Síntese do Poli (ácido láctico) por policondensação direta utilizando um planejamento fatorial

The environmental impact caused by the disposal of non-biodegradable polymer packaging on the environment, as well as the high price and scarcity of oil, caused increase of searches in the area of biodegradable polymers from renewable resources were developed. The poly (lactic acid) (PLA) is a promising polymer in the market, with a large availability of raw material for the production of its monomer, as well as good processability. The aimed of this study was synthesis PLA by direct polycondesation of lactic acid, using the tool of experimental design (DOE) (central composite rotatable design (CCRD)) to optimize the conditions of synthesis. The polymer obtained was characterized by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), viscosimetric analysis, differential scanning calorimeter (DSC) and size exclusion chromatography (SEC). The results confirmed the formation of a poly (lactic acid) semicrystalline in the syntheses performed. Through the central composite rotatable design was possible to optimize the crystallization temperature (Tc) and crystallinity degree (Xc). The crystallization temperature maximum was found for percentage of catalyst around the central point (0,3 (%W)) and values of time ranging from the central point (6h) to the upper level (+1) (8h). The crystallization temperature maximum was found for the total synthesis time of 4h (-1) and percentage of catalyst 0,1(W%) (-1). The results of size exclusion chromatography (SEC) showed higher molecular weights to 0,3 (W%) percent of catalyst and total time synthesis of 3,2h

Migration of Tinuvin P, a UV stabilizer, from PET bottles into fatty-food simulants

Tinuvin P migration from Polyethyleneterephthalate (PET) bottles was investigated using several fatty-food simulants such as olive oil, soybean oil, n-heptane and iso-octane, at exposure conditions of 2-10 days at 40 degrees C (total immersion). The stability of several UV stabilizers (BHT, Cyasorb UV 5411, Tinuvin P, Tinuvin 326 and Tinuvin 327) in n-heptane and iso-octane was also studied. After 10 days at 40 degrees C, losses of 6% and 20% in iso-octane and n-heptane respectively, were verified for Tinuvin P. Other UV stabilizers at the same experimental conditions showed higher losses (up to 30% for Tinuvin 327). These results confirm that, when carrying out specific migration studies, the stability of the substance of interest should be established in the food simulant to avoid underestimating the real migration behaviour. In order to quantify UV stabilizer migration, n-heptane and iso-octane solutions were concentrated and directly analysed by SIM mode GC-MS. For olive and soybean oils, Tinuvin P was isolated using size-exclusion chromatography and quantified by SIM mode GC-MS. Iso-octane proved to be a move suitable fatty-food simulant than n-heptane for the migration study of Tinuvin P from PET. Higher levels of Tinuvin P migrated to olive and soybean oils rather than to n-heptane. These results suggest that the MERCOSUL recommended official methods for specific migration studies should be revised since the migration levels using n-heptane as a fatty-food simulant could be underestimated when compared to edible oils. Copyright (C) 1999 John Wiley & Sons, Ltd.

Hidrólise e caracterização de poliacrilamida hidrofobicamente modificada: avaliação da aplicabilidade na recuperação de petróleo

The hydrolysis reaction in alkaline conditions of the commercial polymer poly(acrylamide-co-metacrylate of 3,5,5-trimethyl-hexane) called HAPAM, containing 0.75 % of hydrophobic groups, was carried out in 0.1 M NaCl and 0.25M NaOH solutions, varying the temperature and reaction time. The polymers were characterized by 1H and 13C Nuclear Magnetic Resonance (NMR), Elemental Analysis and Size Exclusion Chromatography (SEC). The values of the hydrolysis degree were obtained by 13C NMR. The viscosity of HAPAM and HAPAM-10N-R solutions was evaluated as a function of shear rate, ionic strength and temperature. At high polymer concentration (Cp), the viscosity of HAPAM solutions increased with the ionic strength and decreased with the temperature. The viscosity of HAPAM-10N-R solutions increased significantly in distilled water, due to repulsions between the carboxylate groups. At high Cp, with the increase of ionic strength and temperature, occurred a decrease of viscosity, due to mainly the high hydrolysis degree and the low amount of hydrophobic groups. These results indicated that the studied polymers have properties more suitable for the application in Enhanced Oil Recovery (EOR) in low salinity and moderate temperature reservoirs

Structural and functional characteristics of selected soft wheat starches

Starches from eight soft wheat samples (two parent lines and six offspring) were isolated; relationships between their structures and properties were examined. Branch chain-length distributions of amylopectins were determined by using high-performance anion exchange chromatography equipped with an amyloglucosidase reactor and a pulsed amperometric detector (HPAEC-ENZ-PAD). Results showed that the average chain length of the eight samples varied at DP 25.6-26.9. Starch samples of lines 02, 60, 63, 95, and 114 consisted of amylopectins with more long chains (DP greater than or equal to 37) and longer average chain length (DP 26.2-26.9) than that of other samples. These starch samples of longer branch chain length displayed higher gelatinization temperatures (55.3-56.5degreesC) than that of other samples (54.4-54.9degreesC) and higher peak viscosity (110-131 RVU) and lower pasting temperature (86.3-87.6degreesC) than others (83-100 RVU and 88.2-88.9degreesC, respectively). The M-w of amylopectins, determined by using high-performance size exclusion chromatography equipped with multiangle laser-light scattering and refractive index detectors (HPSEC-MALLS-RI), were similar for all samples (6.17 x 10(8) to 6.97 x 10(8)). There were no significant differences in amylose and phosphorus contents between samples. These results indicated that physical properties of wheat starch were affected by the branch-chain length of amylopectin.

