Screening and Production Study of Microbial Xylanase Producers from Brazilian Cerrado

Hemicelluloses are polysaccharides of low molecular weight containing 100 to 200 glycosidic residues. In plants, the xylans or the hemicelluloses are situated between the lignin and the collection of cellulose fibers underneath. The xylan is the most common hemicellulosic polysaccharide in cell walls of land plants, comprising a backbone of xylose residues linked by beta-1,4-glycosidic bonds. So, xylanolytic enzymes from microorganism have attracted a great deal of attention in the last decade, particularly because of their biotechnological characteristics in various industrial processes, related to food, feed, ethanol, pulp, and paper industries. A microbial screening of xylanase producer was carried out in Brazilian Cerrado area in Selviria city, Mato Grosso do Sul State, Brazil. About 50 bacterial strains and 15 fungal strains were isolated from soil sample at 35 A degrees C. Between these isolated microorganisms, a bacterium Lysinibacillus sp. and a fungus Neosartorya spinosa as good xylanase producers were identified. Based on identification processes, Lysinibacillus sp. is a new species and the xylanase production by this bacterial genus was not reported yet. Similarly, it has not reported about xylanase production from N. spinosa. The bacterial strain P5B1 identified as Lysinibacillus sp. was cultivated on submerged fermentation using as substrate xylan, wheat bran, corn straw, corncob, and sugar cane bagasse. Corn straw and wheat bran show a good xylanase activity after 72 h of fermentation. A fungus identified as N. spinosa (strain P2D16) was cultivated on solid-state fermentation using as substrate source wheat bran, wheat bran plus sawdust, corn straw, corncob, cassava bran, and sugar cane bagasse. Wheat bran and corncobs show the better xylanase production after 72 h of fermentation. Both crude xylanases were characterized and a bacterial xylanase shows optimum pH for enzyme activity at 6.0, whereas a fungal xylanase has optimum pH at 5.0-5.5. They were stable in the pH range 5.0-10.0 and 5.5-8.5 for bacterial and fungal xylanase, respectively. The optimum temperatures were 55C and 60 A degrees C for bacterial and fungal xylanase, respectively, and they were thermally stable up to 50 A degrees C.

Production of prostaglandins and leukotrienes by Paracoccidioides brasiliensis

Paracoccidioides brasiliensis is the causative agent of paracoccidioidomycosis, the most prevalent deep mycosis in Latin America. Production of eicosanoids, including prostaglandins and leukotrienes, during fungal infections is theorized to play a critical role on fungal survival and/or growth as well as on host immune response modulation. Host cells are one source of these mediators; however another potential source may be the fungus itself. The purpose of our study was to assess whether P. brasiliensis strains with different degree of virulence (Pb18, Pb265, PbBT79, Pb192) produce both, prostaglandin E(2) (PGE(2)) and leukotriene B(4) (LTB(4)). Moreover, we asked if P. brasiliensis can use exogenous sources of arachidonic acid (AA), as well as metabolic pathways dependent on cyclooxygenase (COX) and lipoxygenase (5-LO) enzymes, for PGE(2) and LTB(4) production, respectively. Finally, a possible association between these eicosanoids and fungus viability was assessed. We demonstrated, using ELISA assays, that all P. brasiliensis strains, independently of their virulence, produce high PGE(2) and LTB(4) levels after a 4-hour culture, which were reduced after 8 hours. However, in both culture times, higher eicosanoids levels were detected when culture medium was supplemented with exogenous AA. Differently, treatment with indomethacin, a COX inhibitor, or MK886, a 5-LO inhibitor, induces a reduction on PGE(2) and LTB(4) levels, respectively, as well as in fungus viability. The data provide evidence that P. brasiliensis is able to metabolize either endogenous or exogenous AA by pathways that depend on COX and 5-LO enzymes for producing, respectively, PGE(2) and LTB(4) that are critical for its viability.

