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Chinese Journal of Biotechnology ; (12): 159-168, 2019.
Article in Chinese | WPRIM | ID: wpr-771390


Two proteins of similar molecular weight (named as ASPR-C-1 and ASPR-C-2) from the crude drug of Angelica sinensis were purified and characterized by 80% ammonium sulfate precipitation, Sephadex G-50 gel filtration chromatography, and DEAE-Sepharose anion exchange chromatography. The molecular weight of ASPR-C-1 and ASPR-C-2 on SDS-PAGE was 17.33 kDa and 17.18 kDa, respectively. They were mainly monomeric in solution, but partially formed dimers and they were glycoproteins with glycosyl content of 2.6% and 8.2%, respectively. Both ASPR-C-1 and ASPR-C-2 were identified to be members of pathogenesis-related 10 family of proteins by matrix-assisted laser desorption ionization time-of-flight mass spectrometry and have ribonuclease activities with the specific activity of 73.60 U/mg and 146.76 U/mg, respectively. The optimum pH of the two isoforms was similar, at about 5.6, while their optimum temperatures were different. The optimum temperature of ASPR-C-1 was 50 ℃, and that of ASPR-C-2 was 60 ℃. Both isoforms presented highest thermal stability at 60 ℃. However, ASPR-C-2 was more thermotolerant than ASPR-C-1. The latter was rapidly inactivated and retained only about 20% residual activity while the former still maintained about 80% of its original activity at a higher treatment temperature (80 to 100 ℃). In addition, Fe²⁺ had an activating effect on the ribonuclease activities of two isoforms while Ca²⁺, Mg²⁺, Zn²⁺, Mn²⁺, Ag⁺, Cu²⁺, EDTA (Elhylene diamine tetraacetic acid), dithiothreitol and sodium dodecylsulphate showed different degrees of inhibition of the enzyme activities. Our findings provide a foundation for further research on the biological function of PR-10 protein from Angelica sinensis.

Angelica sinensis , Chromatography, Gel , Chromatography, Ion Exchange , Electrophoresis, Polyacrylamide Gel , Enzyme Stability , Hydrogen-Ion Concentration , Kinetics , Molecular Weight , Protein Isoforms , Temperature
Univ. sci ; 22(1): 45-70, Jan.-Apr. 2017. tab, graf
Article in English | LILACS, COLNAL | ID: biblio-904705


Abstract Using Response Surface Methodology (RSM) we evaluated the culture conditions (nitrogen source, carbon source, pH and agitation rate) that increase the biomass of Acidocellafaalis strain USBA-GBX-505 and therefore enhance the production of its lipolytic enzyme, 505 LIP. RSM results revealed that yeast extract and agitation were key culture factors that increased the growth-associated lipolytic activity by 4.5-fold (from 0.13 to 0.6 The 505 LIP lipase was partially purified using size-exclusion chromatography and ion-exchange chromatography. Its molecular weight was >77 kDa. The enzyme shows its optimum catalytic activity at 55 °C and pH 7.5. EDTA, PMSF, 1-butanol and DMSO inhibited enzymatic activity, whereas Tween 20, acetone, glycerol and methanol increased it. Metallic ions are not required for the activity of 505 LIP, and even have an inhibitory effect on the enzyme. This study shows the potential use of A. facilis strain USBA- GBX-505 for the production of a newly identified lipolytic enzyme, 505 LIP, which is stable at moderate temperatures and in the presence of organic solvents. These are important characteristics for the synthesis of many useful products.

Resumen Por medio de la Metodología de Respuesta de Superficie (RSM) evaluamos las condiciones de cultivo (fuente de N, fuente de C, pH y tasa de agitación) que incrementan la biomasa de Acidocella facilis cepa USBA-GBX-505 y, como consecuencia, la producción de su enzima lipolítica, llamada 505 LIP. Los resultados de la RSM revelaron que el extracto de levadura y la agitación fueron factores de cultivo claves, que incrementaron de 4 a 5 veces la actividad lipolítica asociada al crecimiento (de 0.13 a 0.6 La lipasa 505 LIP se purificó parcialmente usando cromatografía de exclusión por tamaño y cromatografía de intercambio iónico. Su peso molecular fue > 77 kDa. La enzima muestra su actividad catalítica óptima a 55 °C y pH 7.5. El EDTA, el PMSF, el 1-butanol y el DMSO inhibieron la actividad enzimática, mientras que el Tween 20, la acetona, el glicerol y el metanol la incrementaron. La enzima 505 LIP no requiere iones metálicos para su actividad, e incluso se inhibe en presencia de ellos. Este estudio muestra el uso potencial de A. facilis cepa USBA-GBX-505 para la producción de una nueva enzima lipolítica, 505 LIP, que es estable a temperaturas moderadas y en la presencia de solventes orgánicos. Estas son características importantes en la síntesis de muchos productos útiles.