Refolding and purification of bothropstoxin-1, a Lys49 - Phospholipase A(2) homologue, expressed as inclusion bodies in Escherichia coli

Hydrolysis of phospholipids by Group II phospholipase A(2) enzymes involves a nucleophilic attack on the sn-2 ester bond by the His48 residue and stabilization of the reaction intermediate by a Ca2+ ion cofactor bound to the Asp49 residue in the protein active site region, Bothropstoxin-I (BthTX-I) is a PLA, variant present in the venom of the snake Bothrops jararacussu which shows a Asp49 to Lys substitution and which lacks hydrolytic activity yet damages artificial membranes by a noncatalytic Ca2+-independent mechanism. In order to better characterize this unusual mechanism of membrane damage, we have established an expression system for BthTX-I in Escherichia coli. The DNA-coding sequence for BthTX-I was subcloned into the vector pET11-d, and the BthTX-I was expressed as inclusion bodies in E, coli BL21(DE3). The native BthTX-I contains seven disulfide bonds, and a straightforward protocol has been developed to refold the recombinant protein at high protein concentration in the presence of surfactants using a size-exclusion chromatography matrix. After refolding, recovery yields of 2.5% (corresponding to 4-5 mg of refolded recombinant BthTX-I per liter of bacterial culture) were routinely obtained. After refolding, identical fluorescent and circular dichroism spectra were obtained for the recombinant BthTX-I compared to those of the native protein. Furthermore, the native and refolded recombinant protein demonstrated identical membrane-damaging properties as evaluated by measuring the release of an entrapped fluorescent marker from liposomes, (C) 2001 Academic Press.

A stability transition at mildly acidic pH in the alpha-hemolysin (alpha-toxin) from Staphylococcus aureus

The effects of mildly acidic conditions on the free energy of unfolding (Delta G(u)(buff)) of the pore-forming alpha-hemolysin (alpha HL) from Staphylococcus aureus were assessed between pH 5.0 and 7.5 by measuring intrinsic tryptophan fluorescence, circular dichroism and elution time in size exclusion chromatography during urea denaturation, Decreasing the pH from 7.0 to 5.0 reduced the calculated Delta G(u)(buff) from 8.9 to 4.2 kcal moI(-1), which correlates with an increased rate of pore formation previously observed over the same pH range, It is proposed that the lowered surface pH of biological membranes reduces the stability of alpha HL thereby modulating the rate of pore formation. (C) 1999 Federation of European Biochemical Societies.

Crystal Structures of Xanthomonas Small Heat Shock Protein Provide a Structural Basis for an Active Molecular Chaperone Oligomer

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

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.

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.

UMP kinase from Mycobacterium tuberculosis: Mode of action and allosteric interactions, and their likely role in pyrimidine metabolism regulation

The pyrH-encoded uridine 5′-monophosphate kinase (UMPK) is involved in both de novo and salvage synthesis of DNA and RNA precursors. Here we describe Mycobacterium tuberculosis UMPK (MtUMPK) cloning and expression in Escherichia coli. N-terminal amino acid sequencing and electrospray ionization mass spectrometry analyses confirmed the identity of homogeneous MtUMPK. MtUMPK catalyzed the phosphorylation of UMP to UDP, using ATP-Mg 2+ as phosphate donor. Size exclusion chromatography showed that the protein is a homotetramer. Kinetic studies revealed that MtUMPK exhibits cooperative kinetics towards ATP and undergoes allosteric regulation. GTP and UTP are, respectively, positive and negative effectors, maintaining the balance of purine versus pyrimidine synthesis. Initial velocity studies and substrate(s) binding measured by isothermal titration calorimetry suggested that catalysis proceeds by a sequential ordered mechanism, in which ATP binds first followed by UMP binding, and release of products is random. As MtUMPK does not resemble its eukaryotic counterparts, specific inhibitors could be designed to be tested as antitubercular agents. © 2010 Elsevier Inc. All rights reserved.

