Extraction of Ascorbate Oxidase from Cucurbita maxima by Continuous Process in Perforated Rotating Disc Contactor Using Aqueous Two-Phase Systems

The ascorbate oxidase is the enzyme used to determine the content of ascorbic acid in the pharmaceutical and food industries and clinics analyses. The techniques currently used for the purification of this enzyme raise its production cost. Thus, the development of alternative processes and with the potential to reduce costs is interesting. The application of aqueous two-phase system is proposed as an alternative to purification because it enables good separation of biomolecules. The objective of this study was to determine the conditions to continuously pre-purify the enzyme ascorbate oxidase by an aqueous two-phase system (PEG/citrate) using rotating column provided with perforated discs. Under the best conditions (20,000 g/mol PEG molar mass, 10% PEG concentration, and 25% citrate concentration), the system showed satisfactory results (partition coefficient, 3.35; separation efficiency, 54.98%; and purification factor, 1.46) and proved suitable for the pre-purification of ascorbate oxidase in continuous process.

Liquid-liquid extraction by mixed micellar systems: A new approach for clavulanic acid recovery from fermented broth

This work is the first attempt to apply aqueous two-phase mixed micellar systems (ATPMS) of the nonionic surfactant Triton X-114 and the anionic one AOT to extract clavulanic acid (CA) from broth fermented by Streptomyces clavuligerus. Cloud points were determined in McIlvane buffer pH 6.5 with or without NaCl, and diagram phases/coexistence curves were constructed. CA partition was investigated following a 2(4)-full factorial design in which AOT (0.022, 0.033 and 0.044% w/w), Triton X-114 (1.0, 3.0 and 5.0% w/w) and NaCl (0, 2.85 and 5.70% w/w) concentrations and temperature (24,26 and 28 degrees C) were selected as independent variables, and CA partition coefficient (K(CA)) and yield in the top phase (eta(CA)) as responses. CA partitioned always to the top, micelle-poor phase. The regression analysis pointed out that NaCl concentration and interaction between temperature and Triton X-114 concentration had statistically significant effects on K(CA), while eta(CA) was mainly influenced by temperature, Triton X-114 concentration and their interaction. Different ATPMS compositions were then needed to maximize these responses, specifically 0.022% (w/w) AOT, 5% (w/w) Triton X-114 for K(CA) (2.08), and 0.044% (w/w) AOT, 1% (w/w) Triton X-114 for eta(CA) (98.7%), both at 24 degrees C without NaCl. Since at 0.022% (w/w) AOT, 1% (w/w) Triton X-114 and 28 degrees C without NaCl the system was able to ensure satisfactory intermediate results (K(CA) = 1.48; eta(CA) = 86.3%), these conditions were selected as the best ones. These preliminary results are of concern for possible industrial application, because CA partition to the dilute phase can simplify the subsequent purification protocol. (C) 2011 Elsevier B.V. All rights reserved.

Continuous extraction of alpha-toxin from a fermented broth of Clostridium perfringens Type A in perforated rotating disc contactor using aqueous two-phase PEG-phosphate system

A 2(3-1) factorial experimental design was used to evaluate the performance of a perforated rotating disc contactor to extract alpha-toxin from the fermented broth of Clostridium perfringens Type A by aqueous two-phase system of polyethylene glycol-phosphate salts. The influence of three independent variables, specifically the dispersed phase flowrate, the continuous phase flowrate and the disc rotational speed, was investigated on the hold up, the mass transfer coefficient, the separation efficiency and the purification factor, taken as the response variables. The optimum dispersed phase flowrate was 3.0 mL/min for all these responses. Besides, maximum values of hold up (0.80), separation efficiency (0. 10) and purification factor (2.4) were obtained at this flowrate using the lowest disc rotational speed (35 rpm), while the optimum mass transfer coefficient (0. 165 h(-1)) was achieved at the highest agitation level (140 rpm). The results of this study demonstrated that the dispersed phase flowrate strongly influenced the performance of PRDC, in that both the mass transfer coefficient and hold up increased with this parameter. (c) 2007 Elsevier B. V. All rights reserved.