Resumo Utilizando a Metodologia de Superfície de Resposta (MSR) avaliamos as condições de cultivo (fontes de nitrogénio e carbono, pH e taxa de agitação) que aumentam a biomassa de Acidocella facilis cepa USBA-GBX-505, e, portanto, elevam a produção de sua enzima lipolítica 505 LIP. Os resultados da MSR revelaram que o extrato de levedura e a agitação foram fatores de cultivo chave que permitiram aumentar 4 a 5 vezes a atividade lipolítica associada ao crescimento (de 0,13 a 0,6 A lipase 505 LIP foi parcialmente purificada utilizando cromatografia por exclusão de tamanho e cromatografia de intercambio iónico. Seu peso molecular foi > 77 kDa. A enzima mostra sua atividade catalítica ótima a 55 °C e pH 7,5. EDTA, PMSF, 1-butanol e DMSO inibiram a atividade enzimática, enquanto que Tween 20, acetona, glicerol e metanol aumentaram esta atividade. Íons metálicos não são necessários para a atividade da 505 LIP, apresentando inclusive efeito inibitório da enzima. Este estudo demonstra o potencial uso de A. facilis cepa USBA-GBX-505 para a produção de uma nova enzima lipolítica, 505 LIP, a qual é estável a moderadas temperaturas e na presença de solventes orgánicos. Estas características são importantes para a síntese de diversos produtos úteis.

Chromatography, Ion Exchange/methods
Braz. j. microbiol ; 47(3): 647-657, July-Sept. 2016. tab, graf
Article in English | LILACS | ID: lil-788974


ABSTRACT The purpose of this study was to isolate, purify and optimize the production conditions of an organic solvent tolerant and thermostable lipase from Acinetobacter sp. AU07 isolated from distillery waste. The lipase production was optimized by response surface methodology, and a maximum production of 14.5 U/mL was observed at 30 ºC and pH 7, using a 0.5% (v/v) inoculum, 2% (v/v) castor oil (inducer), and agitation 150 rpm. The optimized conditions from the shake flask experiments were validated in a 3 L lab scale bioreactor, and the lipase production increased to 48 U/mL. The enzyme was purified by ammonium sulfate precipitation and ion exchange chromatography and the overall yield was 36%. SDS-PAGE indicated a molecular weight of 45 kDa for the purified protein, and Matrix assisted laser desorption/ionization time of flight analysis of the purified lipase showed sequence similarity with GDSL family of lipases. The optimum temperature and pH for activity of the enzyme was found to be 50 ºC and 8.0, respectively. The lipase was completely inhibited by phenylmethylsulfonyl fluoride but minimal inhibition was observed when incubated with ethylenediaminetetraacetic acid and dithiothreitol. The enzyme was stable in the presence of non-polar hydrophobic solvents. Detergents like SDS inhibited enzyme activity; however, there was minimal loss of enzyme activity when incubated with hydrogen peroxide, Tween 80 and Triton X-100. The kinetic constants (Km and Vmax) revealed that the hydrolytic activity of the lipase was specific to moderate chain fatty acid esters. The Vmax, Km and Vmax/Km ratio of the enzyme were 16.98 U/mg, 0.51 mM, and 33.29, respectively when 4-nitrophenyl palmitate was used as a substrate.

Organic Chemicals , Solvents , Bacterial Proteins/isolation & purification , Bacterial Proteins/biosynthesis , Acinetobacter/enzymology , Lipase/isolation & purification , Lipase/biosynthesis , Organic Chemicals/chemistry , Solvents/chemistry , Substrate Specificity , Temperature , Bacterial Proteins/chemistry , Enzyme Stability , Kinetics , Chromatography, Ion Exchange , Enzyme Activation , Extracellular Space/enzymology , Hydrogen-Ion Concentration , Ions , Lipase/chemistry , Lipolysis , Metals , Molecular Weight
Article in Chinese | WPRIM | ID: wpr-337397


Recombinant Fl-V (rFl-V) fusion protein is the main ingredient of the current candidate vaccine against Yersinia pestis infection, which has been under investigation in clinical trial in USA. We investigated the soluble expression conditions of rF1-V in Escherichia coli BL21 (DE3) that we constructed before. After scale-up and optimization of fermentation processes, we got the optimized fermentation process parameters: the culture was induced at the middle exponential phase with 50 µmol/L of IPTG at 25 °C for 5 h. Soluble rFl-V protein was isolated to 99% purity by ammonium sulfate precipitation, ion exchange chromatography, hydrophobic chromatography and gel filter chromatography. The protein recovery was above 20%. Protein identity and primary structure were verified by mass spectrometry and Edman sequencing. Results of purity, quality and western blotting analysis indicated that the target protein is a consistent and properly folded product. Furthermore, the immunogenicity of various antigens formulated with aluminum hydroxide adjuvant was evaluated in mice. Serum antibody titers of 4 groups including 20 µg rFl, rV and rFl-V and 10 µg rFl+10 µg rV, were assayed by ELISA after 2 doses. The antibody titers of anti-Fl with 20 µg rFl-V were obviously higher than titers with other groups; meanwhile there were no significant difference of anti-V antibody titers among them. These findings confirm that rFl-V would be the active pharmaceutical ingredient of the plague subunit vaccine.