Prostaglandin E-2 production by high and low virulent strains of Paracoccidioides brasiliensis

The production of prostaglandins (PGs) during fungal infections could be an important suppressor factor of host immune response. Host cells are one source of prostaglandin E-2 (PGE(2)); however another potential source of PGE(2) is the fungal pathogen itself. Thus, both host and fungal PGE2 production is theorized to play a role in pathogenesis, being critical for growth of the fungus and to modulate the host immune response. The purpose of this work was to investigate if high and low virulent strains of Paracoccidioides brasiliensis have the capacity to produce PGE(2) in vitro, and if this production was related to the fungal growth. The results demonstrated that both strains of P. brasiliensis produce high levels of PGE(2) and the treatment with indomethacin, a cyclooxygenase inhibitor, significantly reduced the production of this mediator, as well as the viability of the fungus. Thus, our data indicate that PGE(2) is produced by P. brasiliensis by a cyclooxygenase-dependent metabolic pathway, and its production is required for fungal survival. This discovery reveals an important factor that has potentially great implications for understanding the mechanisms of immune deviation during infection.

Ecological study of Paracoccidioides brasiliensis in soil: growth ability, conidia production and molecular detection

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

Cytokine production in lungs and adrenal glands of high and low antibody producing mice infected with Paracoccidioides brasiliensis

Mice genetically selected for high (H) and low (L) antibody production (HIV-A and L-IV-A) were used in an experimental model of paracoccidioidomycosis. In a previous work, it was observed that male HIV-A animals were more susceptible to the infection due to adrenal gland damage. Male HIV-A and LIV-A animals were intravenously inoculated with Paracoccidioides brasiliensis (strain 18) and sacrificed 2, 4, 6, 8 and 10 weeks after inoculation. At each time interval, lungs and adrenals were removed to estimate recoverability of the fungus, as well as to determine Th1 (IFN-gamma, TNF-alpha) and Th2 (IL-4 and IL-10) cytokine profiles. While viable fungi recoverability from the lungs of HIV-A mice was higher after 4 and 8 weeks, there was less fungal recovery from the adrenals of LIV-A animals after the 2nd week, with total fungal elimination after the 8th week. With regard to Th2 cytokines, there was an inhibition in IL-4 production in the organs from infected animals, the extent of which varied according to the organ and the time period after initiation of infection. IL-10 production was found to be lower in both organs. Determination of Th1 cytokines revealed that IFN-gamma production increased in both organs, mainly in the adrenal of LIV-A after 8 and 10 weeks, when these animals showed a total fungal elimination. A significant difference was observed between HIV-A and LIV-A concerning TNF-alpha production in both organs and at all recovery times, in that LIV-A produced a higher level of this cytokine, mainly in the adrenal. These results may explain the high susceptibility of HIV-A to P. brasiliensis infection, is due, at least in part, to adrenal involvement. The higher production of Th1 cytokines by LIV-A in comparison to HIV-A mice may account for LIV-A resistance to P. brasiliensis infection. Our data reveal the importance of this experimental model in the study of the adrenal involvement in paracoccidioidomycosis, since this gland may be highly compromised in the patients, leading to the development of Addison's Disease.

Cytokine production in experimental paracoccidioidomycosis of murine selected lineages for acute inflammatory response (air)

Paracoccidioidomycosis is a systemic human mycosis caused by Paracoccidioides brasiliensis (P. brasiliensis), an imperfect dimorphic fungus whose conidia are its infective form. Mice genetically selected for maximum (AIRmax) and minimum (AIRmin) acute inflammatory response were used as experimental paracoccidioidomycosis models. The animals were intraperitoneally inoculated with P. brasiliensis (strain 18) and killed 6, 12 and 24 hours or 3, 7 and 14 days after infection. In these periods, fragments from their spleen, liver and lung were removed for evaluation of the infection level by fungal cells, assessment of macrophagic activity by peritoneal and splenic macrophages - through the determination of nitric oxide (NO) concentrations and production of pro- and anti-inflammatory cytokines of lung and spleen homogenate supernatants. In the present study, it was observed that AIRmax lineages presented greater control of the infectious process than the AIRmin ones. Regarding NO production, AIRmax animals produced more metabolites in late periods, what may help control the infectious process. Concerning cytokine production, it was observed that the production of INF-gamma, TNF-alpha, IL-1, IL-6, IL-8 and IL-12 were increased in AIRmax lineages in most analyzed organs and periods, thus contributing to the greater resistance exhibited by such lineages against infection, except for IL-4 and IL-10 that showed decreased production in AIRmax lineage, reproducing its suppressive biological effect. From these results, it was observed that the AIRmax lineage was more effective in controlling the infectious process, with an important involvement of the analyzed cytokines. These findings are probably related to the genetically selected factors involved in the acute inflammatory response.