Separation and partitioning of Green Fluorescent Protein from Escherichia coli homogenate in poly(ethylene glycol)/sodium-poly(acrylate) aqueous two-phase systems

The partitioning of Green Fluorescent Protein (GFP) in poly(ethylene glycol)/Na-poly(acrylate) aqueous two-phase systems (PEG/NaPA-ATPS) has been investigated. The aqueous two-phase systems are formed by mixing the polymers with a salt and a protein solution. The protein partitioning in the two-phase system was investigated at 25 degrees C. The concentration of the GFP was measured by fluorimetry. It was found that the partitioning of GFP depends on the salt type, pH and concentration of PEG. The data indicates that GFP partitions more strongly to the PEG phase in presence of Na2SO4 relative to NaCl. Furthermore, the GFP partitions more to the PEG phase at higher pH. The partition to the PEG phase is strongly favoured in systems with larger tie-line lengths (i.e. systems with higher polymer concentrations). The molecular weight of PEG is important since the partition coefficient (K) of GFP gradually decreases with increasing PEG size, from K ca. 300-400 for PEG 400 to K equal to 1.19 for PEG 8000. A separation process was developed where GFP was separated from a homogenate in two extraction steps: the GFP is first partitioned to the PEG phase in a PEG 3000/NaPA 8000 system containing 3 wt% Na2SO4, where the K value of GFP was 8. The GFP is then re-extracted to a salt phase formed by mixing the previous top-phase with a Na2SO4 solution. The K-value of GFP in this back-extraction was 0.22. The total recovery based on the start material was 74%. (c) 2008 Elsevier B.V. All rights reserved.

Liquid-liquid extraction of proteases from fermented broth by PEG/citrate aqueous two-phase system

This work deals with the use of an aqueous two-phase system (ATPS) of PEG/citrate to remove proteases from a Clostridium perfringens fermentation broth. To plan the experimental tests and evaluate the corresponding results, three successive experimental designs were employed, for which the PEG molar mass (M-PEG) and concentration (C-PEG), the citrate concentration (C-C) and the pH were selected as independent variables, while the purification factor (PF), the partition coefficient (K), the activity yield (Y) and the selectivity (S) were selected as responses. PF of proteases in the top phase was shown to increase with increasing MPEG and decreasing Cc, whereas a completely opposite trend was observed for K. On the other hand, Y was favored by simultaneous decreases in both these variables, while S decreased with increasing Cc. Therefore, selecting a simultaneous increase in PF and Y as the most desirable result, the best performance of the system was obtained using M-PEG = 10-000 g/mol C-PEG = 22% (w/w) and C-c = 8.0% (w/w) at pH 8.5. Under these conditions, the activity yield was very high (131 %) but the purification factor (4.2) and the selectivity (4.3) were lower than those ensured by more selective purification methods. According to these results, the ATPS seems to be an interesting alternative primary concentration/decontamination step for vaccine preparation from C. perfringens fermented broth. (C) 2007 Elsevier B.V. All rights reserved.

Purification of alpha-toxin from Clostridium perfringens type A in PEG-phosphate aqueous two-phase systems: a factorial study

BACKGROUND: Purification of a-toxin produced by Clostridium perfringens type A in aqueous two-phase systems (ATPS) was studied with a full two-level factorial design on two factors (concentrations of 8000 g mol(-1) PEG and phosphate salt at pH 8.0), to estimate the influence of these factors on the purification results. RESULTS: The partition coefficient (K), purification factor (PF) and activity yield (Y) were strongly influenced by the PEG and phosphate concentrations. Raising the levels of the two factors increased these responses. The highest purification factor (5.7) was obtained with PEG and phosphate concentrations of 17.5% and 15%, respectively. CONCLUSION: These results support the proposal that polymer excluded volume and hydrophobic interactions are the factors that drive the alpha-toxin in PEG/phosphate aqueous two-phase systems. (c) 2008 Society of Chemical Industry

Topliss method in the optimization of salicylic acid derivatives as potential antimycobacterial agents

The Topliss method was used to guide a synthetic path in support of drug discovery efforts toward the identification of potent antimycobacterial agents. Salicylic acid and its derivatives, p-chloro, p-methoxy, and m-chlorosalicylic acid, exemplify a series of synthetic compounds whose minimum inhibitory concentrations for a strain of Mycobacterium were determined and compared to those of the reference drug, p-aminosalicylic acid. Several physicochemical descriptors (including Hammett`s sigma constant, ionization constant, dipole moment, Hansch constant, calculated partition coefficient, Sterimol-L and -B-4 and molecular volume) were considered to elucidate structure-activity relationships. Molecular electrostatic potential and molecular dipole moment maps were also calculated using the AM1 semi-empirical method. Among the new derivatives, m-chlorosalicylic acid showed the lowest minimum inhibitory concentration. The overall results suggest that both physicochemical properties and electronic features may influence the biological activity of this series of antimycobacterial agents and thus should be considered in designing new p-aminosalicylic acid analogs.

