Influence of nitrogen sources on the enzymatic activity and grown by Lentinula edodes in biomass Eucalyptus benthamii.

Lignocellulose is the most abundant environmental component and a renewable organic resource in soil. There are some filamentous fungi which developed the ability to break down and use cellulose, hemicellulose and lignin as an energy source. The objective of this research was to analyze the effect of three nitrogen resources (ammonium sulfate, saltpetre, soybean) in the holocellulolitic activity of Lentinula edodes EF 50 using as substrate sawdust E. benthamii. An experimental design mixture was applied with repetition in the central point consisting of seven treatments (T) of equal concentrations of nitrogen in ammonium sulfate, potassium nitrate and soybean. The enzymatic activity of avicelase, carboxymetilcellulase, ß-glucosidase, xylanases and manganese peroxidase was determined. The humidity, pH, water activity (aw) and qualitative analysis of mycelial growth in 8 times of cultivation were evaluated. The results showed negative effect on enzyme production in treatments with maximum concentration of ammonium sulfate and potassium nitrate. The treatments with cooked soybean flour expressed higher enzymatic activities in times of 3, 6 and 9 days of culture, except in the activity of manganese peroxidase. The highest production was observed in the treatment with ammonium sulfate, and soybean (83.86 UI.L-1) at 20 days of cultivation.

Influence of nitrogen sources on the enzymatic activity and grown by Lentinula edodes in biomass Eucalyptus benthamii / Influência das fontes de nitrogênio na atividade enzimática e crescimento de Lentinula edodes na biomassa Eucalyptus benthamii

Abstract Lignocellulose is the most abundant environmental component and a renewable organic resource in soil. There are some filamentous fungi which developed the ability to break down and use cellulose, hemicellulose and lignin as an energy source. The objective of this research was to analyze the effect of three nitrogen resources (ammonium sulfate, saltpetre, soybean) in the holocellulolitic activity of Lentinula edodes EF 50 using as substrate sawdust E. benthamii. An experimental design mixture was applied with repetition in the central point consisting of seven treatments (T) of equal concentrations of nitrogen in ammonium sulfate, potassium nitrate and soybean. The enzymatic activity of avicelase, carboxymetilcellulase, β-glucosidase, xylanases and manganese peroxidase was determined. The humidity, pH, water activity (aw) and qualitative analysis of mycelial growth in 8 times of cultivation were evaluated. The results showed negative effect on enzyme production in treatments with maximum concentration of ammonium sulfate and potassium nitrate. The treatments with cooked soybean flour expressed higher enzymatic activities in times of 3, 6 and 9 days of culture, except in the activity of manganese peroxidase. The highest production was observed in the treatment with ammonium sulfate, and soybean (83.86 UI.L–1) at 20 days of cultivation.

Resumo Lignocelulose é o componente mais abundante do meio ambiente e recurso orgânico renovável no solo. Alguns fungos filamentosos têm desenvolvido a habilidade de degradar e utilizar celulose, hemicelulose e lignina como fonte de energia. O objetivo deste trabalho foi analisar o efeito de três fontes de nitrogênio (sulfato de amônio, nitrato de potássio e farelo de soja) na atividade enzimática de Lentinula edodes EF 50 utilizando como substrato serragem de E. benthamii. Foi aplicado um planejamento experimental de mistura com três repetições no ponto central constituído de sete tratamentos (T) de iguais concentrações em nitrogênio de sulfato de amônia, nitrato de potássio e farinha de soja cozida. Foram determinadas a atividade enzimática da avicelase, carboximetilcelulase, β-glicosidase, xilanases e manganês peroxidase. Foram avaliados o teor de umidade, pH, atividade de água (aw) e análise qualitativa do crescimento micelial em 8 tempos de cultivo. Os resultados mostraram efeito negativo na produção das enzimas nos tratamentos com máxima concentração de sulfato de amônia e nitrato de potássio. Os tratamentos com farinha de soja cozida expressaram maiores atividades enzimáticas, nos tempos de 3, 6 e 9 dias de cultivo exceto na atividade do manganês peroxidase. A maior produção foi observada no tratamento com sulfato de amônia e farinha de soja cozida (83.86 UI.L–1) em 20 dias de cultivo.

Degradation of keratinous materials by the plant pathogenic fungus Myrothecium verrucaria.

