Characterization of ß-glucosidase produced by the white rot fungus Flammulina velutipes.

ß-Glucosidase production by the white rot fungus Flammulina velutipes CFK 3111 was evaluated using different carbon and nitrogen sources under submerged fermentation. Maximal extracellular enzyme production was 1.6 U/ml, corresponding to a culture grown in sucrose 40 g/l and asparagine 10 g/l. High production yield was also obtained with glucose 10 g/l and asparagine 4 g/l medium (0.5 U/ml). Parameters affecting the enzyme activity were studied using p-nitrophenyl-ß-D-glucopyranoside as the substrate. Optimal activity was found at 50°C and pHs 5.0 to 6.0. Under these conditions, ß-glucosidase retained 25% of its initial activity after 12 h of incubation and exhibited a half-life of 5 h. The addition of MgCl2, urea, and ethanol enhanced the ß-glucosidase activity up to 47%, whereas FeCl2, CuSO4, Cd(NO3)2, and cetyltrimethylammonium bromide inflicted a strong inhibitory effect. Glucose and cellobiose also showed an inhibitory effect on the ß-glucosidase activity in a concentration-dependent manner. The enzyme had an estimated molecular mass of 75 kDa. To the best of our knowledge, F. velutipes CFK 3111 ß-glucosidase production is amongst the highest reported to date, in a basidiomycetous fungus.

Optimization of laccase production by two strains of Ganoderma lucidum using phenolic and metallic inducers.

Ganoderma lucidum (Curtis) P. Karst is a white rot fungus that is able to degrade the lignin component in wood. The ability of two strains of this species to produce the ligninolytic enzyme laccase was assessed. After the evaluation of induction with heavy metals and phenolic compounds, it was found that among the tested substances, copper and ferulic acid are the best laccase inducers. It was also observed that the two types of inducers (phenolic and metallic) produce different electrophoretic patterns of laccase activity. Optimized concentrations of inducers were obtained through a factorial design and the thermal stability of optimized supernatants was studied at a wide range of acidic pH. We found that the enzyme is more thermostable at higher pH values.

Optimization of laccase production by two strains of Ganoderma lucidum using phenolic and metallic inducers / Optimización de la producción de lacasa por dos cepas de Ganoderma lucidum utilizando inductores fenólicos y metálicos

Ganoderma lucidum (Curtis) P. Karst es un hongo causante de pudrición blanca, capaz de degradar la lignina de la madera y otros sustratos en los que crece. En este trabajo se evaluó la capacidad de dos cepas de esta especie de producir la enzima ligninolítica lacasa. Asimismo, se ensayó la inducción de esta enzima con diferentes compuestos fenólicos e iones metálicos, y se encontró que el ácido ferúlico y el cobre fueron los mejores inductores de la lacasa entre los agentes evaluados. También se encontró que los dos tipos de inductores (fenólicos y metálicos) producen distintos patrones electroforéticos de actividad lacasa. Las concentraciones óptimas de los inductores fueron establecidas mediante un diseño factorial. Se estimó la estabilidad térmica de la lacasa en un amplio rango de pH ácidos, y se comprobó que a pH más altos la enzima es más termoestable.(AU)

Ganoderma lucidum (Curtis) P. Karst is a white rot fungus that is able to degrade the lignin component in wood. The ability of two strains of this species to produce the ligninolytic enzyme laccase was assessed. After the evaluation of induction with heavy metals and phenolic compounds, it was found that among the tested substances, copper and ferulic acid are the best laccase inducers. It was also observed that the two types of inducers (phenolic and metallic) produce different electrophoretic patterns of laccase activity. Optimized concentrations of inducers were obtained through a factorial design and the thermal stability of optimized supernatants was studied at a wide range of acidic pH. We found that the enzyme is more thermostable at higher pH values.(AU)

Optimization of laccase production by two strains of Ganoderma lucidum using phenolic and metallic inducers / Optimización de la producción de lacasa por dos cepas de Ganoderma lucidum utilizando inductores fenólicos y metálicos

