The potential of plant and fungal proteins in the control of gastrointestinal nematodes from animals / Potencial de proteínas de plantas e fungos no controle de nematoides gastrintestinais de animais

Abstract Gastrointestinal nematode infection is an important cause of high economic losses in livestock production. Nematode control based on a synthetic chemical approach is considered unsustainable due to the increasing incidence of anthelmintic resistance. Control alternatives such as the use of natural products are therefore becoming relevant from an environmental and economic point of view. Proteins are macromolecules with various properties that can be obtained from a wide range of organisms, including plants and fungi. Proteins belonging to different classes have shown great potential for the control of nematodes. The action of proteins can occur at specific stages of the nematode life cycle, depending on the composition of the external layers of the nematode body and the active site of the protein. Advances in biotechnology have resulted in the emergence of numerous protein and peptide therapeutics; however, few have been discussed with a focus on the control of animal nematodes. Here, we discuss the use of exogenous proteins and peptides in the control of gastrointestinal.

Resumo A infecção por nematoides gastrintestinais é uma importante causa de grandes perdas econômicas na pecuária. O controle de nematoides com compostos químicos sintéticos é considerado insustentável devido ao aumento da resistência anti-helmíntica. Alternativas de controle, como o uso de produtos naturais, estão se tornando relevantes do ponto de vista ambiental e econômico. As proteínas são macromoléculas com várias propriedades que podem ser obtidas de uma ampla gama de organismos, incluindo plantas e fungos. Proteínas pertencentes a diferentes classes têm mostrado grande potencial para o controle de nematoides. A ação das proteínas pode ocorrer em estágios específicos do ciclo de vida do nematoide, dependendo da composição das camadas externas do parasito e do sítio ativo da proteína. Avanços na biotecnologia resultaram no surgimento de numerosas terapias de proteínas e peptídeos; no entanto, pouco foi discutido com foco no controle de nematoides parasitos de animais. Na presente revisão foi discutido o uso de proteínas exógenas e peptídeos no controle de nematoides gastrintestinais, os mecanismos sugeridos de ação, e os desafios e perspectivas para o uso dessas biomoléculas como uma classe de anti-helmínticos.

Characterization of succinic semialdehyde dehydrogenase from Aspergillus niger.

The catabolism of fungal 4-aminobutyrate (GABA) occurs via succinic semialdehyde (SSA). Succinic semialdehyde dehydrogenase (SSADH) from the acidogenic fungus Aspergillus niger was purified from GABA grown mycelia to the highest specific activity of 277 nmol min-1 mg-1, using phenyl Sepharose and DEAE Sephacel chromatography. The purified enzyme was specific for its substrates SSA and NAD+. The substrate inhibition observed with SSA was uncompetitive with respect to NAD+. While product inhibition by succinate was not observed, NADH inhibited the enzyme competitively with respect to NAD+ and noncompetitively with respect to SSA. Dead-end inhibition by AMP and p-hydroxybenzaldehyde (pHB) was analyzed. The pHB inhibition was competitive with SSA and uncompetitive with NAD+; AMP competed with NAD+. Consistent with the kinetic data, a sequential, ordered Bi Bi mechanism is proposed for this enzyme.

Development of bioprocess for the production of laccase by Pleurotus ostreatus MTCC 1802 using evolutionary optimization technique.

For cost effective production of laccase enzyme (benzenediol: oxygen oxidoreductase) from P. ostreatus MTCC 1802 through solid sate fermentation, physico-chemical parameters such as temperature (20-35 ºC), incubation period (9-17 days) and substrate (Neem bark and wheat bran, in various ratios, w/w) were optimized first by one parameter at time approach and then obtained optimum conditions were considered as zero level in evolutionary optimization factorial design technique. At statistically optimized conditions yield of laccase was found 303.59+16.8) U/gds after 13 days of incubation at 25 ºC taking wheat bran and neem bark as substrate at a ratio of 3:2 (w/w). The results obtained could be a base line for industrial scale production of laccase.

