Biotechnological potential of an exo-polygalacturonase of the new strain Penicillium janthinellum VI2R3M: biochemical characterization and clarification of fruit juices.

AIMS: The aim of this work was to characterize and apply a polygalacturonase of Penicillium janthinellum new strain VI2R3M. METHODS AND RESULTS: The polygalacturonase obtained from P. janthinellum VI2R3M was incubated in cultures of passion fruit peel and was partially purified by ion-exchange chromatography and gel filtration. The enzyme showed a relative molecular mass of 102·0 kDa, maximum activity at pH 5·0, temperature of 50°C, 100% stablity at 50°C and 80% stablity at pH 3·0-5·0. The apparent Km , Vmax and Kcat values for hydrolyzing polygalacturonic acid were 2·56 mg ml-1 , 163·1 U mg-1 and 277 s-1 respectively. The polygalacturonase presented exo activity and was activated by Mg2+ . The juices treated with polygalacturonase presented increases in transmittance with reduction in colour. CONCLUSIONS: The results suggest that the new lineage P. janthinellum VI2R3M presents a high yield of an exo-polygalacturonase induced by agro-industrial residues, with excellent activity and stability in acidic pH and at 50°C. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of agro-industrial residue to obtain the polygalacturonase can contribute to a decrease enzyme production cost. The results of the activity, stability to acidic pH and excellent performance in the clarification of juices show that the enzyme is promising for industrial application.

GH11 xylanase from Aspergillus tamarii Kita: Purification by one-step chromatography and xylooligosaccharides hydrolysis monitored in real-time by mass spectrometry.

The present study describes the one-step purification and biochemical characterization of an endo-1,4-ß-xylanase from Aspergillus tamarii Kita. Extracellular xylanase was purified to homogeneity 7.43-fold through CM-cellulose. Enzyme molecular weight and pI were estimated to be 19.5kDa and 8.5, respectively. The highest activity of the xylanase was obtained at 60°C and it was active over a broad pH range (4.0-9.0), with maximal activity at pH 5.5. The enzyme was thermostable at 50°C, retaining more than 70% of its initial activity for 480min. The K0.5 and Vmax values on beechwood xylan were 8.13mg/mL and 1,330.20µmol/min/mg of protein, respectively. The ions Ba2+ and Ni2+, and the compounds ß-mercaptoethanol and DTT enhanced xylanase activity, while the heavy metals (Co2+, Cu2+, Hg+, Pb2+ and Zn2+) strongly inhibited the enzyme, at 5mM. Enzymatic hydrolysis of xylooligosaccharides monitored in real-time by mass spectrometer showed that the shortest xylooligosaccharide more efficiently hydrolyzed by A. tamarii Kita xylanase corresponded to xylopentaose. In agreement, HPLC analyzes did not detect xylopentaose among the hydrolysis products of xylan. Therefore, this novel GH11 endo-xylanase displays a series of physicochemical properties favorable to its application in the food, feed, pharmaceutical and paper industries.

Expression and characterization of an N-truncated form of the NifA protein of Azospirillum brasilense

Azospirillum brasilense is a nitrogen-fixing bacterium associated with important agricultural crops such as rice, wheat and maize. The expression of genes responsible for nitrogen fixation (nif genes) in this bacterium is dependent on the transcriptional activator NifA. This protein contains three structural domains: the N-terminal domain is responsible for the negative control by fixed nitrogen; the central domain interacts with the RNA polymerase σ54 co-factor and the C-terminal domain is involved in DNA binding. The central and C-terminal domains are linked by the interdomain linker (IDL). A conserved four-cysteine motif encompassing the end of the central domain and the IDL is probably involved in the oxygen-sensitivity of NifA. In the present study, we have expressed, purified and characterized an N-truncated form of A. brasilense NifA. The protein expression was carried out in Escherichia coli and the N-truncated NifA protein was purified by chromatography using an affinity metal-chelating resin followed by a heparin-bound resin. Protein homogeneity was determined by densitometric analysis. The N-truncated protein activated in vivo nifH::lacZ transcription regardless of fixed nitrogen concentration (absence or presence of 20 mM NH4Cl) but only under low oxygen levels. On the other hand, the aerobically purified N-truncated NifA protein bound to the nifB promoter, as demonstrated by an electrophoretic mobility shift assay, implying that DNA-binding activity is not strictly controlled by oxygen levels. Our data show that, while the N-truncated NifA is inactive in vivo under aerobic conditions, it still retains DNA-binding activity, suggesting that the oxidized form of NifA bound to DNA is not competent to activate transcription.

Expression and characterization of an N-truncated form of the NifA protein of Azospirillum brasilense.

