnp. The protease is active in the presence of CaCl2, KCl, and BaCl, and partially inhibited by CuCl2, CoCl2 and totally inhibited by AlCl3 and LiCl. In the presence of 1 M urea, the protease remains 50 % active. The activity of the protease increases 60 % when it is exposed to 0.4 % nonionic surfactant-Triton X-100 and loses 10 % activity in the presence of 0.4 % Tween-80. Using fluorescence resonance energy transfer analysis, the protease showed the most specificity for the peptide Abz-KIRSSKQ-EDDnp with k cat/K m of 10,666 mM-1 s-1, followed by the peptide Abz-GLRSSKQ-EDDnp with a k cat/K m of 7,500 mM -1 s-1. Basic and acidic side chain-containing amino acids performed best at subsite S1. Subsites S2, S3, S′ 2, and S′ 1, S ′ 3 showed a preference for binding for amino acids with hydrophobic and basic amino acid side chain, respectively. High values of k cat/K m were observed for the subsites S2, S3, and S′ 2. The sequence of the N-terminus (ANVVQSNVPSWGLARLSSKKTGTTDYTYD) showed high similarity to the fungi Penicillium citrinum and Penicillium chrysogenum, with 89 % of identity at the amino acid level. © 2012 Springer Science+Business Media New York.

Rational use of residential digesters for sewage treatment with Carbon Credits

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Hyaluronidase from the venom of the social wasp Polybia paulista (Hymenoptera, Vespidae): Cloning, structural modeling, purification, and immunological analysis

In this study, we describe the cDNA cloning, sequencing, and 3-D structure of the allergen hyaluronidase from Polybia paulista venom (Pp-Hyal). Using a proteomic approach, the native form of Pp-Hyal was purified to homogeneity and used to produce a Pp-specific polyclonal antibody. The results revealed that Pp-Hyal can be classified as a glycosyl hydrolase and that the full-length Pp-Hyal cDNA (1315 bp; GI: 302201582) is similar (80-90%) to hyaluronidase from the venoms of endemic Northern wasp species. The isolated mature protein is comprised of 338 amino acids, with a theoretical pI of 8.77 and a molecular mass of 39,648.8 Da versus a pI of 8.13 and 43,277.0 Da indicated by MS. The Pp-Hyal 3D-structural model revealed a central core (α/β)7 barrel, two sulfide bonds (Cys 19-308 and Cys 185-197), and three putative glycosylation sites (Asn79, Asn187, and Asn325), two of which are also found in the rVes v 2 protein. Based on the model, residues Ser299, Asp107, and Glu109 interact with the substrate and potential epitopes (five conformational and seven linear) located at surface-exposed regions of the structure. Purified native Pp-Hyal showed high similarity (97%) with hyaluronidase from Polistes annularis venom (Q9U6V9). Immunoblotting analysis confirmed the specificity of the Pp-Hyal-specific antibody as it recognized the Pp-Hyal protein in both the purified fraction and P. paulista crude venom. No reaction was observed with the venoms of Apis mellifera, Solenopsis invicta, Agelaia pallipes pallipes, and Polistes lanio lanio, with the exception of immune cross-reactivity with venoms of the genus Polybia (sericea and ignobilis). Our results demonstrate cross-reactivity only between wasp venoms from the genus Polybia. The absence of cross-reactivity between the venoms of wasps and bees observed here is important because it allows identification of the insect responsible for sensitization, or at least of the phylogenetically closest insect, in order to facilitate effective immunotherapy in allergic patients. © 2013 Elsevier Ltd.

Concentrations of Cu, Fe, Mn, and Zn in tropical soils amended with sewage sludge and composted sewage sludge

Sewage sludge may be used as an agricultural fertilizer, but the practice has been criticized because sludge may contain trace elements and pathogens. The aim of this study was to compare the effectiveness of total and pseudototal extractants of Cu, Fe, Mn, and Zn, and to compare the results with the bioavailable concentrations of these elements to maize and sugarcane in a soil that was amended with sewage sludge for 13 consecutive years and in a separate soil that was amended a single time with sewage sludge and composted sewage sludge. The 13-year amendment experiment involved 3 rates of sludge (5, 10, and 20 t ha-1). The one-time amendment experiment involved treatments reflecting 50, 100, and 200 % of values stipulated by current legislation. The metal concentrations extracted by aqua regia (AR) were more similar to those obtained by Environmental Protection Agency (EPA) 3052 than to those obtained by EPA3051, and the strongest correlation was observed between pseudo(total) concentrations extracted by AR and EPA3052 and bioavailable concentrations obtained by Mehlich III. An effect of sewage sludge amendment on the concentrations of heavy metals was only observed in samples from the 13-year experiment. © 2012 Springer Science+Business Media B.V.