Development of bioconjugate from Streptomyces tyrosinase and gold nanoparticles for rapid detection of phenol constituents.

Most of the phenol compounds are toxic and have been considered as hazardous pollutants. Several physicochemical and biological methods are available to detect and monitor the phenol pollutants in water and soil. In the present study, phenol constituents of winery, paper and plastic industrial effluents were successfully detected employing tyrosinase-gold nanoparticles bioconjugate. The synthesis of extracellular tyrosinase and gold nanoparticles was achieved by a single isolate of Streptomyces sp. DBZ-39. Enhanced production (369.41 IU) of tyrosinase was produced in submerged bioprocess employing response surface method with central composite design. Extracellular gold nanoparticles synthesized (12-18 nm) by Streptomyces sp. DBZ-39 were characterized with TEM, EDAX and FTIR analysis. A rapid detection (within 10 min) of phenol constituents from winery effluents was achieved by bioconjugate, when compared to tyrosinases and gold nanoparticles independently. Streptomyces tyrosinase could exhibit relatively a better performance than commercially available mushroom tyrosinase in the detection of phenol constituents. Winery effluent has shown much higher content (0.98 O.D) of phenol constituents than paper and plastic effluents based on the intensity of color and U.V absorption spectra.

Purified dextransucrase from Pediococcus pentosaceus CRAG3 as food additive.

The extracellular crude dextransucrase (0.67 U/mg) from P. pentosaceus CRAG3 (GenBank accession number JX679020) after PEG-1500 fractionation gave specific activity, 20.0 U/mg which by gel filtration resulted in 46.0 U/mg. The purified dextransucrase displayed molecular size of approximately, 224 kDa. The optimum assay conditions for dextransucrase activity were 5% sucrose in 20 mM sodium acetate buffer (pH 5.4) and 30 oC. The dextransucrase was stable up to 40 oC and at pH range of 5.4-7.0. The metal ions such as Co2+, Ca2+, Mg2+ and Zn2+ stimulated the dextransucrase activity by 56, 44, 14 and 12%, respectively. It was most stable at -20 oC with half-life of 307 days. Amongst various additives used, glycerol and Tween 80 provided significant stability to the enzyme with half-life 15.5 and 85.5 h, respectively as compared to control (6.9 h). The solidification of sucrose supplemented milk by purified dextransucrase due to dextran synthesis displayed its application as additive for improving the texture of dairy products.

Production, purification and properties of fungal chitinases—A review.

After cellulose, chitin is the second most abundant organic and renewable polysaccharide in nature. This polymer is degraded by enzymes called chitinases which are a part of the glycoside hydrolase family. Chitinases have many important biophysiological functions and immense potential applications especially in control of phytopathogens, production of chito-oligosaccharides with numerous uses and in treatment and degradation of chitinous biowaste. At present many microbial sources are being explored and tapped for chitinase production which includes potential fungal cultures. With advancement in molecular biology and gene cloning techniques, research on fungal chitinases have made fast progress. The present review focuses on recent advances in fungal chitinases, containing a short introduction to types of chitinases, their fermentative production, purification and characterization and molecular cloning and expression.

Purification and characterization of a ribonuclease from the wild edible mushroom Armillaria luteo-virens.

A 15 kDa ribonuclease (RNase) was purified from dried fruiting bodies of the wild edible mushroom Armillaria luteo-virens. The simple 4-step purification protocol involved ion-exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, ion-exchange chromatography on SP-Sepharose and a final gel filtration by FPLC on Superdex-75. The RNase was unadsorbed on Affi-gel blue gel, but adsorbed on DEAE-cellulose and SP-Sepharose. The N-terminal amino acid sequence of purified RNase was AGVQYKLTILLV, which showed low sequence homology to those of previously reported RNases. The optimal pH and temperature of the enzyme were very close to 4.0 and 70°C, respectively. The enzyme showed considerably high ribonucleolytic activity and broad specificity towards polyhomoribonucleotides, with a specificity of poly(U)>poly(C)>poly (G)>poly(A). The ribonucleolytic activities towards poly(U), poly(C), poly(G) and poly(A) were 279.5, 184.1, 69.9 and 52.3 U/mg, respectively.