Quantitative Determination of Amlodipine Besylate in Tablets by High Performance Liquid Chromatography and UV Spectrophotometry

The purpose of this study was to develop and validate analytical methods for determination of amlodipine besylate in tablets. Simple, accurate and precise liquid chromatographic and spectrophotometric methods are proposed. For the chromatographic method, the conditions were: a LiChrospher (R) 100 RP-18 Merck (R) (125 mm x 4.6 mm, 5 mu m) column; methanol/water containing 1 % of trietylamine adjusted to pH 5.0 with phosphoric acid (35:65) as mobile phase; a flow rate of 1.0 mL/min and UV detector at 238 nm. Linearity was in the range of 50.0 - 350.0 mu g/mL with a correlation coefficient (r) = 0.9999. For the spectrophotometric method, the first dilutions of samples were performed in methanol and the consecutives in ultrapure water. The quantitation was made at 364.4 nm. Linearity was determined within the range of 41.0 - 61.0 mu g/mL with a correlation coefficient (r) = 0.9996. Our results demonstrate that both methods can be used in routine analysis for quality control of tablets containing amlodipine besylate.

Development and Validation of a Simple Method for Routine Analysis of Ractopamine Hydrochloride in Raw Material and Feed Additives by HPLC

A simple method was optimized and validated for determination of ractopamine hydrochloride (RAC) in raw material and feed additives by HPLC for use in quality control in veterinary industries. The best-optimized conditions were a C8 column (250 x 4.6 mm id, 5.0 mu m particle size) at room temperature with acetonitrile-100 mM sodium acetate buffer (pH 5.0; 75 + 25, v/v) mobile phase at a flow rate of 1.0 mL/min and UV detection at 275 nm. With these conditions, the retention time of RAC was around 5.2 min, and standard curves were linear in the concentration range of 160-240 mu g/mL (correlation coefficient >= 0.999). Validation parameters, such as selectivity, linearity, limit of detection (ranged from 1.60 to 2.05 mu g/mL), limit of quantification (ranged from 4.26 to 6.84 mu g/mL), precision (relative standard deviation <= 1.87%), accuracy (ranged from 96.97 to 100.54%), and robustness, gave results within acceptable ranges. Therefore, the developed method can be successfully applied for the routine quality control analysis of raw material and feed additives.

Lead in saliva from lead-exposed and unexposed children

Introduction: Whole blood is used for diagnosis of lead exposure. A non-invasive method to obtain samples for the biomonitoring of lead contamination has become a necessity. This study 1) compares the lead content in whole saliva samples (Pb-saliva) of children from a city with no reported lead contamination (Ribeirao Preto, Sao Paulo State, Brazil) and children of a region notoriously contaminated with lead (Bauru, Sao Paulo State, Brazil), and 2) correlates Pb-saliva with the lead content in the enamel microbiopsy samples (Pb-enamel) in the case of these two populations. Methods: From a population of our previous study that had included 247 children (4- to 6-year-old) from Ribeirao Preto, and 26 children from Bauru, Pb-saliva was analyzed in 125 children from Ribeirdo Preto and 19 children from Bauru by inductively coupled plasma mass spectrometry (ICPMS). To correlate Pb-saliva with Pb-enamel, we used Pb-enamel data obtained in our previous study. The Mann-Whitney test was employed to compare the Pb-saliva data of the two cities. Pb-saliva and Pb-enamel values were then Log(10) transformed to normalize data, and Pb-saliva and Pb-enamel were correlated using Pearson`s correlation coefficient. Results: Median Pb-saliva from the Ribeirao Preto population (1.64 mu g/L) and the Bauru population (5.85 mu g/L) were statistically different (p<0.0001). Pearson`s correlation coefficient for Log(10) Pb-saliva versus Log(10) Pb-enamel was 0.15 (p=0.08) for Ribeirao Preto and 0.38 (p=0.11) for Bauru. Conclusions: A clear relationship between Pb-saliva and environmental contamination by lead is shown. Further studies on Pb-saliva should be undertaken to elucidate the usefulness of saliva as a biomarker of lead exposure, particularly in children. (C) 2008 Elsevier B.V. All rights reserved.

