Evaluation of hollocelulase production by Lentinula edodes (Berk.) Pegler during the submerged fermentation growth using RSM / Avaliação da produção de enzimas holocelulolíticas por Lentinula edodes (Berk.) Pegler durante o crescimento em fermentação submersa usando MSR

The cellulase proteins have a great importance in the enzymatic hydrolysis of woody biomass. Despite of costs being a major concern, it has been a stimulus to study basidiomycetes biochemical properties which degrade lignocellulosic material and have prompted the processes' study for obtaining cellulolytic enzymes in fungi. The objective of this research was to evaluate the effects of the initial nitrogen content on (ammonium sulfate) and on sugar cane bagasse, which hereby, acts as an inducer of hydrolytic enzymes to produce cellulases and xylanases, using three Lentinula edodes (Berk.) Pegler strains as a transformation agent. A factorial design with 22 replications in the central point was conducted, varying concentrations of ammonium sulfate and sugar cane bagasse. The submerged cultures carried out in synthetic culture medium and incubated at 25°C for 7 days on an orbital shaker at 150 rpm. The total protein and cellulase activity as endoglucanase, exoglucanase and β-glucosidase and the xylanase was also determined. The results showed that the production of hydrolytic enzymes was stimulated by the presence of high concentrations of sugar cane bagasse (30g/L), characterizing it as an inducer due to the demonstrated proportional relationship. Thus, ammonium sulfate acted as a reducing agent in the synthesis of enzymes, being the low concentrations (0.1g/L) indicated for the enzyme production system under study. Among the studied strains, the EF52 showed higher activity for xylanase, endoglucanases, β-glucosidase and also protein.

As celulases são proteínas de grande importância na hidrólise enzimática de biomassa florestal. No entanto, seu custo elevado tem estimulado o estudo de processos de obtenção de enzimas celulolíticas por fungos filamentosos, tais como os basidiomicetos que apresentam propriedades bioquímicas para degradação de material lignocelulósico. O objetivo deste trabalho foi avaliar os efeitos do teor inicial de nitrogênio (sulfato de amônia) e de um indutor de enzimas hidrolíticas (bagaço de cana de açúcar) na produção de xilanases e celulases utilizando três isolados de Lentinula edodes (Berk.) Pegler como agente de transformação. Foi realizado um planejamento fatorial 22 com repetição no ponto central, variando as concentrações de sulfato de amônia e bagaço de cana de açúcar. O cultivo submerso realizado em meio de cultivo sintético e incubado a 25°C por 7 dias em agitador orbital a 150 rpm. Foram determinados o teor de proteínas totais e a atividade de celulase como: endoglucanase, exoglucanase e β-glucosidase e ainda xilanase. Os resultados demonstraram que a produção das enzimas hidrolíticas foi estimulada pela presença de alta concentração de bagaço de cana (30g/L), caracterizando-o como agente indutor devido à relação de proporcionalidade demonstrada. Por sua vez, o sulfato de amônio atuou como redutor da síntese de enzimas, sendo as baixas concentrações (0,1g/L) indicadas para o sistema de produção das enzimas em estudo. Quanto às linhagens, a EF52 mostrou maior atividade para xilanase, endoglucanases, β-glucosidase e proteínas.

Eliciadores proteicos e glicídicos de Adiantum capillus-veneris L. para fitoalexinas em sorgo Sorghum bicolor (L. ) Moench / Proteinaceous and carbohydrate elicitors from Adiantum capillus-veneris inducing phytoalexins in sorghum

Este trabalho objetivou a purificação parcial, por precipitação com sulfato de amônio (SA) e cromatografia de filtração em gel (CFG), de compostos presentes no decocto de Adiantum capillus-veneris (avenca) eficientes na indução de fitoalexinas em mesocótilos de Sorghum bicolor sorgo. Decocto de A. capillus-veneris a 1% (peso seco/volume) foi precipitado com concentrações de SA variando de 0 a 100% (em intervalos de 20%), e essas frações foram submetidas à CFG. Para o decocto não precipitado foram obtidos nove picos proteicos e um pico glicídico com massas moleculares variando de 0,61 à 0,01 KDa. Para a precipitação fracionada obteve-se: na fração 0-20% dois picos proteicos (menores que 0,01 KDa) e dois glicídicos com concentração de açúcares variando de 4,1 a 17,5 µg mL-1; na fração 20-40% três picos proteicos (111,5 à 0,98 KDa) e cinco glicídicos (11,3 a 73,7 µg de açúcares mL-1); na fração 40-60% dois picos proteicos (111,5 à 0,09 KDa) e dois glicídicos (5,6 a 7, 5 µg de açúcares mL-1); na fração 60-80% seis picos proteicos (menores que 0,02 KDa) e dois glicídicos (16,5 a 51,3 µg de açúcares mL-1); e na fração 80-100% três picos proteicos (menores que 0,09 KDa). Mesocótilos de sorgo foram tratados com as frações provenientes da CFG, além do decocto a 1%, acibenzolar-S-metil (ASM) (125 mg L-1 do i.a. como eliciador de referência) e tampão fosfato de sódio 10 mM pH 6,0. O pico proteico II (0,09 KDa) do decocto não precipitado induziu fitoalexinas, 6,68% superior a ASM. Entre os precipitados, a fração 60-80% de SA induziu 76% mais que ASM. Dessa forma, pôde-se obter frações proteicas e/ou glicídicas indutoras de fitoalexinas em sorgo de maneira superior ao extrato (decocto) do qual é originária, indicando o potencial dessas moléculas para trabalhos futuros sobre indução de resistência.