Grape stalks as substrate for white rot fungi, lignocellulolytic enzyme production and dye decolorization / Uso del escobajo como sustrato para el crecimiento de hongos de la pudrición blanca, la producción de enzimas ligninolíticas y la decoloración de tinturas

The aim of this work was to evaluate the potential of grape stalks, an agroindustrial waste, for growth and lignocellulolytic enzyme production via solid-state fermentation, using the following three white rot fungi: Trametes trogii, Stereum hirsutum and Coriolus antarcticus. The decolorization of several dyes by the above mentioned cultures was also investigated. Similar values of dry weight loss of the substrate were measured after 60 days (33-43 %). C. antarcticus produced the highest laccase and Mn-peroxldase activities (33.0 and 1.6 U/g dry solid). The maximum endoglucanase production was measured in S. hirsutum cultures (10.4 U/g), while the endoxylanase peak corresponded to T. trogii (14.6 U/g). The C. antarcticus/grape stalk system seems potentially competitive in bioremediation of textile processing effluents, attaining percentages of decolorization of 93, 86, 82, 82, 77, and 58 % for indigo carmine, malachite green, azure B, remazol brilliant blue R, crystal violet and xylidine, respectively, in 5 h.

El objetivo de este trabajo fue evaluar el potencial del escobajo, un residuo agroindustrial, como sustrato para el crecimiento y la producción de enzimas lignocelulósicas de tres hongos causantes de pudrición blanca en la madera: Trametes trogii, Stereum hirsutum y Coriolus antarcticus. Para ello se utilizaron técnicas de fermentación en estado sólido. También se ensayó la decoloración de colorantes industriales sobre estos cultivos. La pérdida de peso seco del sustrato fue similar después del día 60 (33-43 %). C. antarcticus produjo las mayores actividades de lacasa y Mn-peroxidasa (33,0 y 1,6 U/g peso seco). La mayor actividad endoglucanasa fue medida en cultivos de S. hirsutum (10,4 U/g), y la mayor actividad endoxilanasa en T. trogii (14,6 U/g). El sistema C. antarcticus/escobap mostró un importante potencial para su aplicación en la biorremediación de efluentes textiles, con porcentajes de decoloración de 93, 86, 82, 82, 77 y 58 % para índigo carmín, verde de malaquita, azure B, azul R brillante de remazol, cristal violeta y xilidina, respectivamente, en 5 h.

Extraction and characterization of polygalacturonase of Fomes sclerodermeus produced by solid-state fermentation / Extracción y caracterización de poligaracturonasa de Fomes sclerodermeus producida por fermentación en estado sólido

Polygalacturonase (PG) production by Fomes sclerodermeus using solid-state fermentation (SSF) was carried out. Maximal PG activity (26 U/gdw) was obtained between days 11 and 13 at the end of exponential growth. PG activity in the crude extract was more stable at pH 5-6 and 30 °C and had optimum activity at pH 5 and 50 °C. Optimal conditions for PG extraction were: one time extraction with Na2SO4 as solvent with 10 min. of agitation. In a scale-up system, PG activity per gram of dry substrate decreased about 60% compared with the activity obtained in an Erlenmeyer flask; however, high total PG activity was obtained.

Se estudió la producción de poligalacturonasa (PG) por Fomes sclerodermeus usando técnicas de fermentación en estado sólido. La actividad PG máxima (26 U/g ps) fue observada entre los días 11 y 13. La actividad PG en los extractos crudos fue más estable a pH 5-6 y 30 °C, con una actividad óptima a pH 5 y a 50 °C. Las condiciones óptimas para la extracción de PG se lograron con una única extracción empleando Na2SO4 como solvente, con 10 minutos de agitación. En el escalado del sistema, la actividad PG por gramo de peso seco de sustrato disminuyó cerca de 60% comparada con la obtenida en frascos Erlenmeyer, pero la actividad total fue mayor.

