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.

Subcellular localization and kinetic characterization of a gill (Na +, K+)-ATPase from the giant freshwater prawn macrobrachium rosenbergii

The stimulation by Mg2+, Na+, K+, NH 4 +, and ATP of (Na+, K+)-ATPase activity in a gill microsomal fraction from the freshwater prawn Macrobrachium rosenbergii was examined. Immunofluorescence labeling revealed that the (Na +, K+)-ATPase α-subunit is distributed predominantly within the intralamellar septum, while Western blotting revealed a single α-subunit isoform of about 108 kDa M r. Under saturating Mg2+, Na+, and K+ concentrations, the enzyme hydrolyzed ATP, obeying cooperative kinetics with V M = 115.0 ± 2.3 U mg-1, K 0.5 = 0.10 ± 0.01 mmol L-1. Stimulation by Na+ (V M = 110.0 ± 3.3 U mg-1, K 0.5 = 1.30 ± 0.03 mmol L -1), Mg2+ (V M = 115.0 ± 4.6 U mg -1, K 0.5 = 0.96 ± 0.03 mmol L-1), NH4 + (V M = 141.0 ± 5.6 U mg -1, K 0.5 = 1.90 ± 0.04 mmol L-1), and K+ (V M = 120.0 ± 2.4 U mg-1, K M = 2.74 ± 0.08 mmol L-1) followed single saturation curves and, except for K+, exhibited site-site interaction kinetics. Ouabain inhibited ATPase activity by around 73 % with K I = 12.4 ± 1.3 mol L-1. Complementary inhibition studies suggest the presence of F0F1-, Na+-, or K +-ATPases, but not V(H+)- or Ca2+-ATPases, in the gill microsomal preparation. K+ and NH4 + synergistically stimulated enzyme activity (≈25 %), suggesting that these ions bind to different sites on the molecule. We propose a mechanism for the stimulation by both NH4 +, and K+ of the gill enzyme. © 2013 Springer Science+Business Media New York.

Systemic CA-MRSA infection following trauma during soccer match in inner Brazil: Clinical and molecular characterization

Even though community-acquired methicillin resistant Staphylococcus aureus (CA-MRSA) was described a decade ago, reports from Brazil are scarce and cases occurred in large urban centers. We report MRSA sepsis in a 16-year-old male from a small town and who had no history of exposure to healthcare or recent travel. After trauma during a soccer match, he presented swelling in the right thigh, which evolved in a month to cellulitis complicated by local abscess, orchitis and pneumonia. The patient presented severe sepsis, with fever and respiratory failure. Laboratory findings included blood leukocyte counts above 40,000/mm3 and thrombocytopenia. He was submitted to mechanical ventilation and therapy with vancomycin and imipenem. He had a slow but favorable response to therapy and was discharged after six weeks of hospitalization. MRSA grew from blood cultures and respiratory aspirates obtained before antimicrobial therapy. The isolate belonged to sequence type 5, spa type t311, harbored SCCmec type IV and genes for Panton-Valentine leukocidin and Enterotoxin A. The pulsed-field gel electrophoresis pattern was distinct from North American classic CA-MRSA clones. However, the sequence type and the spa type revealed that the clone belong to the same clonal complex isolated in Argentina. This is the first CA-MRSA infection reported in that region, with significant epidemiologic and clinical implications. © 2013 Elsevier Inc.

The characterization of a thermostable and cambialistic superoxide dismutase from thermus filiformis

The superoxide dismutase (TfSOD) gene from the extremely thermophilic bacterium Thermus filiformis was cloned and expressed at high levels in mesophilic host. The purified enzyme displayed approximately 25 kDa band in the SDS-PAGE, which was further confirmed as TfSOD by mass spectrometry. The TfSOD was characterized as a cambialistic enzyme once it had enzymatic activity with either manganese or iron as cofactor. TfSOD showed thermostability at 65, 70 and 80°C. The amount of enzyme required to inhibit 50% of pyrogallol autoxidation was 0·41, 0·56 and 13·73 mg at 65, 70 and 80°C, respectively. According to the circular dichroism (CD) spectra data, the secondary structure was progressively lost after increasing the temperature above 70°C. The 3-dimensional model of TfSOD with the predicted cofactor binding corroborated with functional and CD analysis. © 2013 The Society for Applied Microbiology.