In this paper it is described for the first time the capability of Myrothecium verrucaria to grow in submerged and solid state cultures using poultry feathers as the only substrate. The fungus produced a protease with an unusual keratinolytic activity among plant pathogenic fungi. Its crude protease hydrolyzed keratinous substrates at pH 9.0 and 40 degrees C in the following order: poultry feather keratin > sheep wool keratin > human nail keratin > human hair keratin. Protease activity was highly sensitive to phenylmethyl sulphonyl fluoride (PMSF) indicating that the enzyme belonged to the serine protease family.

Effect of pepstatin A on the virulence factors of Candida albicans strains isolated from vaginal environment of patients in three different clinical conditions.

The aspartate proteinase inhibitor pepstatin A was used to study a possible correlation among proteinase activity and other virulence factors of Candida albicans strains isolated from the vaginal environment of patients in three different clinical conditions: asympthomatic, vulvovaginal candidiasis (VVC) and recurrent vulvovaginal candidiasis (RVVC). The addition of 1.0 muM pepstatin A did not have any significant effect on hyphae formation, biofilm production and in the cell surface hydrofobicity of isolates in the three different clinical conditions. However, pepstatin A reduced the adherence of C. albicans to vaginal mucosa epithelial cells (53.1, 48.7 and 59.9%, respectively to isolates from asymptomatic, VVC and RVVC patients). This result suggests that the secreted aspartate proteinases (Saps) of this fungal pathogen may have auxiliary roles in cellular adhesion.

Vulvovaginal candidiasis is associated with the production of germ tubes by Candida albicans.

Twenty Candida albicans strains isolated from women attended at the Teaching and Research in the Laboratory of Teaching and Research in Clinical Analysis of the State University of Maringa, Paraná, Brazil, have been analyzed. Yeasts were identified by classical methods and patients subdivided into asymptomatic, vulvovaginal candidiasis(VVC) and recurrent vulvovaginal candidiasis (RVVC) groups. Yeasts were incubated in RPMI + fetal calf serum to analyze germ tubes every two hours, up to 10 h. In vitro sensitivity to fluconazole, itraconazole, ketoconazole, amphotericin B and nystatin was analyzed according to NCCLS-M27-A microdilution assay. Yeast isolated from symptomatic women produced significantly more germ tubes than asymptomatic women (P < 0.05). However, no significant difference between yeasts from VVC and RVVC occurred (P > 0.05). Variation between MIC50 and MIC90 of tested antifungal agents was slight among isolated yeasts, while no resistant yeasts were detected. Nevertheless, VVC yeasts were more DDS (reduced dose-dependent susceptibility) for nystatin and RVVC were more DDS for ketoconazole. Results suggest that colonization by yeast in the vagina and lack of symptoms may be partially explained by the yeast's sparse capacity to form germ tubes, On the other hand, RVVC was not associated with antimicrobial resistance. DDS high frequency for nystatin and ketoconazole indicates that identification, and susceptibility of antifungals tests are important to management of VVC.

Decolorization of industrial dyes by a Brazilian strain of Pleurotus pulmonarius producing laccase as the sole phenol-oxidizing enzyme.

The ability of a Brazilian strain of Pleurotus pulmonarius to decolorize structurally different synthetic dyes (including azo, triphenylmethane, heterocyclic and polymeric dyes) was investigated in solid and submerged cultures. Both were able to decolorize completely or partially 8 of 10 dyes (Amido Black, Congo Red, Trypan Blue, Methyl Green, Remazol Brilliant Blue R, Methyl Violet, Ethyl Violet, Brilliant Cresyl Blue). No decolorization of Methylene Blue and Poly R 478 was observed. Of the four phenol-oxidizing enzymes tested in culture filtrates (lignin peroxidase, manganese peroxidase, aryl alcohol oxidase, laccase), P. pulmonarius produced only laccase. Both laccase activity and dye decolorization were related to glucose and ammonium starvation or to induction by ferulic acid. The decolorization in vivo was tested using three dyes--Remazol Brilliant Blue R, Trypan Blue and Methyl Green. All of them were completely decolorized by crude extracellular extracts. Decolorization and laccase activity were equally affected by pH and temperature. Laccase can thus be considered to be the major enzyme involved in the ability of P. pulmonarius to decolorize industrial dyes.

A new species of Fusarium producer of galactose oxidase.