Purification and characterization of mycoferritin from Aspergillusflavus MTCC 873.

The fungus Aspergillus flavus MTCC 873, a non-toxigenic isolate demonstrated its capability to synthesize mycoferritin (MF) upon induction with iron in yeast extract sucrose (YES) medium. The molecular mass, yield, iron and carbohydrate contents of the MF were 440 kDa, 0.015 mg/g of wet mycelia, 0.8 and 30.4%, respectively. Native gel-electrophoresis revealed a band corresponding to dimeric form of equine spleen ferritin (ESF). Subunit analysis by SDS-PAGE revealed a single protein band with an apparent molecular mass of 24 kDa, suggesting similar sized subunits in the structure of apoferritin shell. Immunological cross-reactivity was observed with the anti-fish liver ferritin. Transmission electron microscopy (TEM) revealed an apparent particle size of 100 Å. N-terminal amino acid sequence of MF revealed a sequence of SLPLQDYA, which showed identities with other eukaryotic ferritin sequences. The spectral characteristics (UV/VIS, fluorescence and circular dichroic spectra) were similar to ESF. The fungus, unlike A. parasiticus 255 (non-toxigenic) was incapable of producing aflatoxins, when grown in YES media.

New mechanical disruption method for extraction of whole cell protein from Candida albicans.

Cell disruption or lysis is a crucial step to obtain cellular components for various biological studies. We subjected different concentrations of Candida albicans to 5, 10, 15 and 20 cycles of disruption. The degree of cell lysis was observed using light microscopy and the yields obtained were measured and analysed. The optimum extraction with 1 x 10(10) yeast cells/ml was achieved after 5 cycles of disruption with 1.0 mm diameter glass beads at 5,000 rpm. Approximately 80% of the cells were lysed and the protein yield was 6,000 microg/ml. SDS-PAGE analysis revealed approximately 25 distinct protein bands with molecular weights ranging from 8 kDa to 220 kDa. We conclude that this mechanical disruption of fungal cells is a rapid, efficient and inexpensive technique for extracting whole cell proteins from yeast cells.

Kinetics of IFN-gamma, TNF-alpha, IL-10 and IL-4 production by mononuclear cells stimulated with gp43 peptides, in patients cured of paracoccidioidomycosis

We analyzed the kinetics of cytokine production by mononuclear cells from 17 patients who had been treated for paracoccidioidomycosis, using the stimulus of gp43 peptide groups (43kDa glycoprotein of Paracoccidioides brasiliensis) at 0.1 and 1µM, gp43 (1µg/ml) and crude Paracoccidioides brasiliensis antigen (PbAg; 75µg/ml). IFN-gamma production was a maximum at 144 hours in relation to the G2 and G8 peptide groups at 1µM and was greatest at 144 hours when stimulated by gp43 and by PbAg. The maximum TNF-alpha production was at 144 hours for the G2 group (0.1µM) and for gp43. IL-10 production was highest after 48 and 72 hours for G7 and G6 at 1µM, respectively. We also suggest the best time for analysis of IL4 production. These results may contribute towards future studies with gp43 peptides and encourage further investigations with the aim of understanding the influence of these peptides on the production of inflammatory and regulatory cytokines.

Analisamos a cinética da produção de citocinas de células mononucleares de 17 pacientes com paracoccidioidomicose tratada, usando como estímulo: grupos de peptídeos da gp43 (glicoproteina de 43kDa de Paracoccidioides brasiliensis) a 0,1 e 1µM, gp43 (1µg/mL) e antígeno bruto de Paracoccidioides brasiliensis - AgPb (75µg/mL). A produção de IFN-gama foi máxima em 144 horas frente aos grupos de peptídeos G2 e G8 a 1µM e maior em 144 horas quando estimuladas por gp43 e por AgPb. A produção de TNF-alfa foi máxima em 144 horas para G2 (0,1µM) e para gp43. A produção de IL-10 foi maior após 48 e 72 horas para G7 e G6 a 1µM, respectivamente. Sugerimos também o melhor período para a análise da produção de IL4. Tais resultados podem contribuir para estudos com peptídeos da gp43, estimulando investigações posteriores visando entender a influência de tais peptídeos na produção de citocinas inflamatórias e regulatórias.