Azospirillum brasilense is a nitrogen-fixing bacterium associated with important agricultural crops such as rice, wheat and maize. The expression of genes responsible for nitrogen fixation (nif genes) in this bacterium is dependent on the transcriptional activator NifA. This protein contains three structural domains: the N-terminal domain is responsible for the negative control by fixed nitrogen; the central domain interacts with the RNA polymerase σ(54) co-factor and the C-terminal domain is involved in DNA binding. The central and C-terminal domains are linked by the interdomain linker (IDL). A conserved four-cysteine motif encompassing the end of the central domain and the IDL is probably involved in the oxygen-sensitivity of NifA. In the present study, we have expressed, purified and characterized an N-truncated form of A. brasilense NifA. The protein expression was carried out in Escherichia coli and the N-truncated NifA protein was purified by chromatography using an affinity metal-chelating resin followed by a heparin-bound resin. Protein homogeneity was determined by densitometric analysis. The N-truncated protein activated in vivo nifH::lacZ transcription regardless of fixed nitrogen concentration (absence or presence of 20 mM NH(4)Cl) but only under low oxygen levels. On the other hand, the aerobically purified N-truncated NifA protein bound to the nifB promoter, as demonstrated by an electrophoretic mobility shift assay, implying that DNA-binding activity is not strictly controlled by oxygen levels. Our data show that, while the N-truncated NifA is inactive in vivo under aerobic conditions, it still retains DNA-binding activity, suggesting that the oxidized form of NifA bound to DNA is not competent to activate transcription.

Qualidade microbiológica do leite cru produzido na região oeste do Paraná e ocorrência de Listeria monocytogenes / Microbiologic quality of raw milk produced in the west region of Paraná and occurrence of Listeria monocytogenes

Foram estudadas 34 propriedades rurais, no período de outubro a dezembro de 2006, com 68 amostras de leite coletadas em triplicata diretamente dos tanques resfriadores ou vasilhames equivalentes. Foi pesquisada a presença de Listeria spp., comparando-se com o nível de contagem bacteriana total no leite de tanques. Foi utilizado agar-Aloa para isolamento de Listeria spp e de Listeria monocytogenes, com confirmação por PCR. Das amostras de leite cru submetidas a pesquisa de Listeria spp. observou-se que 19 (27,9%) estavam contaminadas, e duas (2,9%) apresentaram resultado positivo para L. monocytogenes. A contagem bacteriana total apresentou média de 5,41 log UFC/mL (±0,57), e 29,3% das amostras estavam acima do limite de 7,5 x 103 UFC/mL. Os resultados permitem afirmar que este patógeno deve ser pesquisado em amostras de leite cru por constituir um importante indicador de segurança alimentar e que os produtores rurais não têm conhecimento adequado sobre os riscos a que estão sujeitos ao manipularem e consumirem este produto.

The presence of Listeria spp., correlated at the level of total bacterial count in bulk tank milk was investigated, about 34 farms in the period of October to December 2006, with 68 triplicate samples of milk collected directly from the bulk tanks or equivalent containers. Aloa-agar was used for isolation of Listeria spp. and Listeria monocytogenes, with confirmation by PCR (polymerase chain reaction). In the samples of raw milk subjected to search of Listeria spp., it was observed that 19 (27.9%) were contaminated, and two (2.9%) were positive for L. monocytogenes. The total bacterial count showed an average of 5.41 log CFU/mL (± 0.57), and 29.3% of samples were above the limit of 7.5 x 10 CFU/mL. The results indicate that this pathogen should be studied in samples of raw milk cause it's an important indicator of food security and farmers don't have adequate knowledge about the risks they are subject to manipulate and consume this product.

QUALIDADE MICROBIOLÓGICA DO LEITE CRU PRODUZIDO NA REGIÃO OESTE DO PARANÁ E OCORRÊNCIA DE Listeria monocytogenes

Foram estudadas 34 propriedades rurais, no período de outubro a dezembro de 2006, com 68 amostras de leite coletadas em triplicata diretamente dos tanques resfriadores ou vasilhames equivalentes. Foi pesquisada a presença de Listeria spp., comparando-se com o nível de contagem bacteriana total no leite de tanques. Foi utilizado agar-Aloa para isolamento de Listeria spp e de Listeria monocytogenes, com confirmação por PCR. Das amostras de leite cru submetidas a pesquisa de Listeria spp. observou-se que 19 (27,9%) estavam contaminadas, e duas (2,9%) apresentaram resultado positivo para L. monocytogenes. A contagem bacteriana total apresentou média de 5,41 log UFC/mL (± 0,57), e 29,3% das amostras estavam acima do limite de 7,5 x 105 UFC/mL. Os resultados permitem afirmar que este patógeno deve ser pesquisado em amostras de leite cru por constituir um importante indicador de segurança alimentar e que os produtores rurais não têm conhecimento adequado sobre os riscos a que estão sujeitos ao manipularem e consumirem este produto.PALAVRAS-CHAVE: Boas práticas. Leite cru. Segurança alimentar

