Biotechnological Potential of Agro-Industrial Wastes as a Carbon Source to Thermostable Polygalacturonase Production in Aspergillus niveus

Agro-industrial wastes are mainly composed of complex polysaccharides that might serve as nutrients for microbial growth and production of enzymes. The aim of this work was to study polygalacturonase (PG) production by Aspergillus niveus cultured on liquid or solid media supplemented with agro-industrial wastes. Submerged fermentation (SbmF) was tested using Czapeck media supplemented with 28 different carbon sources. Among these, orange peel was the best PG inducer. On the other hand, for solid state fermentation (SSF), lemon peel was the best inducer. By comparing SbmF with SSF, both supplemented with lemon peel, it was observed that PG levels were 4.4-fold higher under SSF. Maximum PG activity was observed at 55°C and pH 4.0. The enzyme was stable at 60°C for 90 min and at pH 3.0–5.0. The properties of this enzyme, produced on inexpensive fermentation substrates, were interesting and suggested several biotechnological applications

Biotechnological potential of alternative carbon sources for production of pectinases by Rhizopus microsporus var. rhizopodiformis

Fungi collected from Brazilian soil and decomposing plants were screened for pectinase production. R. microsporus var. rhizopodiformis was the best producer and was selected to evaluate the pectic enzyme production under several nutritional and environmental conditions. The pectinase production was studied at 40ºC, under 28 carbon sources-supplemented medium. The inducer effect of several agro-industrial residues such as sugar cane bagasse, wheat flour and corncob on polygalacturonase (PG) activity was 4-, 3- and 2-fold higher than the control (pectin). In glucose-medium, a constitutive pectin lyase (PL) activity was detected. The results demonstrated that R. microsporus produced high levels of PG (57.7 U/mg) and PL (88.6 U/mg) in lemon peel-medium. PG had optimum temperature at 65 ºC and was totally stable at 55 ºC for 90 min. Half-life at 70 ºC was 68 min. These results suggested that the versatility of waste carbon sources utilization by R. microsporus, produce pectic enzymes, which could be useful to reduce production costs and environmental impacts related to the waste disposal.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)(CNPq) Conselho de Desenvolvimento Científico e Tecnológic

Toxoplasma gondii chitinase induces macrophage activation

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Toxoplasma gondii is an obligate intracellular protozoan parasite found worldwide that is able to chronically infect almost all vertebrate species, especially birds and mammalians. Chitinases are essential to various biological processes, and some pathogens rely on chitinases for successful parasitization. Here, we purified and characterized a chitinase from T. gondii. The enzyme, provisionally named Tg_chitinase, has a molecular mass of 13.7 kDa and exhibits a Km of 0.34 mM and a Vmax of 2.64. The optimal environmental conditions for enzymatic function were at pH 4.0 and 50 degrees C. Tg_chitinase was immunolocalized in the cytoplasm of highly virulent T. gondii RH strain tachyzoites, mainly at the apical extremity. Tg_chitinase induced macrophage activation as manifested by the production of high levels of pro-inflammatory cytokines, a pathogenic hallmark of T. gondii infection. In conclusion, to our knowledge, we describe for the first time a chitinase of T. gondii tachyzoites and provide evidence that this enzyme might influence the pathogenesis of T. gondii infection.Toxoplasma gondii is an obligate intracellular protozoan parasite found worldwide that is able to chronically infect almost all vertebrate species, especially birds and mammalians. Chitinases are essential to various biological processes, and some pathoge1012112FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNQP - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)FAPESP [2013/10741-8]2013/10741-8SEM INFORMAÇÃOThis study was supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo (Grant number 2013/10741-8). Additional financial help was provided by Conselho Nacional de Desenvolvimento Científico e Tecnológico, and Fundação de Apoio ao Ensino, Pe

Toxoplasma gondii Chitinase Induces Macrophage Activation.