Adjuvants, Immunologic , Animals , Antibodies, Bacterial , Blood , Antibody Formation , Antigens, Bacterial , Allergy and Immunology , Blotting, Western , Chromatography, Ion Exchange , Enzyme-Linked Immunosorbent Assay , Mice , Plague , Plague Vaccine , Allergy and Immunology , Recombinant Fusion Proteins , Allergy and Immunology , Vaccines, Subunit , Allergy and Immunology , Yersinia pestis
Braz. j. microbiol ; 46(4): 1265-1268, Oct.-Dec. 2015. graf
Article in English | LILACS | ID: lil-769661


Abstract Noroviruses (NVs) are responsible for most cases of human nonbacterial gastroenteritis worldwide. Some parameters for the purification of NV virus-like particles (VLPs) such as ease of production and yield were studied for future development of vaccines and diagnostic tools. In this study, VLPs were produced by the expression of the VP1 and VP2 gene cassette of the Brazilian NV isolate, and two purification methods were compared: cesium chloride (CsCl) gradient centrifugation and ion-exchange chromatography (IEC). IEC produced more and purer VLPs of NV compared to CsCl gradient centrifugation.

Child , Humans , Centrifugation, Density Gradient/methods , Chromatography, Ion Exchange/methods , Norovirus/genetics , Viral Structural Proteins/genetics , Virosomes/isolation & purification , Brazil , Viral Structural Proteins/metabolism , Virosomes/genetics , Virosomes/metabolism
Braz. j. microbiol ; 46(1): 251-260, 05/2015. tab, graf
Article in English | LILACS | ID: lil-748253


An Aspergillus niger UFV-1 phytase was characterized and made available for industrial application. The enzyme was purified via ultrafiltration followed by acid precipitation, ion exchange and gel filtration chromatography. This protein exhibited a molecular mass of 161 kDa in gel filtration and 81 kDa in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), indicating that it may be a dimer. It presented an optimum temperature of 60 °C and optimum pH of 2.0. The KM for sodium phytate hydrolysis was 30.9 mM, while the kcat and kcat/KM were 1.46 ×105 s−1 and 4.7 × 106 s−1.M−1, respectively. The purified phytase exhibited broad specificity on a range of phosphorylated compounds, presenting activity on sodium phytate, p-NPP, 2- naphthylphosphate, 1- naphthylphosphate, ATP, phenyl-phosphate, glucose-6-phosphate, calcium phytate and other substrates. Enzymatic activity was slightly inhibited by Mg2+, Cd2+, K+ and Ca2+, and it was drastically inhibited by F−. The enzyme displayed high thermostability, retaining more than 90% activity at 60 °C during 120 h and displayed a t1/2 of 94.5 h and 6.2 h at 70 °C and 80 °C, respectively. The enzyme demonstrated strong resistance toward pepsin and trypsin, and it retained more than 90% residual activity for both enzymes after 1 h treatment. Additionally, the enzyme efficiently hydrolyzed phytate in livestock feed, liberating 15.3 μmol phosphate/mL after 2.5 h of treatment.

/isolation & purification , /metabolism , Aspergillus niger/enzymology , /chemistry , Chemical Precipitation , Chromatography, Gel , Chromatography, Ion Exchange , Electrophoresis, Polyacrylamide Gel , Enzyme Stability , Enzyme Inhibitors/analysis , Hydrogen-Ion Concentration , Hydrolysis , Kinetics , Molecular Weight , Protein Multimerization , Proteolysis , Peptide Hydrolases/metabolism , Phytic Acid/metabolism , Substrate Specificity , Temperature , Ultrafiltration
Salud pública Méx ; 57(1): 38-49, ene.-feb. 2015. ilus, tab
Article in Spanish | LILACS | ID: lil-736460


Objetivo. Comparar la salud, uso de servicios sanitarios y necesidad insatisfecha de atención médica (NIAM) entre inmigrantes y nativos del sureste español. Material y métodos. Estudio transversal de dos muestras representativas de población: inmigrante (n=1150) y nativa (n=1303; Encuesta Nacional de Salud). Se creó una única base de datos con ponderación específica para cada muestra y se estimaron razones de prevalencia (RP) mediante regresión multivariante. Resultados. Marroquíes, ecuatorianos y europeos del este (EE) declararon peor salud que los nativos (RPs [IC95%]: 2.45 [1.91-3.15]; 1.51 [1.28-1.79] y 1.44 [1.08-1.93], respectivamente). Los inmigrantes hicieron mayor uso de las urgencias (excepto EE) y consumieron menos fármacos. Los marroquíes mostraron la mayor diferencia en la frecuencia de NIAM (RP [IC95%]: 12.20 [5.25-28.37]), principalmente por razones laborales (46%). Conclusiones. La salud y el uso de servicios sanitarios difirieron significativamente entre inmigrantes y nativos. Destaca la NIAM alta en marroquíes por causa laboral.

Objective. To compare the self-perceived health, use of health services and unmet need for health care (UNHC) among immigrants and native populations of Southeast Spain. Materials and methods. Cross-sectional study of two representative samples of 1150 immigrants, and 1303 native participants from the National Health Survey. A single database was created with specific weights for each sample, and prevalence ratios (PR) were estimated by multivariate regression. Results. Moroccans, Ecuadorians and Eastern Europeans (EE) reported poorer health than the native population (PRs [CI95%]: 2.45 [1.91-3.15]; 1.51 [1.28-1.79] and 1.44 [1.08-1.93], respectively). Immigrants made greater use of emergencies that natives (except for EE) and had lower use of medication. Moroccan showed the greatest difference in the frequency of UNHC (PR [CI95%]:12.20 [5.25 - 28.37]), mainly because of working limitations (46%). Conclusions. The health status and use of health services among immigrants differ significantly from those of natives. Results highlight the higher frequency of UNHC among immigrants, especially high in Moroccans.