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.

Purification and structural characterization of snake venom proteins

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

Characterization of mannose-binding lectin plasma levels and genetic polymorphisms in HIV-1-infected individuals

INTRODUÇÃO: O presente estudo investigou a associação entre o polimorfismo no gene da lectina ligante de manose (MBL) e os níveis séricos da proteína com a infecção pelo HIV-1. MÉTODOS: As amostras de sangue (5mL) foram coletadas de 97 indivíduos infectados pelo HIV-1 residentes em Belém, Estado do Pará, Brasil, que frequentavam a Unidade de Referência Especial para Doenças Infecciosas e Parasitárias Especiais (URE-DIPE). Os níveis de linfócitos T CD4+ e da carga viral plasmática foram quantificados. Um fragmento de 349pb do exon 1 da MBL foi amplificado via PCR, utilizando DNA genômico extraído das amostras controles e dos indivíduos portadores do HIV-1, seguindo protocolos previamente estabelecidos. O nível plasmático de MBL nos pacientes foi quantificado usando kit de ensaio imunoenzimático. RESULTADOS: Dois alelos foram observados - MBL*O, com uma frequência de 26,3% em indivíduos infectados e o alelo selvagem MBL*A (73,7%). Frequências similares foram observadas no grupo controle (p > 0,05). As frequências genotípicas estavam em equilíbrio de Hardy-Weinberg em ambos os grupos. A média dos níveis plasmáticos MBL variou por genótipo, com diferenças significativas entre os genótipos AA e AO (p < 0,0001), e AA e OO (p < 0,001), mas não entre AO e OO (p=0,17). Além disso, os linfócitos T CD4+ e os níveis plasmáticos de carga viral não diferiram significativamente de acordo com o genótipo (p>0,05). CONCLUSÕES: Os resultados deste estudo não apoiam a hipótese de que o polimorfismo no gene MBL ou baixa concentração plasmática de MBL poderia ter uma influência direta sobre a infecção pelo HIV-1, embora um estudo com número maior de pacientes seja necessário.

Molecular characterization of human T-cell lymphotropic virus coinfecting human immunodeficiency virus 1 infected patients in the Amazon region of Brazil

ABSTRACT: The present work evaluated the epidemiology of human immunodeficiency virus 1/human T-cell lymphotropic virus (HIV-1/HTLV) coinfection in patients living in Belém (state of Pará) and Macapá (state of Amapá), two cities located in the Amazon region of Brazil. A total of 169 blood samples were collected. The sera were tested by enzyme-linked immunosorbent assay to determine the presence of antibodies anti-HTLV-1/2. Confirmation of infection and discrimination of HTLV types and subtypes was performed using a nested polymerase chain reaction targeting the pX and 5' LTR regions, followed by restriction fragment length polymorphism and sequencing analysis. The presence of anti-HTLV1/2 was detected in six patients from Belém. The amplification of the pX region followed by RFLP analysis, demonstrated the presence of HTLV-1 and HTLV-2 infections among two and four patients, respectively. Sequencing HTLV-1 5' LTR indicated that the virus is a member of the Cosmopolitan Group, Transcontinental subgroup. HTLV-2 strains isolated revealed a molecular profile of subtype HTLV-2c. These results are a reflex of the epidemiological features of HIV-1/HTLV-1/2 coinfection in the North region of Brazil, which is distinct from other Brazilian regions, as reported by previous studies.

Design, synthesis and characterization of a hexapeptide bio-inspired by acetylcholinesterase and its interaction with pesticide dichlorvos

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

Phytase production by Rhizopus microsporus var. microsporus biofilm: characterization of enzymatic activity after spray drying in presence of carbohydrates and nonconventional adjuvants

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

Production and Characterization of a Milk-clotting Protease Produced in Submerged Fermentation by the Thermophilic Fungus Thermomucor indicae-seudaticae N31

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Biophysical and Structural Characterization of the Recombinant Human eIF3L

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

Characterization of a Hexameric Exo-Acting GH51 alpha-l-Arabinofuranosidase from the Mesophilic Bacillus subtilis

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

PURIFICATION AND CHARACTERIZATION OF SOY COTYLEDON -GLUCOSIDASE

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Molecular and Kinetic Characterization of Two Extracellular Xylanases Isolated from Leucoagaricus gongylophorus

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