Paracoccidioides brasiliensis Uses Endogenous and Exogenous Arachidonic Acid for PGE(x) Production

Paracoccidioides brasiliensis is the agent of paracoccidioidomycosis, the most prevalent deep mycosis in Latin America. Production of eicosanoids during fungal infections plays a critical role on fungal biology as well as on host immune response modulation. The purpose of our study was to assess whether P. brasiliensis strains with different degree of virulence (Pb18, Pb265, Bt79, Pb192) produce prostaglandin E-x (PGE(x)). Moreover, we asked if P. brasiliensis could use exogenous sources of arachidonic acid (AA), as well as metabolic pathways dependent on cyclooxygenase (COX) enzyme, as reported for mammalian cells. A possible association between this prostanoid and fungus viability was also assessed. Our results showed that all strains, independently of their virulence, produce high PGE(x) levels on 4 h culture that were reduced after 8 h. However, in both culture times, higher prostanoid levels were detected after supplementation of medium with exogenous AA. Treatment with indomethacin, a COX inhibitor, induced a reduction on PGEx, as well as in fungus viability. The data provide evidence that P. brasiliensis produces prostaglandin-like molecules by metabolizing either endogenous or exogenous AA. Moreover, the results suggest the involvement of these mediators on fungal viability.

Interleukin-10 but not Transforming Growth Factor beta inhibits murine activated macrophages Paracoccidioides brasiliensis killing: Effect on H2O2 and NO production

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

High expression of human monocyte iNOS mRNA induced by Paracoccidioides brasiliensis is not associated with increase in NO production

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

Production of leukotriene B4 by Paracoccidioides brasiliensis

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

Morphophysiological Differences between the Metapleural Glands of Fungus-Growing and Non-Fungus-Growing Ants (Hymenoptera, Formicidae)

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

Production of extracellular acid proteases by Aspergillus clavatus

The production of extracellular acid proteases from Aspergillus clavatus was evaluated in a culture filtrate medium, with different carbon and nitrogen sources. The fungus was cultivated at three different temperatures during 10 days. The proteolytic activity was determined on haemoglobin pH 5.0 at 37 degreesC. The highest acid proteolytic activity (80 U/ml) was observed in culture medium containing glucose and gelatin at 1% (w/v) at 30 degreesC at the third day of incubation. Cultures developed in Vogel medium with glucose at 2% (w/v) showed at about 45% of proteolytic activity when compared to the cultures with 1% of the same sugar. The optimum pH of enzymatic activity was 2.0 and the enzyme was stable at pH values ranging from 2.0 to 4.0. The optimum temperature was 40 degreesC and the half-lives at 40, 45 and 50 degreesC were 30, 10 and 5 min, respectively.

Xylanase production by Aspergillus versicolor

The xylanolytic system of Aspergillus versicolor is controlled by induction and carbon catabolite repression. Carboxymethylcellulose and wheat bran were the best inducers of xylanolytic activity. When the fungus was grown for 5 days on VOGEL's liquid medium with wheat bran, the optimal pH and temperature for xylanase production were 6.5 and 30 degrees C, respectively. Optimal conditions for the xylanolytic activity assay were at pH 6.0 and 55 degrees C. The half-life at 60 degrees C of the crude enzyme was 6.5 and 21 minutes, in the absence or presence of substrate, respectively.Xylan is the main hemicellulosic component of plant biomass being present in appreciable quantities in agricultural and several agroindustrial wastes. From the products of xylan enzymatic hydrolysis it is possible to obtain cell protein, fuels and other chemicals. Xylanases combined with cellulase could have applications in food processing. Cellulase-free xylanases can be also utilized for preparation of cellulose pulps and liberation of textile fibres (WOODWARD 1984; BIELY 1985, WONG et al. 1988). In view of the potential applications of xylanases, a study of these enzymes from various sources and their multiplicity is desirable.Among xylanolytic microorganisms, filamentous fungi have been more extensively studied and the genus Aspergillus has been shown to be an efficient producer of xylanases. Preliminary observations from our laboratory have demonstrated that a strain of Aspergillus versicolor, isolated from Brazilian soil, produced high xylanase and low cellulase levels, which is an interesting characteristic for some industrial applications. In this report we describe the production and some properties of xylanase obtained from this fungus.

Production and characteristics comparison of crude beta-glucosidases produced by microorganisms Thermoascus aurantiacus e Aureobasidium pullulans in agricultural wastes

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

Production of xylanolytic enzymes by Penicillium janczewskii

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)