Production and purification of an Endoter1.4-beta-Xylanase from Humicola grisea var. thermoidea by electroelution

Humicola grisea var. thermoidea produces two forms of extracellular xylanase. The component form 1 was purified using the electroelution method, due to the very small production of this extracellular enzyme. The apparent molecular mass was 61.8 kDa by SDS-PAGE.

Extraction and characterization of proteins from cell walls of two strains of paracoccidioides brasiliensis

En el presente trabajo se analizaron las proteínas de la pared celular de 2 cepas de paracoccidioides brasiliensis en fase levaduriforme (PbHC-PE y Pb 18). Las proteínas fueron extraidas por tres difrentes métodos y estudiadas por electroféresis SDS-PAGE. Los resultados de los perfiles de las dos cepas fueron diferentes, permitiendo la posibilidad de su uso como marcadores quimiotaxonómicos. Se observó una secreción transitoria de la proteína gp 43 a través de la pared celular de las cepas de p. brasiliensis.

Effect of 6-MFA, an interferon inducer obtained from fungus Aspergillus ochraceus on hepatic mixed function oxidase system in rats.

Effect of 6-MFA (sixth mycelial fraction of acetone), an interferon inducer obtained from fungus A. ochraceus on hepatic mixed function oxidase system (MFO) of rat has been investigated. Treatment with 6-MFA, 100 mg/kg/day, ip for 1-5 days to adult rats inhibited significantly the different indices of MFO system, viz. hepatic cytochrome P-450, cytochrome b5 content, cytochrome c reductase, aminopyrine-N-demethylase and acetanilide hydroxylase activities. Similar treatment for 3 days in young growing rats significantly inhibited MFO system's components except acetanilide hydroxylase activity which showed marked elevation. These effects seem to be specific as in vitro experiments suggested that 6-MFA does not compete with subsdtrates nor it acts as a sponge reacting with the end product to give false inhibitory effect. It is concluded from the present study that 6-MFA like other interferon inducers depresses MFO system in rats. Its possible clinical implications are discussed.

Characterization of glycoprotein gp43, the major laminin-binding protein of Paracoccidioides brasiliensis

We have demonstrated that laminin mediates the adhesion of P. brasiliensis to monolayers of epithelial cells through specific binding to the surface glycoprotein gp43. This binding seems to be related to the fungal pathogenesis. We now report the confirmation of these findings by scanning electron microscopy and show that some isolates that do not secrete gp43 do express the protein as seen studying whole cell extracts. This results confirm the ability of these strains to produce paracoccidioidomycosis but should not be used for serological purposes since the absence of gp43 in exoantigens may lead to false negative results

Chemical composition of a mixture of single-cell protein obtained from Kluyveromyces fragilis and whey proteins

De la fermentación de suero de leche entera se obtuvo un producto consistente en una mezcla de biomasa de Kluyveromyces fragilis y proteínas coaguladas del suero. El producto tuvo una composición similar a la de productos lavados de que se informa en la literatura, con un alto contenido de proteína cruda y un bajo contenido de cenizas. Asimismo, acusó tambiém un alto contenido de aminoácidos azufrados y de triptofano, los que usualmente son limitantes en al biomasa de levadura. El contenido de lisina fue inexplicablemente más bajo de lo esperado resusltando ser el aminoácido limitante. La calificación química de la protéina fue 91%. Del producto de biomasa com proteínas de suero se obtuvo un concentrado proteínico con un rendimento de 80%. El contenido de proteína del aislado fue de 75% y el contenido de ácidos nucleícos se redujó en 90.8%. Los restos de pared celular también se redujeron considerablemente