Further characterization of the subunits of the giant extracellular hemoglobin of Glossoscolex paulistus (HbGp) by SDS-PAGE electrophoresis and MALDI-TOF-MS

Further characterization of hemoglobin of Glossoscolex paulistus (HbGp) subunits was performed based on SDS-PAGE, size exclusion chromatography (SEC) and MALDI-TOF-MS analysis. SDS-PAGE has shown a total of four linker chains, two quite intense and two of lower intensity. HbGp fractions (I-VI), obtained by size exclusion chromatography (SEC), from oligomeric dissociation at alkaline pH 9.6, were monitored. Fraction I is identical to the whole protein. The monomeric chains c, obtained from the trimer abc reduction, present four isoforms with MM 17,336 Da, 17,414 Da, 17,546 Da and 17,620 Da. Furthermore, the trimer subunit presents two isoforms, T 1 and T 2, with MM 51,200 ± 60 and 51,985 ± 50 Da, respectively. Based on SDS-PAGE, the linker chains seem to be distributed along the different fractions of the SEC chromatogram, appearing along the peaks corresponding to fractions I-V. The fraction IV contains, predominantly, trimers with some linkers contamination. The strong interaction of linker chains L with the trimers abc, makes it difficult to obtain these subunits in pure form. The monomer d in fraction VI appears to be quite pure, in agreement with previous studies. © 2011 Elsevier Ltd. All rights reserved.

EIF5A dimerizes not only in vitro but also in vivo and its molecular envelope is similar to the EF-P monomer

The protein eukaryotic initiation factor 5A (eIF5A) is highly conserved among archaea and eukaryotes, but not in bacteria. Bacteria have the elongation factor P (EF-P), which is structurally and functionally related to eIF5A. eIF5A is essential for cell viability and the only protein known to contain the amino acid residue hypusine, formed by post-translational modification of a specific lysine residue. Although eIF5A was initially identified as a translation initiation factor, recent studies strongly support a function for eIF5A in the elongation step of translation. However, the mode of action of eIF5A is still unknown. Here, we analyzed the oligomeric state of yeast eIF5A. First, by using size-exclusion chromatography, we showed that this protein exists as a dimer in vitro, independent of the hypusine residue or electrostatic interactions. Protein-protein interaction assays demonstrated that eIF5A can form oligomers in vitro and in vivo, in an RNA-dependent manner, but independent of the hypusine residue or the ribosome. Finally, small-angle X-ray scattering (SAXS) experiments confirmed that eIF5A behaves as a stable dimer in solution. Moreover, the molecular envelope determined from the SAXS data shows that the eIF5A dimer is L-shaped and superimposable on the tRNAPhe tertiary structure, analogously to the EF-P monomer. © 2012 Springer-Verlag.

Biophysical characterization of the recombinant importin-α from neurospora crassa

Neurospora crassa has been widely used as a model organism and contributed to the development of biochemistry and molecular biology by allowing the identification of many metabolic pathways and mechanisms responsible for gene regulation. Nuclear proteins are synthesized in the cytoplasm and need to be translocated to the nucleus to exert their functions which the importin-α receptor has a key role for the classical nuclear import pathway. In an attempt to get structural information of the nuclear transport process in N. crassa, we present herein the cloning, expression, purification and structural studies with N-terminally truncated IMPα from N. crassa (IMPα-Nc). Circular dichroism analysis revealed that the IMPα-Nc obtained is correctly folded and presents a high structural conservation compared to other importins-α. Dynamic light scattering, analytical size-exclusion chromatography experiments and molecular dynamics simulations indicated that the IMPα-Nc unbound to any ligand may present low stability in solution. The IMPα-Nc theoretical model displayed high similarity of its inner concave surface, which binds the cargo proteins containing the nuclear localization sequences, among IMPα from different species. However, the presence of non-conserved amino acids relatively close to the NLS binding region may influence the binding specificity of IMPα-Nc to cargo proteins. Copyright © 2012 Bentham Science Publishers. All Rights Reserved.

Structural and Phylogenetic Studies with MjTX-I Reveal a Multi-Oligomeric Toxin - a Novel Feature in Lys49-PLA2s Protein Class

The mortality caused by snakebites is more damaging than many tropical diseases, such as dengue haemorrhagic fever, cholera, leishmaniasis, schistosomiasis and Chagas disease. For this reason, snakebite envenoming adversely affects health services of tropical and subtropical countries and is recognized as a neglected disease by the World Health Organization. One of the main components of snake venoms is the Lys49-phospholipases A2, which is catalytically inactive but possesses other toxic and pharmacological activities. Preliminary studies with MjTX-I from Bothrops moojeni snake venom revealed intriguing new structural and functional characteristics compared to other bothropic Lys49-PLA2s. We present in this article a comprehensive study with MjTX-I using several techniques, including crystallography, small angle X-ray scattering, analytical size-exclusion chromatography, dynamic light scattering, myographic studies, bioinformatics and molecular phylogenetic analyses.Based in all these experiments we demonstrated that MjTX-I is probably a unique Lys49-PLA2, which may adopt different oligomeric forms depending on the physical-chemical environment. Furthermore, we showed that its myotoxic activity is dramatically low compared to other Lys49-PLA2s, probably due to the novel oligomeric conformations and important mutations in the C-terminal region of the protein. The phylogenetic analysis also showed that this toxin is clearly distinct from other bothropic Lys49-PLA2s, in conformity with the peculiar oligomeric characteristics of MjTX-I and possible emergence of new functionalities inresponse to environmental changes and adaptation to new preys. © 2013 Salvador et al.