QUALIDADE MICROBIOLÓGICA DO LEITE CRU PRODUZIDO NA REGIÃO OESTE DO PARANÁ E OCORRÊNCIA DE Listeria monocytogenes

Foram estudadas 34 propriedades rurais, no período de outubro a dezembro de 2006, com 68 amostras de leite coletadas em triplicata diretamente dos tanques resfriadores ou vasilhames equivalentes. Foi pesquisada a presença de Listeria spp., comparando-se com o nível de contagem bacteriana total no leite de tanques. Foi utilizado agar-Aloa para isolamento de Listeria spp e de Listeria monocytogenes, com confirmação por PCR. Das amostras de leite cru submetidas a pesquisa de Listeria spp. observou-se que 19 (27,9%) estavam contaminadas, e duas (2,9%) apresentaram resultado positivo para L. monocytogenes. A contagem bacteriana total apresentou média de 5,41 log UFC/mL (± 0,57), e 29,3% das amostras estavam acima do limite de 7,5 x 105 UFC/mL. Os resultados permitem afirmar que este patógeno deve ser pesquisado em amostras de leite cru por constituir um importante indicador de segurança alimentar e que os produtores rurais não têm conhecimento adequado sobre os riscos a que estão sujeitos ao manipularem e consumirem este produto.PALAVRAS-CHAVE: Boas práticas. Leite cru. Segurança alimentar

QUALIDADE MICROBIOLÓGICA DO LEITE CRU PRODUZIDO NA REGIÃO OESTE DO PARANÁ E OCORRÊNCIA DE Listeria monocytogenes

Foram estudadas 34 propriedades rurais, no período de outubro a dezembro de 2006, com 68 amostras de leite coletadas em triplicata diretamente dos tanques resfriadores ou vasilhames equivalentes. Foi pesquisada a presença de Listeria spp., comparando-se com o nível de contagem bacteriana total no leite de tanques. Foi utilizado agar-Aloa para isolamento de Listeria spp e de Listeria monocytogenes, com confirmação por PCR. Das amostras de leite cru submetidas a pesquisa de Listeria spp. observou-se que 19 (27,9%) estavam contaminadas, e duas (2,9%) apresentaram resultado positivo para L. monocytogenes. A contagem bacteriana total apresentou média de 5,41 log UFC/mL (± 0,57), e 29,3% das amostras estavam acima do limite de 7,5 x 105 UFC/mL. Os resultados permitem afirmar que este patógeno deve ser pesquisado em amostras de leite cru por constituir um importante indicador de segurança alimentar e que os produtores rurais não têm conhecimento adequado sobre os riscos a que estão sujeitos ao manipularem e consumirem este produto.PALAVRAS-CHAVE: Boas práticas. Leite cru. Segurança alimentar

Characterization of trehalase activities from the thermophilic fungus Scytalidium thermophilum.

The thermophilic fungus Scytalidium thermophilum produced large amounts of intracellular and extracellular trehalase activity when grown on starch as the sole carbon source. The specific activity of the purified proteins: 1700 U (mg protein)-1 (extracellular) and 3700 U (mg protein)-1 (intracellular), was many times higher than the values reported for other microbial sources. The apparent molecular mass of the native enzymes was estimated to be 370 kDa (extracellular trehalase) and 398 kDa (intracellular trehalase) by gel-filtration chromatography. Analysis by SDS-PAGE showed unique polypeptide bands of approx. 82 kDa (extracellular trehalase) and 85 kDa (intracellular trehalase), suggesting that the native enzymes were composed of five subunits. The carbohydrate content of extracellular and intracellular trehalases was estimated to be 81% and 51%, respectively. Electrofocusing indicated a pI of 3.7 and 3.4, respectively, for the extracellular and intracellular enzymes. Both trehalases were highly specific for trehalose and were stimulated by calcium and manganese. Calcium and manganese also protected both trehalases from thermoinactivation. Inhibition was observed in the presence of aluminium, mercurium, copper, zinc, EDTA, ADP, and ATP. Apparent Km values, for the extracellular and intracellular trehalases, were 3.58 mM and 2.24 mM, respectively. The optimum of pH for the extracellular and the intracellular trehalase was 6.0, and the optimum of temperature 60 degrees C and 65 degrees C, respectively.