This study aimed to partially purify the compounds present in decoction of Adiantum capillus-veneris, which are efficient in the induction of phytoalexins in sorghum mesocotyl, by ammonium sulphate (AS) fractionation and gel filtration chromatography (GFC). The decoction of A. capillus-veneris at 1% (weight/volume) was precipitated with AS at the concentration of 0-20%, 20-40%, 40-60%, 60-80% and 80-100%, and these fractions were subjected to GFC. For the decoction not precipitated with AS, nine protein peaks and one carbohydrate peak were obtained with molecular weights ranging from 0.61 to 0.01 KDa. For the AS precipitation, we obtained: for the fraction 0-20%, two protein peaks (0.01 KDa) and two carbohydrate peaks with concentration of sugars ranging from 4.1 to 17.5 µg of sugar mL-1; for the 20-40%, three protein peaks (0.98 to 111.5 KDa) and five carbohydrate peaks (11.3 to 73.7 µg sugar mL-1); for the 40-60%, two protein peaks (0.09 to 111.5 KDa) and two carbohydrate peaks (5.6 to 7.5 µg of sugar mL-1); for the 60-80%, six protein peaks (lower than 0.02 KDa) and two carbohydrate peaks (16.5 to 51.3 µg of sugar mL-1); and for the 80-100%, three protein peaks with molecular weight equivalent to 0.09 KDa. The sorghum mesocotyls were treated with GFC fractions, decoction (1%), acibenzolar-S-methyl (ASM) (125 mg L-1 a.i. as elicitor reference) and sodium phosphate buffer (10 mM, pH 6.0). The protein peak II (0.09 KDa) from the decoction not precipitated was effective in inducing phytoalexin, exceeding in 6.68% the ASM. Among the fractions, the one with 60-80% of AS increased in 76% the induction of phytoalexin compared to ASM. According to the results, we could obtain protein and/or carbohydrate fractions capable of inducing phytoalexins in sorghum better than the decoction from which they are derived from, showing the potential of these molecules for future research studies on the induction of resistance.

Laboratory studies on the effect of inorganic fertilizers on survival and development of immature Culex quinquefasciatus (Diptera: Culicidae).

BACKGROUND & OBJECTIVES: Studies were conducted to determine the effect of ammonium sulfate (AM) and muriate of potash (MOP) fertilizers on survival and development of immature stages of Culex quinquefasciatus Say, a major vector of Bancroftian filariasis in Africa. METHODS: Twenty I instar larvae each were added in four doses of each fertilizer dissolved in one litre of deionised water and in one litre of deionised water as a control in replicates of five. The larvae were monitored every morning throughout their life and the numbers of each instar surviving were recorded. The experiments were discontinued when all the larvae had died or emerged into adults. RESULTS: An analysis of variance test and Tukey's HSD test revealed a significant impact of fertilizers on survival and development of aquatic stages of Cx. quinquefasciatus. Ammonium sulfate accounted for up to 40% mortality rate, and one week delay in development time and this effect was both instar and dose dependent. None of the MOP dosages had significant impact on survival of immatures of Cx. quinquefasciatus and only the higher dosages showed significant impact on development time but in significantly lower magnitudes compared with similar dosages of ammonium sulfate. INTERPRETATION & CONCLUSION: These findings demonstrate the toxic effect of fertilizers on immature stages of Cx. quinquefasciatus contrary to field observations.