Fifty-two isolates of Fusarium species and one of Gibberella fujikuroi were tested for galactose oxidase (GO) production. Five Fusarium isolates contained GO activity in the culture filtrate: three F. graminearum and one each F. moniliforme f. sp. subglutinans and F. acuminatum. This is the first time F. acuminatum is reported to be a producer of GO enzyme. GO enzyme activity produced by isolates was assayed through a time course. Moreover, GO protein was partially purified from the most productive four isolates to show that the activity measured in the culture filtrates was due to the presence of GO protein.

Influence of growth conditions on the production of xylanolytic enzymes by Aspergillus flavus.

Investigations were carried out to optimize the culture conditions for the production of xylanase and beta-xylosidase by Aspergillus flavus, a filamentous fungus isolated from soil. The production of enzymes was tolerant to a wide range of initial culture pH values. Maximum xylanase (190 U/ml) and beta-xylosidase (35 U/ml) production was obtained when the strain was grown on mineral medium supplemented with 3% (w/v) corn cob powder as the carbon source. The enzymes had optimal activities at pH values between 5.5 and 6.0 and exhibited high activity and stability under alkaline conditions.

Production of lipase by soil fungi and partial characterization of lipase from a selected strain (Penicillium wortmanii).

Filamentous fungi from soil were screened for their ability to produce lipase. Among 56 filamentous fungi tested, one strain identified as Penicillium wortmanii was selected as the highest lipase producer. Maximum lipase production (12.5 U/ml) was obtained in 7-days cultures utilizing 5% (w/v) olive oil as the carbon source. Optimum pH and temperature for crude lipase were 7.0 and 45 degrees C, respectively. The enzyme was stable at 40 and 45 degrees C and it retained about 55% of its activity when heated at 50 degrees C for 1 hour.

Production of amylase by Aspergillus fumigatus utilizing alpha-methyl-D-glycoside, a synthetic analogue of maltose, as substrate.

A strain of Aspergillus fumigatus isolated from soil was able to produce biomass and high amylase activities in media containing alpha-methyl-D-glucoside (alphaMG), a synthetic analogue of maltose, as the only carbon source. alphaMG was a more effective inducer than starch and maltose at the same concentration: alphaMG cultures produced about 3 times more alpha-amylase and glucoamylase activity than starch cultures. Maximum production of alpha-amylase (60 U/mg) and glucoamylase (130 U/mg) was obtained in 8-10 days alphaMG cultures.

Beta-D-glycosidase activities of Humicola grisea: biochemical and kinetic characterization of a multifunctional enzyme.

A beta-D-glycosidase activity was purified from mycelium of Humicola grisea var. thermoidea grown on avicel as the main carbon source. The purified enzyme was a glycoprotein and migrated as a single polypeptide band on polyacrylamide gel electrophoresis under native or denaturing conditions. The apparent molecular weight of the enzyme was estimated to be 55 kDa by gel filtration and SDS-PAGE. The enzyme was active against o-nitrophenyl beta-D-galactoside; p-nitrophenyl beta-D-glucoside, p-nitrophenyl beta-D-fucoside, lactose and cellobiose, PNP fucoside (synthetic substrate) and cellobiose (natural substrate) being the best utilized. A comparison of the properties of beta-D-galactosidase, beta-D-glucosidase and beta-D-fucosidase showed that three activities exhibited similar pH and temperature optima and the same thermostability. The hydrolysis rate of substrate mixtures suggests that the enzyme possesses a common catalytic site for all the substrates assayed.

An analysis of developmental timing in Blastocladiella emersonii sporulation.

We propose a model of time regulation for the expression of the Blastocladiella emersonii sporulation phenotypes based on new methods (Soll, 1986) which analyse the effect of temperature on the rate limiting processes, i.e., "timers" of certain events during development. By using reciprocal shift experiments (transferring sporulating cells from 22 to 27 degrees C and vice versa) we characterized the timers of the phenotypes: septate zoosporangium, papillate zoosporangium, cleavage zoosporangium, and empty zoosporangium, considering the number of the components, sensitivity, duration, and the mutual dependency of each limiting factor. The timers for the first three phenotypes started at zero time of sporulation induction and acted in parallel. The fourth phenotype, empty zoosporangium, has a timer which appears to act sequentially to that of the papillate zoosporangium. We also studied the effects of polyoxin D, calcofluor white, and congo red on sporulation. The first drug prevents the appearance of the septate zoosporangium and the other two prevent the expression of the papillate zoosporangium. In spite of the morphological blockage, the zoosporogenesis proceeds, resulting in the formation of normal zoospores. These results are interpreted as additional evidence for the parallel model of control proposed here.