Animals , Humans , Cysteine Endopeptidases/isolation & purification , Taenia solium/enzymology , Chromatography, Gel , Chromatography, Ion Exchange , Collagen/metabolism , Cysteine Endopeptidases/chemistry , Cysteine Endopeptidases/metabolism , Cysteine Proteinase Inhibitors/pharmacology , Immunoglobulin G/metabolism , Iodoacetic Acid/pharmacology , Leucine/analogs & derivatives , Leucine/pharmacology , Serum Albumin, Bovine/metabolism
Electron. j. biotechnol ; 18(1): 20-28, Jan. 2015. ilus, graf, tab
Article in English | LILACS | ID: lil-736981


Background Oil and grease laden wastewaters pose hindrance to the treatment units and further threaten the receiving water bodies. Lipase-producing microbial strains are increasingly being exploited for the remediation of such effluents. Results When bacterial strains isolated from oil mill effluent were screened for their lipolytic activity, two isolates, COM-4A and COM-6B showed significant extracellular lipase activity. They were identified to be Staphylococcus pasteuri and Bacillus subtilis, respectively. S. pasteuri COM-4A was cultivated in nutrient media based on coconut oil mill waste (CMW), in which it showed good growth at concentrations up to 20 g/L. While growing in such media, it was capable of producing lipase and other important extracellular hydrolytic enzymes. Furthermore, the isolate was able to effectively biodegrade the CMW supplemented in the medium. Applying the Box Behnken Design of Response Surface Methodology, lead to a 1.4-fold increase in both lipase production and oil removal by the isolate. The lipase was purified 9.02-fold and the molecular weight of the monomeric enzyme was deduced to be around 56 kDa. Characterization of the enzyme revealed it to be alkaliphilic and moderately thermophilic in nature, with pH and temperature optima of 9.0 and 50°C, respectively. The enzyme was also quite stable in the presence of water-miscible organic solvents. Conclusion Hence, the COM-4A lipase could be considered to be suitable for a variety of industrial applications such as in detergent formulations and in biodiesel production as well, apart from the possibility of applying it for bioremediation of fat and oil contaminants.

Staphylococcus/enzymology , Palm Oil/metabolism , Lipase/isolation & purification , Lipase/biosynthesis , Temperature , Bacillus subtilis/enzymology , Biodegradation, Environmental , Chromatography, Ion Exchange , Biomass , Detergents , Biofuels , Waste Water , Hydrogen-Ion Concentration
SQUMJ-Sultan Qaboos University Medical Journal. 2015; 15 (4): 418-425
in English | IMEMR | ID: emr-173874


Objectives: Afzal is a common smokeless tobacco product [STP] available illegally in Oman. This study aimed to assess pH and moisture levels and determine cancer-enhancing factors in a randomly selected sample of Afzal

Methods: This study was carried out at the Sultan Qaboos University in Muscat, Oman, between April and December 2013. A package of Afzal was purchased from a single provider and divided into samples. The pH and moisture content of the samples were measured according to the protocols of the Centers for Disease Control and Prevention. Gas chromatography-mass spectrometry was used to analyse nicotine levels and ionexchange chromatography [IC] was used to determine concentrations of nitrate, nitrite, chloride, fluoride, bromide, sulphate and phosphate anions

Results: The samples had an alkaline pH of 10.46 with high levels of total [48,770.00 micro per g of STP [microg/g]] and unionised [48,590.00 microg/g] nicotine. The concentration of nitrate [8,792.20 microg/g] was alarmingly high. The chloride concentration [33,170.80 microg/g] showed a surge on IC chromatography. The moisture content percentage was 52.00%

Conclusion: The moisture content percentage and chloride concentration of Afzal was consistent with those of other STPs. In contrast, nitrite, sulphate and phosphate concentrations were below reported levels of other STPs. All anion concentrations were below the maximum daily limit set by international health organisations. However, the high concentrations of nitrite, nitrate and nicotine and the elevated alkaline pH observed in the analysed Afzal samples suggest that STP users will face health risks as a result of their use

Nicotine , Neoplasms , Anions , Gas Chromatography-Mass Spectrometry , Chromatography, Ion Exchange , Hydrogen-Ion Concentration , Carcinogens
Article in Chinese | WPRIM | ID: wpr-326002


<p><b>OBJECTIVE</b>To develop a method for determination of mandelic acid (MA) and phenylglyoxylic acid (PGA) in urine by reagent-free ion chromatography.</p><p><b>METHODS</b>Ion chromatography was performed on an AS19 column with a gradient elution solution containing 10-35 mmoL/L KOH at a flow rate of 1.00 ml/min, and MA and PGA were detected at ultraviolet wavelengths of 225 nm and 254 nm, respectively. The samples were diluted 10 times with purified water, then purified on a silver column to remove high concentrations of chloride ion, and injected after being filtered through a 0.2-µm m filter membrane.</p><p><b>RESULTS</b>The recoveries of standard addition of MA and PGA were 96.5% and 99.3%, respectively, with both relative standard deviations less than 5.0%. Good linear relationships were noted in the range of 1.0-100.0 mg/L for both MA and PGA (r >0.9995). The detection limits of MA and PGA were 0.02 mg/L and 0.05 mg/L, respectively; the minimum detectable concentrations of MA and PGA were 0.2 mg/L and 0.5 mg/L (when the sampling amount was 5.0 ml and diluted to 50.0 ml with water, and the injection volume was 300 µL).</p><p><b>CONCLUSIONS</b>This method is fast, convenient, and highly sensitive and selective. It can be used for the analysis of MA and PGA in the urine of styrene-exposed workers.</p>