A chemometric study on the analgesic activity of cannabinoid compounds using SDA, KNN and SIMCA methods

The supervised pattern recognition methods K-Nearest Neighbors (KNN), stepwise discriminant analysis (SDA), and soft independent modelling of class analogy (SIMCA) were employed in this work with the aim to investigate the relationship between the molecular structure of 27 cannabinoid compounds and their analgesic activity. Previous analyses using two unsupervised pattern recognition methods (PCA-principal component analysis and HCA-hierarchical cluster analysis) were performed and five descriptors were selected as the most relevants for the analgesic activity of the compounds studied: R (3) (charge density on substituent at position C(3)), Q (1) (charge on atom C(1)), A (surface area), log P (logarithm of the partition coefficient) and MR (molecular refractivity). The supervised pattern recognition methods (SDA, KNN, and SIMCA) were employed in order to construct a reliable model that can be able to predict the analgesic activity of new cannabinoid compounds and to validate our previous study. The results obtained using the SDA, KNN, and SIMCA methods agree perfectly with our previous model. Comparing the SDA, KNN, and SIMCA results with the PCA and HCA ones we could notice that all multivariate statistical methods classified the cannabinoid compounds studied in three groups exactly in the same way: active, moderately active, and inactive.

Heterogeneity, saving-investment dynamics and capital mobility in Latin America

The purpose of this paper is to analyze the dynamics of national saving-investment relationship in order to determine the degree of capital mobility in 12 Latin American countries. The analytically relevant correlation is the short-term one, defined as that between changes in saving and investment. Of special interest is the speed at which variables return to the long run equilibrium relationship, which is interpreted as being negatively related to the degree of capital mobility. The long run correlation, in turn, captures the coefficient implied by the solvency constraint. We find that heterogeneity and cross-section dependence completely change the estimation of the long run coefficient. Besides we obtain a more precise short run coefficient estimate compared to the existent estimates in the literature. There is evidence of an intermediate degree of capital mobility, and the coefficients are extremely stable over time.

Non-Spatial Government Policies and Regional Income Inequality in Brazil

Silveira Neto R. Da M. and Azzoni C. R. Non-spatial government policies and regional income inequality in Brazil, Regional Studies. This paper uses both macro- and micro-data to analyse the role of social programmes in the recent reduction in Brazilian regional income inequality. Convergence indicators are presented for different sources of regional income in the period 1995-2006. A decomposition of the Gini indicator allows the identification of the role of each of these income sources with respect to the reduction of regional inequality during the period. The results point out that both labour productivity and government non-spatial policies - mainly minimum wage changes and income transference programmes - do have a role in explaining regional inequality reduction during the period. [image omitted] Silveira Neto R. Da M. et Azzoni C. R. Les politiques gouvernementales non-spatiales et l`ecart des revenus regionaux au Bresil, Regional Studies. Cet article emploie des donnees a la fois macroeconomiques et microeconomiques afin d`analyser le role des programmes d`actions sociales quant a la baisse recente de l`ecart des revenus regionaux au Bresil. On presente des indicateurs de convergence pour diverses sources des revenus regionaux pour la periode allant de 1995 a 2006. Une decomposition du coefficient de Gini permet d`identifier le role de chacune de ces sources des revenus par rapport a la baisse de l`ecart des revenus pendant cette periode. Les resultats indiquent que la productivite du travail et les politiques gouvernementales non-spatiales - notamment la modification du salaire minimum et les programmes visant le transfert des revenus - ont un role a jouer pour expliquer la baisse de l`ecart des revenus regionaux pendant la periode en question. Convergence Productivite du travail Transfert des revenus Salaire minimum Effets spatiaux des politiques non-spatiales Silveira Neto R. Da M. und Azzoni C. R. Nicht raumliche Regierungspolitiken und das regionale Einkommensungleichgewicht in Brasilien, Regional Studies. In diesem Beitrag analysieren wir mit Hilfe von Makro- und Mikrodaten die Rolle von sozialen Programmen bei der unlangst erzielten Verringerung des regionalen Einkommensungleichgewichts in Brasilien. Wir stellen Konvergenz-Indikatoren fur verschiedene regionale Einkommensquellen im Zeitraum von 1995 bis 2006 vor. Eine Dekomposition des Gini-Indikators ermoglicht die Identifizierung der jeweiligen Rolle dieser Einkommensquellen fur die Verringerung des regionalen Ungleichgewichts im betreffenden Zeitraum. Die Ergebnisse weisen darauf hin, dass sowohl die Produktivitat der Arbeitskrafte als auch die nicht raumlichen Regierungspolitiken - in erster Linie Veranderungen beim Mindestlohn und Programme fur Einkommenstransfers - als Grunde fur die Verringerung des regionalen Ungleichgewichts in dieser Periode durchaus eine Rolle spielen. Konvergenz Arbeitsproduktivitat Einkommenstransfer Mindestlohn Raumliche Auswirkungen nicht raumlicher Politiken Silveira Neto R. Da M. y Azzoni C. R. Politicas gubernamentales no espaciales y desigualdades de ingresos regionales en Brasil, Regional Studies. En este articulo utilizamos datos macro y micro para analizar el papel de los programas sociales en la reciente reduccion en las desigualdades de ingresos regionales de Brasil. Presentamos los indicadores de convergencia para diferentes fuentes de ingresos regionales en el periodo de 1995 a 2006. Una descomposicion del indice Gini permite identificar el papel de cada una de estas fuentes de ingresos con respecto a la reduccion de las desiguadades regionales durante este periodo. Los resultados destacan que tanto la productividad laboral como las politicas no espaciales del gobierno - principalmente los cambios de salario minimo y los programas de transferencias de ingresos - desempenan una funcion a la hora de explicar la reduccion de las desigualdades regionales durante este periodo. Convergencia Productividad laboral Transferencias de ingresos Salario minimo Efectos espaciales de politicas no espaciales.