Toxoplasma gondii is an obligate intracellular protozoan parasite found worldwide that is able to chronically infect almost all vertebrate species, especially birds and mammalians. Chitinases are essential to various biological processes, and some pathogens rely on chitinases for successful parasitization. Here, we purified and characterized a chitinase from T. gondii. The enzyme, provisionally named Tg_chitinase, has a molecular mass of 13.7 kDa and exhibits a Km of 0.34 mM and a Vmax of 2.64. The optimal environmental conditions for enzymatic function were at pH 4.0 and 50 °C. Tg_chitinase was immunolocalized in the cytoplasm of highly virulent T. gondii RH strain tachyzoites, mainly at the apical extremity. Tg_chitinase induced macrophage activation as manifested by the production of high levels of pro-inflammatory cytokines, a pathogenic hallmark of T. gondii infection. In conclusion, to our knowledge, we describe for the first time a chitinase of T. gondii tachyzoites and provide evidence that this enzyme might influence the pathogenesis of T. gondii infection

Characterization of PbPga1, an antigenic GPI-protein in the pathogenic fungus Paracoccidioides brasiliensis.

Paracoccidioides brasiliensis is the etiologic agent of paracoccidioidomycosis (PCM), one of the most prevalent mycosis in Latin America. P. brasiliensis cell wall components interact with host cells and influence the pathogenesis of PCM. Cell wall components, such as glycosylphosphatidylinositol (GPI)-proteins play a critical role in cell adhesion and host tissue invasion. Although the importance of GPI-proteins in the pathogenesis of other medically important fungi is recognized, little is known about their function in P. brasiliensis cells and PCM pathogenesis. We cloned the PbPga1 gene that codifies for a predicted GPI-anchored glycoprotein from the dimorphic pathogenic fungus P. brasiliensis. PbPga1 is conserved in Eurotiomycetes fungi and encodes for a protein with potential glycosylation sites in a serine/threonine-rich region, a signal peptide and a putative glycosylphosphatidylinositol attachment signal sequence. Specific chicken anti-rPbPga1 antibody localized PbPga1 on the yeast cell surface at the septum between the mother cell and the bud with stronger staining of the bud. The exposure of murine peritoneal macrophages to rPbPga1 induces TNF-α release and nitric oxide (NO) production by macrophages. Furthermore, the presence of O-glycosylation sites was demonstrated by β-elimination under ammonium hydroxide treatment of rPbPga1. Finally, sera from PCM patients recognized rPbPga1 by Western blotting indicating the presence of specific antibodies against rPbPga1. In conclusion, our findings suggest that the PbPga1gene codifies for a cell surface glycoprotein, probably attached to a GPI-anchor, which may play a role in P. brasiliensis cell wall morphogenesis and infection. The induction of inflammatory mediators released by rPbPga1 and the reactivity of PCM patient sera toward rPbPga1 imply that the protein favors the innate mechanisms of defense and induces humoral immunity during P. brasiliensis infection

Chitinase activity from <i>T</i>. <i>gondii</i>.

<p>Soluble <i>T</i>. <i>gondii</i> antigens were purified by affinity with an IgY-Sepharose 4B column, and the chitinase activity of the delayed fractions was measured. (A) Electrophoresis analysis of purified chitinase from <i>T</i>. <i>gondii</i>. First lane—MW (Molecular Weight Ladder), second lane—purified chitinase in 12% SDS-PAGE. (B) Crude extract and purified chitinase activity from <i>T</i>. <i>gondii</i> was measured. Error bars represent standard errors calculated from three replicates.</p

Tg_chitinase subcellular localization.

<p>(A) <i>T</i>. <i>gondii</i> tachyzoite DNA was stained with DAPI (blue), and (B) immunostained for Tg_chitinase with an anti-chitinase antibody, which was biotinylated with an antibody conjugated to anti-streptavidin-FITC (green). Superimposition of images stained for Tg_chitinase and DAPI (Merge and phase microscopy, C and D, respectively). Tg_chitinase is localized throughout the cytosol of <i>T</i>. <i>gondii</i> tachyzoites. Scale bar = 2.5 μm.</p

Amino acid sequence and predicted 3D structure of Tg_chitinase from <i>T</i>. <i>gondii</i>.

<p>Tg_chitinase from <i>T</i>. <i>gondii</i> was identified by mass spectrometry using the amino acid sequences of tryptic peptides listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144507#pone.0144507.t002" target="_blank">Table 2</a> (indicated in bold). (A) The amino acid sequence obtained from analysis of the peptide (SEIYQGDSVR) identified by BLASTP <i>T</i>. <i>gondii</i> GT1 (TGGT1_286465). (B) Predicted 3D structure of Tg_chitinase using I-TASSER.</p