Chromatography, Ion Exchange , Glyoxylates , Urine , Humans , Mandelic Acids , Urine , Styrene
J. bras. nefrol ; 36(4): 430-436, Oct-Dec/2014. tab, graf
Article in Portuguese | LILACS | ID: lil-731139


Introdução: Atualmente, é descrita elevada prevalência de hipovitaminose D no Lúpus Eritematoso Sistêmico (LES), a qual se associa a algumas manifestações clínicas e maior atividade inflamatória. Objetivo: Avaliar a associação entre insuficiência de vitamina D com LES e marcadores inflamatórios. Métodos: Estudo transversal, tendo sido avaliados 45 pacientes com LES e 24 controles sem a doença. Níveis de 25-hidroxivitamina D [25(OH)D] menores que 30 ng/mL foram considerados insuficientes. A atividade da doença foi avaliada pelo Systemic Lupus Erythematosus Disease Activity Index (SLEDAI). Foram avaliados, ainda, proteína C reativa ultrassensível (PCRus) e interleucina-6 (IL-6) para verificação do status inflamatório. Para avaliação do envolvimento renal, foram realizados análise de elementos anormais e sedimentoscopia urinárias (EAS), hematúria e piúria quantitativas, proteinúria e depuração de creatinina em urina de 24 horas e anti-DNA de dupla hélice sérico. Resultados: A prevalência de insuficiência de 25(OH)D foi de 55% nos pacientes lúpicos e 8% nos participantes controles (p = 0,001). A mediana da 25(OH)D foi menor nos pacientes do que no grupo controle. Os pacientes com insuficiência de 25(OH)D apresentaram níveis mais elevados de IL-6 e maior prevalência de hematúria ao EAS. Não houve correlação entre vitamina D, nefrite lúpica e SLEDAI. Conclusão: Em nosso estudo, a insuficiência de vitamina D foi mais prevalente em pacientes com LES e se associou com níveis mais elevados de IL-6 e presença de hematúria. .

Introduction: Nowadays it is described a high prevalence of hypovitaminosis D in Systemic Lupus Erythematosus (SLE), which is associated with some clinical manifestations and increased inflammatory activity. Objective: To evaluate the association between vitamin D insufficiency with SLE and inflammatory markers. Methods: Cross-sectional study, in which have been evaluated 45 SLE patients and 24 controls without the disease. Levels of 25-hydroxyvitamin D [25(OH) D] less than 30 ng/mL were considered inadequate. Disease activity was assessed by the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI). High sensitivity C reactive protein (hsCRP) and interleukin-6 (IL-6) were evaluated for verification of the inflammatory status. For assessment of renal involvement, analysis of abnormal elements and urinay sediment (AES), quantitative hematuria and pyuria, proteinuria and creatinine clearance in 24-hour urine and serum anti-double stranded DNA were performed. Results: The prevalence of 25(OH)D insufficiency was 55% in SLE patients and 8% in the controls participants (p = 0.001). The median of 25(OH)D was lower in patients than in controls. Patients with insufficient 25(OH)D had higher levels of IL-6 and higher prevalence of hematuria in the AES. There was no correlation between vitamin D and SLEDAI or lupus nephritis. Conclusion: In our study, vitamin D deficiency was more prevalent in patients with SLE and was associated with higher levels of IL-6 and hematuria. .

Animals , Rabbits , Antigens, Protozoan/immunology , Membrane Proteins/immunology , Protein Folding , Plasmodium falciparum/immunology , Protozoan Proteins/immunology , Sarcosine/analogs & derivatives , Antibodies, Protozoan/immunology , Antigens, Protozoan/biosynthesis , Antigens, Protozoan/genetics , Antigens, Protozoan/isolation & purification , Cysteine , Chromatography, Affinity/methods , Chromatography, Ion Exchange/methods , Edetic Acid , Endotoxins , Escherichia coli , Fermentation , Gene Expression , Membrane Proteins/biosynthesis , Membrane Proteins/genetics , Membrane Proteins/isolation & purification , Nickel , Protein Structure, Tertiary , Plasmodium falciparum/genetics , Protozoan Proteins/biosynthesis , Protozoan Proteins/genetics , Protozoan Proteins/isolation & purification , Sucrose
Hist. ciênc. saúde-Manguinhos ; 21(4): 1283-1300, Oct-Dec/2014.
Article in Portuguese | LILACS | ID: lil-732517


Este estudo investiga as práticas de produção de conhecimento sobre a menopausa no Caism/Unicamp, centro de referência para políticas públicas em saúde da mulher. Foram realizadas observações de consultas ginecológicas, entrevistas com mulheres e médicos e observação de reuniões de apoio psicológico, buscando identificar os discursos que circulam no lugar e o processo de alistamento de diferentes atores para que os conhecimentos ali produzidos alcancem credibilidade e “viajem” além dos limites do hospital-escola, tornando-se “universais”. A análise baseia-se nos “estudos localistas”, alinhados aos estudos sociais de ciência e tecnologia.