Testing extractants for Cu, Fe, Mn, and Zn in tropical soils treated with sewage sludge for 13 consecutive years

In this paper, we report on a field experiment being carried out in a Typic Eutrorthox. The experiment was initiated in the 1997-98 agricultural season as a randomized block design with four treatments (0, 5, 10, and 20 t ha -1) of sewage sludge and five replicates. Compound soil samples were obtained from 20 subsamples collected at depths of 0-0.1 and 0.1-0.2 m. Cu, Fe, Mn, and Zn concentrations were extracted with DTPA pH 7.3; 0.1 mol L -1 HCl, Mehlich-I, Mehlich-III, and 0.01 mol L-1 CaCl 2. Metal concentrations were determined via atomic absorption spectrometry. Diagnostic leaves and the whole above-ground portion of plants were collected to determine Cu, Fe, Mn, and Zn concentrations extracted by nitric-perchloric digestion and later determined via atomic absorption spectrometry. Sewage sludge application caused increases in the concentrations of soil Cu, Fe, and Mn in samples taken from the 0-0.1 m depth evaluated by the extractants Mehlich-I, Mehlich-III, 0.01 mol L-1 HCl and DTPA pH 7.3. None of the extractants provided efficient estimates of changes in Mn concentrations. The acid extractants extracted more Cu, Fe, Mn, and Zn than the saline and chelating solutions. The highest concentrations of Cu, Fe, and Zn were obtained with Mehlich-III, while the highest concentrations of Mn were obtained with HCl. We did not observe a correlation between the extractants and the concentrations of elements in the diagnostic leaves nor in the tissues of the whole maize plant (Zea mays L.). © 2013 Springer Science+Business Media Dordrecht.

Production, purification, and characterization of a major penicillium glabrum xylanase using brewer's spent grain as substrate

In recent decades, xylanases have been used in many processing industries. This study describes the xylanase production by Penicillium glabrum using brewer's spent grain as substrate. Additionally, this is the first work that reports the purification and characterization of a xylanase using this agroindustrial waste. Optimal production was obtained when P. glabrum was grown in liquid medium in pH 5.5, at 25 °C, under stationary condition for six days. The xylanase from P. glabrum was purified to homogeneity by a rapid and inexpensive procedure, using ammonium sulfate fractionation and molecular exclusion chromatography. SDS-PAGE analysis revealed one band with estimated molecular mass of 18.36 kDa. The optimum activity was observed at 60 °C, in pH 3.0. The enzyme was very stable at 50 °C, and high pH stability was verified from pH 2.5 to 5.0. The ion Mn2+ and the reducing agents β-mercaptoethanol and DTT enhanced xylanase activity, while the ions Hg2+, Zn2+, and Cu2+ as well as the detergent SDS were strong inhibitors of the enzyme. The use of brewer's spent grain as substrate for xylanase production cannot only add value and decrease the amount of this waste but also reduce the xylanase production cost. © 2013 Adriana Knob et al.

Evaluation of biochemical and redox parameters in rats fed with corn grown in soil amended with urban sewage sludge

The increased production of urban sewage sludge requires alternative methods for final disposal. A very promising choice is the use of sewage sludge as a fertilizer in agriculture, since it is rich in organic matter, macro and micronutrients. However, urban sewage sludge may contain toxic substances that may cause deleterious effects on the biota, water and soil, and consequently on humans. There is a lack of studies evaluating how safe the consumption of food cultivated in soils containing urban sewage sludge is. Thus, the aim of this paper was to evaluate biochemical and redox parameters in rats fed with corn produced in a soil treated with urban sewage sludge for a long term. For these experiments, maize plants were grown in soil amended with sewage sludge (rates of 5, 10 and 20. t/ha) or not (control). Four different diets were prepared with the corn grains produced in the field experiment, and rats were fed with these diets for 1, 2, 4, 8 and 12 weeks. Biochemical parameters (glucose, total cholesterol and fractions, triglycerides, aspartate aminotransferase and alanine aminotransferase) as well the redox state biomarkers such as reduced glutathione (GSH), malondialdehyde (MDA), catalase, glutathione peroxidase and butyrylcholinesterase (BuChE) were assessed. Our results show no differences in the biomarkers over 1 or 2 weeks. However, at 4 weeks BuChE activity was inhibited in rats fed with corn grown in soil amended with sewage sludge (5, 10 and 20. t/ha), while MDA levels increased. Furthermore, prolonged exposure to corn cultivated in the highest amount per hectare of sewage sludge (8 and 12 weeks) was associated with an increase in MDA levels and a decrease in GSH levels, respectively. Our findings add new evidence of the risks of consuming food grown with urban sewage sludge. However, considering that the amount and type of toxic substances present in urban sewage sludge varies considerably among different sampling areas, further studies are needed to evaluate sludge samples collected from different sources and/or undergoing different types of treatment. © 2013 Elsevier Inc.

Purification and Characterization of an Ethanol-Tolerant β-Glucosidase from Sporidiobolus pararoseus and Its Potential for Hydrolysis of Wine Aroma Precursors

An extracellular ethanol-tolerant β-glucosidase from Sporidiobolus pararoseus was purified to homogeneity and characterized, and its potential use for the enhancement of wine aroma was investigated. The crude enzymatic extract was purified in four steps (concentration, dialysis, ultrafiltration, and chromatography) with a yield of around 40 % for total activity. The purified enzyme (designated Sp-βgl-P) showed a specific activity of approximately 20.0 U/mg, an estimated molecular mass of 63 kDa after sodium dodecyl sulfate polyacrylamide gel electrophoresis, and isoelectric point of 5.0 by isoelectric focusing. Sp-βgl-P has optimal activity at pH 4.0 and at 55 °C. It was stable in a broad pH range at low temperatures and it was tolerant to ethanol and glucose, indicating suitable properties for winemaking. The hydrolysis of glycosidic terpenes was analyzed by adding Sp-βgl-P directly to the wines. The released terpene compounds were evaluated by gas chromatography/mass spectrometry. The enzymatic treatment significantly increased the amount of free terpenes, suggesting that this enzyme could potentially be applicable in wine aroma improvement. © 2013 Springer Science+Business Media New York.