Stakeholders Theory and Theory of the Firm: a study on the hierarchy of objective-functions in Brazilian companies

Discussion opposing the Theory of the Firm to the Theory of Stakeholders are contemporaneous and polemical. One focal point of such debates refers to which objective-function companies, should choose, whether that of the shareholders or that of the stakeholders, and whether it is possible to opt for both simultaneously. Several empirical studies. have attempted-to test a possible correlation between both functions, and there has not been any consensus-so far. The objective of the present research is to examine a gap in such discussions: is there (or not) a subordination of the stakeholders` objective-function to that of the shareholders? The research is empirical,and analytical and employs quantitative methods. Hypotheses were tested and data analyzed by using non-parametrical (chi-square test) and parametrical procedures (frequency. correlation `coefficient). Secondary data was collected from he Economitica database and from the Brazilian Institute of Social and-Economic Analyses (IBASE) website, relative to public companies that have published their Social Balance Statements following the IBASE model from 1999 to 2006, whose sample amounted to 65 companies; In order to assess the objective-function of shareholders a proxy was created based on the following three indices: ROE (return on equity), EnterpriseValue and Tobin`s Q. In order to assess the objective-function of stakeholders a proxy was created by employing the following IBASE social balance indices: internal ones (ISI), external ones (ISE), and environmental ones (IAM). The results have shown no evidence of subordination of stakeholders` objective-function to that of the shareholders in analyzed companies, negating initial expectations and calling for deeper investigation of results. Its main conclusion, which states that the attempted subordination does not take place, is limited to the sample herein investigated and calls for ongoing research aiming at improvements which may lead to sample enlargement and, as a consequence, may make feasible the application of other statistical techniques which may yield a more thorough, analysis of the studied phenomehon.

Solid-phase microextraction using poly(pyrrole) film and liquid chromatography with UV detection for analysis of antidepressants in plasma samples

Poly(pyrrole) (PPY) coating was prepared on a stainless-steel (SS) wire for solid-phase microextraction (SPME) by electrochemical deposition (cyclic voltammetric). The PPY was evaluated by analyzing new-generation antidepressants (mirtazapine, citalopram, paroxetine, duloxetine, fluoxetine, and sertraline) in plasma sample by SPME and liquid chromatography with UV detection (LC-UV). The effect of electrolyte Solution (lithium perchlorate or tetrabutylammonium perchlorate) and the number of cycles (50, 100 or 200) applied during the polymerization process on the SPME performance was evaluated. Important factors in the optimization of SPME efficiency such as extraction time, temperature, pH, influence of plasma proteins on sorption mechanisms, and desorption conditions are discussed. The SPME-PPY/LC method showed to be linear in concentrations ranging from the limit of quantification (LOQ) to 1200 ng mL(-1). The LOQ values range from 16 to 25 ng mL-1. The inter-day precision of the SPME-PPY/LC method presented coefficient of variation (CV) lower than 15%. Based on analytical validation results, the SPME-PPY/LC methodology showed to be adequate for antidepressant analysis, from therapeutic to toxic levels. In order to evaluate the proposed method for clinical use, the SPME-PPY/LC method was applied to the analysis of plasma samples from elderly depressed patients. (c) 2009 Elsevier B.V. All rights reserved,