This study investigates the practices involved in the production of knowledge about menopause at Caism, Unicamp, a reference center for public policies for women’s health. Gynecological appointments and psychological support meetings were observed, and women and doctors were interviewed in order to identify what discourse circulates there and how different actors are brought in to ensure that the knowledge produced attains credibility and “travels” beyond the boundaries of the teaching hospital to become “universal”. The analysis is based on localized studies aligned with social studies of science and technology.

Animals , Male , Mice , /genetics , Major Histocompatibility Complex , Odorants , Benzoic Acid , Benzoates/isolation & purification , Benzoates/urine , Butyrates/isolation & purification , Butyrates/urine , Chromatography, Gas , Chromatography, Ion Exchange , Cresols/isolation & purification , Cresols/urine , Dimethyl Sulfoxide , Discrimination, Psychological , Maze Learning , Mice, Inbred Strains , Phenols/isolation & purification , Phenols/urine , Phenylacetates/isolation & purification , Phenylacetates/urine , Sulfones/isolation & purification , Sulfones/urine , Ultrafiltration
Rev. bras. enferm ; 67(6): 957-964, Nov-Dec/2014.
Article in Portuguese | LILACS, BDENF | ID: lil-732825


Objetivo: identificar as necessidades e as preocupações prioritárias, manifestadas pelos pais no desempenho do seu papel, em três etapas do ciclo vital: adolescência, idade produtiva e idade madura. Metodologia: estudo exploratório com abordagem qualitativa, desenvolvido com quatorze pais residentes em um município no extremo sul do Brasil. Os dados foram coletados entre maio e agosto de 2011, por meio de entrevista em profundidade. Através da técnica da análise textual discursiva e da matriz construída com base na teoria bioecológica de Bronfenbrenner, foram construídas três categorias: Necessidades/preocupações do pai, geradas em sua relação com o mundo do trabalho; Necessidades/preocupações que emergem da relação de cuidado com os filhos e Preocupações dos pais com relação ao futuro dos filhos. Conclusão: identificou-se que a preocupação com o futuro dos filhos foi apontada por pais de todas as faixas-etárias investigadas. .

Objective: this study aimed to identify priority needs and concerns expressed by fathers in the performance of their role in three stages of the life cycle: adolescence, productive age, and mature age. Methodology: this is an exploratory study with a qualitative approach, conducted with fourteen fathers residing in a municipality in the extreme south of Brazil. The data were collected between May and August 2011 by means of the in-depth interview. Through the technique of written discourse analysis and the array built upon Bronfenbrenner's bioecological theory, we obtained three categories: fathers' needs/concerns, generated in their relationship with the world of work; needs/concerns that emerged from the relationship of care with the children; and fathers' concerns about the future of the children. Conclusions: we identified that the concern with the future of the children was pointed out by fathers of all age groups investigated. .

Objetivo: identificar las necesidades y preocupaciones prioritarias, manifestadas por los padres en el desempeño de su función, en tres etapas del ciclo de vida: adolescencia, edad productiva y edad madura. Metodología: estudio exploratorio con abordaje cualitativo, desarrollado con catorce padres residentes en un municipio en el extremo sur de Brasil. Los datos fueran colectados entre mayo y agosto de 2011, a través de entrevistas en profundidad. A través de la técnica de análisis textual y discursiva e de la matriz construida basada en la teoria bioecologica de Bronfenbrenner, fueran construidas tres categorías: Necesidades/ preocupaciones de lo padre, generado en suya relación con el mundo de lo trabajo; Necesidades/preocupaciones que emergen de la relación de cuidado con hijos e preocupaciones de los padres con lo futuro de los hijos. Conclusión: Se identifico que la preocupación con el futuro de los hijos fue apuntado por los padres de todas las edades averiguadas. .

Coenzyme A Ligases/isolation & purification , Phenylacetates/metabolism , Pseudomonas/enzymology , Amino Acids/analysis , Chromatography, Gel , Chromatography, High Pressure Liquid , Chromatography, Ion Exchange , Coenzyme A Ligases/biosynthesis , Coenzyme A Ligases/metabolism , Electrophoresis, Polyacrylamide Gel , Kinetics , Molecular Weight , Pseudomonas/growth & development , Substrate Specificity , Thermodynamics , Ultracentrifugation
Bol. latinoam. Caribe plantas med. aromát ; 13(4): 359-365, jul. 2014. ilus
Article in English | LILACS | ID: lil-785454


The exopolysaccharide (EPS) separated from the entomopathogenic fungus Metarhizium anisopliae was determined by gel permeation chromatography to be homogeneous. The high-performance anion-exchange chromatography with pulsed-amperometric detection (HPAE-PAD) showed a content of monosaccharides D-galactosamine and D-fucose at a molar ratio of about 2:1. The results obtained from Fourier transform-infrared spectroscopy (FT-IR) and second derivative FT-IR spectrum confirmed the proposed structure.