Efeitos da adubação nitrogenada em algodoeiro sobre a biologia de Aphis gossypii Glover (Hemiptera: Aphididae) / Effects of nitrogen fertilization in cotton crop on Aphis gossypii Glover (Hemiptera: Aphididae) biology

O pulgão Aphis gossypii Glover é uma das pragas do algodoeiro e suas relações com o hospedeiro são dependentes da quantidade de nitrogênio disponível para a planta. A biologia do A. gossypii, em função do regime de adubação nitrogenada no algodoeiro, foi estudada em condições de casa-de-vegetação, em Dourados, MS. Para isto foi utilizado o delineamento experimental inteiramente casualizado com nove repetições, com os tratamentos arranjados em fatorial (2 x 4 x 2) + 1, com duas fontes de adubo nitrogenado, quatro doses de nitrogênio (50, 100, 150 e 200 kg ha-1), duas épocas de aplicação do nitrogênio em cobertura e um tratamento adicional sem a adição do nitrogênio. Foram avaliadas as durações dos estádios ninfais e da fase ninfal, os períodos pré-reprodutivo, reprodutivo e pós-reprodutivo, a longevidade, o ciclo biológico e a fecundidade dos pulgões. Concluiu-se que apenas as doses de nitrogênio influenciaram a biologia do pulgão-do-algodoeiro, independente da fonte e época de aplicação, favorecendo seu desenvolvimento e fecundidade.

The cotton aphid, Aphis gossypii Glove, is one of the pests of cotton crop and its relation with the host seem to depend on the amount of nitrogen available to the plant. The biology of A. gossypii using different cotton nitrogen fertility regimes was studied under greenhouse conditions, in Dourados, MS. A completely randomized design with nine replications in a factorial scheme (2x4x2)+1 was used. Two nitrogen sources (sulphate of ammonium and urea), four doses of nitrogen (50, 100, 150 and 200 kg ha-1), two different times of nitrogen application and one additional treatment without nitrogen were taken as factors. The nymphal phases, the pre-reproductive, reproductive and pos-reproductive periods, longevity, the life cycle and fecundity of the cotton aphid were evaluated. The doses of nitrogen influenced the cotton aphid biology in both sources and times of application, favoring its development and fecundity.

A simple method for efficient extraction and purification of C-phycocyanin from Spirulina platensis Geitler.

Phycocyanin is a major light harvesting accessory pigment of red algae and cyanobacteria. In the light of its many commercial applications in food and pharmaceutical industry, purity of the pigment plays a major role. Pharmaceutical industry demands a highly pure phycocyanin with A620/280 ratio of 4 and food industry a ratio of 2. In the present study phycocyanin was extracted in sodium phosphate buffer (pH 7) after macerating in liquid nitrogen. The crude phycocyanin thus extracted was precipitated with 50% ammonium sulphate, purified by dialysis and finally by gel filtration chromatography. Pure phycocyanin was finally obtained with an A620/A280 value of 4.98.

Aerobic growth of motile and non-motile bacteria an the surface of solid culture medium in the presence of the heavy metal and ionic salts at two temperatures

Threshold concentrations of heavy metal and ionic salts defining the range of Concentrations, that do not suppress growth, were determined in tryptic soya agar medium. It has been found that the salts of NH4, Mg, Cd and Hg at low concentrations disrupt the symbiotic relationship between the solid surface culture medium and the microorganisms, but do not suppress the growth of the bacteria. These compounds have been included under two different groups as infection inhibitors [Cd and Hg] and as an infection stimulators [NH4, Mg and K]

Fluoride and phosphatidylserine induced inhibition of cytosolic insulin-degrading activity

Cytosol (C) (100,000 x g/60 min, supernatant) from liver, brain and testis (Wistar male rats) are shown to contain insulin degrading activity (C-IDA). The regulation of C-IDA in these fractions by ligands that activate G protein and PKC were examined C-IDA from liver, brain and testis was inhibited 76%; 64% and 50% by 50 mM F- respectively. Chromatography of C fraction from liver on Sephadex G-50 in presence of 1 M (NH4)2SO4 and 20% (v/v) glycerol (experimental condition to remove guanine nucleotides from G proteins) decreased in about 3-fold aluminum fluoride effect on C-IDA. Mg++ (from 5mM to 10 mM) enhanced fluoride effects by inhibiting fully C-IDA. Phosphatidylserine in presence of ATP completely inhibited C-IDA; this inhibition was 31.3% mediated by a phosphorylation reaction. It is concluded that cytosol from different tissues contain proteins capable to associate ligands as aluminum fluoride and PS to regulate C-IDA. It is proposed a mechanism of protein-protein interaction to modulate C-IDA

Efecto de iones amonio sobre la entrada de L-leucina en Saccharomyces cerevisiae: represíon e inhibicíon of transport systems / Effect of ammoniun ions on the uptake of L-leucine: repression and inhibition of transport systems