El exopolisacárido (EPS) separado desde el hongo entomopatogénico Metarhizium anisopliae determinado por cromatografía de exclusión en gel ser homogéneo. La cromatografía iónica de alto rendimiento con detección de pulso amperométrico (HPAE-PAD) mostró un contenido de monosacáridos D-galactosamina y D-fucosa en una relación molar de alrededor de 2:1. Los resultados obtenidos desde la espectroscopía infrarroja con transformada de Fourier (FT-IR) y la segunda derivada del espectro FT-IR confirmaron la estructura propuesta.

Metarhizium , Fungal Polysaccharides/analysis , Chromatography, Ion Exchange , Spectroscopy, Fourier Transform Infrared
Braz. j. microbiol ; 45(2): 677-687, Apr.-June 2014. ilus, graf, tab
Article in English | LILACS | ID: lil-723134


A mesophilic Enterobacter sp. Bn12 producing an alkaline thermostable lipase was isolated from soil in Tehran, Iran. The lipase gene (ELBn12) was identified from a genomic library. Sequence analysis of the DNA fragment revealed an open reading frame of 879 bp encoding a lipase with a molecular mass of 31.3 kDa. The deduced amino acid sequence showed 96% identity with a lipase of Enterobacter sp. Ag1 and the identity of their DNA sequences was 88.9%. ELBn12 belongs to the lipase subfamily I.1 and its catalytic triad consists of Ser82, Asp237 and His259. The lipase was expressed in Escherichia coli (BL21) pLysS and partially purified by anion exchange chromatography. The maximum activity of ELBn12 was obtained at temperature of 60 °C and pH 8.0 towards tricaprylin (C8) and its specific activity was around 2900 U/mg. ELBn12 was stable within a broad pH range from 6.0 to 11.0. The enzyme showed high stability in both polar and nonpolar organic solvents at 50% (v/v). The lipase activity was enhanced in the presence of 10 mM of Ca2+, Mg2+ and K+, while heavy metals (Fe3+ and Zn2+) had strong inhibitory effect. ELBn12 showed high activity in the presence of 1% (w/v) nonionic surfactants, however ionic surfactants inhibited the lipolytic activity. ELBn12 characteristics show that it has a potential to be used in various industrial processes.

Enterobacter/enzymology , Lipase/isolation & purification , Lipase/metabolism , Amino Acid Sequence , Bacterial Typing Techniques , Base Sequence , Chromatography, Ion Exchange , Cloning, Molecular , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , Enzyme Stability , Enterobacter/classification , Enterobacter/genetics , Enterobacter/isolation & purification , Enzyme Activators/analysis , Enzyme Inhibitors/analysis , Escherichia coli/genetics , Gene Expression , Hydrogen-Ion Concentration , Iran , Lipase/chemistry , Lipase/genetics , Molecular Sequence Data , Molecular Weight , Open Reading Frames , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Sequence Homology, Nucleic Acid , Soil Microbiology , Temperature
Braz. j. microbiol ; 45(1): 127-134, 2014. ilus, graf, tab
Article in English | LILACS | ID: lil-709465


Manganese peroxidase (MnP) was produced from white rot edible mushroom Pleurotus ostreatus on the culture filtrate. The enzyme was purified to homogeneity using (NH4)2SO4 precipitation, DEAE-Sepharose and Sephadex G-100 column chromatography. The final enzyme activity achieved 81UmL-1, specific activity 78 U mg-1 with purification fold of 130 and recovery 1.2% of the crude enzyme. SDS-PAGE indicated that the pure enzyme have a molecular mass of approximately 42 kDa. The optimum pH was between 4-5 and the optimum temperature was 25 ºC. The pure MnP activity was enhanced by Mn2+,Cu2+,Ca2+ and K+ and inhibited by Hg+2 and Cd+2.H2O2 at 5 mM enhanced MnP activity while at 10 mM inhibited it significantly. The MnP-cDNA encoding gene was sequenced and determined (GenBank accession no. AB698450.1). The MnP-cDNA was found to consist of 497 bp in an Open Reading Frame (ORF) encoding 165 amino acids. MnP from P. ostreatus could detoxify aflatoxin B1 (AFB1) depending on enzyme concentration and incubation period. The highest detoxification power (90%) was observed after 48 h incubation at 1.5 U mL-1 enzyme activities.

Aflatoxins/metabolism , Peroxidases/isolation & purification , Peroxidases/metabolism , Pleurotus/enzymology , Biotransformation , Chemical Precipitation , Chromatography, Gel , Chromatography, Ion Exchange , DNA, Fungal/chemistry , DNA, Fungal/genetics , Electrophoresis, Polyacrylamide Gel , Enzyme Activators/metabolism , Enzyme Inhibitors/metabolism , Hydrogen-Ion Concentration , Molecular Sequence Data , Molecular Weight , Metals/metabolism , Open Reading Frames , Peroxidases/chemistry , Sequence Analysis, DNA , Temperature
Article in English | WPRIM | ID: wpr-812271


AIM@#To prepare high-purity ginseng total saponins from a water decoction of Chinese ginseng root.@*METHOD@#Total saponins were efficiently purified by dynamic anion-cation exchange following the removal of hydrophilic impurities by macroporous resin D101. For quality control, ultrahigh-performance liquid chromatography with a charged aerosol detector (CAD) was applied to quantify marker components. The total saponin content was estimated by a colorimetric method using a vanillin-vitriol system and CAD response.@*RESULTS@#D201, which consisted of a cross-linked polystyrene matrix and -N(+)(CH3)3 functional groups, was the best of the four anion exchange resins tested. However, no significant difference in cation exchange ability was observed between D001 (strong acid) and D113 (weak acid), although they have different functional groups and matrices. After purification in combination with D101, D201, and D113, the estimated contents of total saponins were 107% and 90% according to the colorimetric method and CAD response, respectively. The total amount of representative ginsenosides Re, Rd, Rg1, and compound K was approximately 22% based on ultrahigh-performance liquid chromatography-CAD quantitative analysis.@*CONCLUSION@#These findings suggest that an ion exchange resin, combined with macroporous adsorption resin separation, is a promising and feasible purification procedure for neutral natural polar components.

Adsorption , Chromatography, Ion Exchange , Methods , Drugs, Chinese Herbal , Chemistry , Ion Exchange Resins , Chemistry , Panax , Chemistry , Plant Roots , Chemistry , Porosity , Saponins , Chemistry
Article in English | WPRIM | ID: wpr-14498


Several studies have reported that the citrus red mites Panonychus citri were an important allergen of citrus-cultivating farmers in Jeju Island. The aim of the present study was to purify and assess properties of a cysteine protease from the mites acting as a potentially pathogenic factor to citrus-cultivating farmers. A cysteine protease was purified using column chromatography of Mono Q anion exchanger and Superdex 200 HR gel filtration. It was estimated to be 46 kDa by gel filtration column chromatography and consisted of 2 polypeptides, at least. Cysteine protease inhibitors, such as trans poxy-succinyl-L-leucyl-amido (4-guanidino) butane (E-64) and iodoacetic acid (IAA) totally inhibited the enzyme activities, whereas serine or metalloprotease inhibitors did not affect the activities. In addition, the purified enzyme degraded human IgG, collagen, and fibronectin, but not egg albumin. From these results, the cysteine protease of the mites might be involved in the pathogenesis such as tissue destruction and penetration instead of nutrient digestion.

Animals , Chromatography, Gel , Chromatography, Ion Exchange , Collagen/metabolism , Cysteine Proteases/chemistry , Cysteine Proteinase Inhibitors/metabolism , Fibronectins/metabolism , Humans , Immunoglobulin G/metabolism , Molecular Weight , Protein Subunits/chemistry , Proteolysis , Substrate Specificity , Tetranychidae/enzymology
Singapore medical journal ; : e126-8, 2014.
Article in English | WPRIM | ID: wpr-274210


Most haemoglobin (Hb) variants are clinically silent. However, some Hb variants may interfere with the measurement of haemoglobin A1c (HbA1c), resulting in spurious values depending on the assays used. We herein report the case of a 53-year-old Taiwanese man with type 2 diabetes mellitus, who presented with an abnormal HbA1c peak on ion-exchange chromatography. Additional investigations, including intensified self-monitored blood glucose tests, an alternative HbA1c assay, and a glycaemic indicator based on a different method, revealed that the HbA1c values were falsely elevated. Subsequent DNA analysis confirmed that the patient was heterozygous for the insertion of an isoleucine residue at codons 117/118 of the a1-globin gene, Hb Phnom Penh. Clinical laboratorians should be aware of the interfering factors in their HbA1c analysis. Cautious inspection of the chromatogram may provide a valuable clue to the presence of an Hb variant.

Chromatography, High Pressure Liquid , Chromatography, Ion Exchange , Diabetes Complications , Blood , Diabetes Mellitus, Type 2 , Blood , Dyslipidemias , Blood , Hemoglobins, Abnormal , Humans , Hypertension , Blood , Hypoglycemia , Blood , Male , Middle Aged , Reproducibility of Results , Sequence Analysis, DNA , Taiwan
Chinese Journal of Biotechnology ; (12): 404-411, 2014.
Article in Chinese | WPRIM | ID: wpr-279509


Ethyl carbamate (EC) is a carcinogenic substance in many fermented foods. Enzymatic removal of ethyl carbamate from fermented foods is an important way to eliminate its potential health damage to consumers. To study the enzymatic properties of an ethyl carbamate hydrolase (urethanase) from Klebsiella pneumoniae, a strain isolated from murine somach, we purified the enzyme using ammonium sulfate precipitation, ion exchange chromatography and gel filtration chromatography. The molecular mass of this enzyme was estimated to be 55 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Its K(m) was 74 mmol/L when EC was used as the substrate. Moreover, its optimal reaction temperature was 55 degrees C, and the optimum pH was 7.0. The activity was enhanced by ethylene diamine tetraacetic acid (EDTA) and dithiothreitol (DTT), but strongly inhibited by Cu2+ and Zn2+. The enzyme was halophilic and tolerant to low concentration of ethanol. Therefore, it has the potential to remove EC from fermented foods.

Amidohydrolases , Chemistry , Bacterial Proteins , Chemistry , Chromatography, Gel , Chromatography, Ion Exchange , Electrophoresis, Polyacrylamide Gel , Hydrogen-Ion Concentration , Klebsiella pneumoniae , Molecular Weight , Substrate Specificity , Temperature