En Saccharomyces cerevisiae la entrada de L-leucina es mediada por la permeasa general de aminoácidos, GAP, y dos sistemas cinéticamente caracterizados, uno de alta afinidad y baja velocidad, S1, para concentraciones externas de L-leucina 0,05-01mM y otro de baja afinidad, alta velocidad, S2, para concentraciones externas 1.0mM. En células crecidas en medios suplementados con amonio, como única fuente de nitrógeno, los valores de entrada e incorporación son menores que en células crecidas en medios suplementados con L-prolina. En condiciones de represión de la GAP por iones amonio, la entrada de L-leucina es mediada por los sistemas S1 y S2. Los dos sistemas son parcialmente inhibidos por efecto de iones amonio. En condiciones de depresion de la GAP, por crecimiento en L-prolina, la entrada de L-leucina es mediada por los sistemas S1 y GAP, bajas concentraciones externas de L-leucina mediada por centraciones externas. El amonio inhibe en mayor extensión la entrada de L-leucina mediada por La GAP

Tratamiento de la paja de trigo con urea, hidroxido de calcio, o sulfato de amonio / Treatment of wheat straw with urea, calcium hydroxide or ammonium sulfate

Se llevó a cabo un experimento con la finalidad de evaluar la efectividad de la urea, sulfato de amonio o hidróxido de calcio en el mejoramiento del valor nutritivo de la paja de trigo para la alimentación de ruminantes. La paja se sometió a tratamiento con tres soluciones que contenían 5% de urea 50% H2O (5U); 2% de urea-2% CaOH-50% H2O (2U) o con sulfato de amonio, 2% CaOH-50% H2O (5S), ensilándose luego la paja tratada con cada solución, por triplicado, en bolsas de poletileno (5Kg/bolsa) durante 30 días a temperatura ambiente (15-22-C a excepción de la paja sin tratar, que se usó como testigo (T). Posteriormente se hicieron las siguientes determinaciones en las pajas sujetas a los tres tratamientos: pH, humedad, proteína cruda, N amoniacal, cenizas, paredes celulares, hemicelulosa, celulosa, lignina, sílice, energía bruta y digestibilidad in vitro de la materia seca. El valor de pH de 5U fue mayor (P < 0.01) que el de 2U y 5S, y el pocentaje de humedad aumentó en todas las pajas tratadas, en comparación con la testigo. Tanto el contenido de proteína cruda como el de nitrégeno amoniacal fue mayor en las pajas tratadas con 5U (14.66; 1.06%), 2U (5.41; 75%), 5S (9.54; 94%), en comparación con la testigo. Tanto el contenido de proteína cruda como el de nitógeno amoniacal fue mayor en las pajas tratadas con 5U (14.66; 1.06%), 2U (5.41; 75%), 5S (9.54; 94%), en comparación con t (2.63; 50%). Asimismo, el porcentaje de cenizas con 5U, 2U y 5S, aumentó significativamente (P < 0.01) en relación con T y 5U. El contenido de paredes celulares disminuyó al tratar la paja con 5U, 2U y 5S, al igual que el de hemicelulosa, lignina y sílice, mientras que el de celulosa aumentó. Los valores de energía bruta fueron mayores en las pajas tratadas, y el porcentaje de digestibilidad de la materia seca fue mayor, aunque no en grado significativo (P < 0.01) en la paja tratada con 5% de urea (45.03) que en 2U (42.43), 5S (34.82) y T (42.36)

A culture medium for cultivation of mycobacteria, probably Mycobacterium leprae from Mycobacterium leprae infected tissues.

Mycobacterium leprae suspensions were prepared from infected armadillos. The M. leprae cells were inoculated into culture media containing KH2PO4 4.7. g. Na2HPO4 2 g, sodium thioglycolate 1 g, (NH4)2SO4 2 g, MgSO4 0.1 g, ferric ammonium citrate 0.05 g, and lipoic acid (thioctic acid) 0.1 g in one liter distilled water. The solution was enriched with heat killed, sonicated leprosy derived Mycobacterium X or crude mycobactin extract from M. phlei to contain + 0.2 micrograms mycobactin per 1 ml in the final medium. Twenty ml media was distributed into each of 25 ml screw cap tubes and autoclaved for 30 minutes. Positive growth was obtained from seven out of ten specimens when incubated at 34 degrees C. The cultures developed as a sediment in the liquid media, suggesting preference for microaerophylic conditions. No growth was seen on the surface of the semi-solid agar media containing the same ingredients. Latency period of growth was estimated as 10-16 days and time of division as 6 days. Subcultures were obtained. Cells were long, acid fast, arranged side by side or end to end, with a tendency to form long spiral cords or clumps when sedimented on siliconized slides. Pyridine extraction eliminated acid fastness, but not gram positivity. Cultures did not grow on Dubos, Lowenstein or 7H10 media. They produce the disease in the foot pads of mice characteristic of M. leprae. Subcultures remain dependent on the heat killed sonicated mycobacteria, or crude mycobactin extract, and reduced oxygen tension in the media. Results suggest that cultures might be identical to M. leprae.