Ganoderma lucidum (Curtis) P. Karst es un hongo causante de pudrición blanca, capaz de degradar la lignina de la madera y otros sustratos en los que crece. En este trabajo se evaluó la capacidad de dos cepas de esta especie de producir la enzima ligninolítica lacasa. Asimismo, se ensayó la inducción de esta enzima con diferentes compuestos fenólicos e iones metálicos, y se encontró que el ácido ferúlico y el cobre fueron los mejores inductores de la lacasa entre los agentes evaluados. También se encontró que los dos tipos de inductores (fenólicos y metálicos) producen distintos patrones electroforéticos de actividad lacasa. Las concentraciones óptimas de los inductores fueron establecidas mediante un diseño factorial. Se estimó la estabilidad térmica de la lacasa en un amplio rango de pH ácidos, y se comprobó que a pH más altos la enzima es más termoestable.

Ganoderma lucidum (Curtis) P. Karst is a white rot fungus that is able to degrade the lignin component in wood. The ability of two strains of this species to produce the ligninolytic enzyme laccase was assessed. After the evaluation of induction with heavy metals and phenolic compounds, it was found that among the tested substances, copper and ferulic acid are the best laccase inducers. It was also observed that the two types of inducers (phenolic and metallic) produce different electrophoretic patterns of laccase activity. Optimized concentrations of inducers were obtained through a factorial design and the thermal stability of optimized supernatants was studied at a wide range of acidic pH. We found that the enzyme is more thermostable at higher pH values.

Optimization of laccase production by two strains of Ganoderma lucidum using phenolic and metallic inducers. / Optimization of laccase production by two strains of Ganoderma lucidum using phenolic and metallic inducers.

Ganoderma lucidum (Curtis) P. Karst is a white rot fungus that is able to degrade the lignin component in wood. The ability of two strains of this species to produce the ligninolytic enzyme laccase was assessed. After the evaluation of induction with heavy metals and phenolic compounds, it was found that among the tested substances, copper and ferulic acid are the best laccase inducers. It was also observed that the two types of inducers (phenolic and metallic) produce different electrophoretic patterns of laccase activity. Optimized concentrations of inducers were obtained through a factorial design and the thermal stability of optimized supernatants was studied at a wide range of acidic pH. We found that the enzyme is more thermostable at higher pH values.

Protective effect of vanilloids against chemical stress on the white-rot fungus Ganoderma lucidum.

Bioremediation of contaminated sites by biosorption of pollutants onto a wide range of materials has emerged as a promising treatment for recalcitrant aromatic compounds or heavy metals. When adsorption occurs on living white-rot fungi mycelia, the pollutants may be degraded by ligninolytic enzymes. However, the survival of mycelia in harsh conditions is one of the drawbacks of those methodologies. In this study, it was demonstrated that culture media supplemented with several guaiacol derivatives (vanilloids) increased the resistance of Ganoderma lucidum E47 cultures to chemical stress by enhancing the adsorptive capacity of the extracellular mucilaginous material (ECMM). The toxicity of the fungicides gentian violet (GV), malachite green (MG) and clotrimazole, and the heavy metal Cadmium was noticeably diminished in fungal cultures supplemented with the guaiacol derivative vanillic acid (VA). No degradation of the tested compounds was detected. The activity of the oxidative enzymatic systems like laccase, a well-known oxidase associated to dye degradation, was only detectable after complete growth on plates. Extremely low concentrations of VA caused a significant protective effect, radial extension of the growth halo in plates supplemented with 0.0001 mM of VA plus GV was up to 20% to that obtained in control plates (without addition of GV and VA). Therefore, the protective effect could not be attributable to VA per se. ECMM separated from the mycelium exhibited a much higher increase in the adsorptive capacity when isolated from liquid cultures containing VA, while that obtained from unsupplemented cultures showed an almost null adsorptive capacity.

Black liquor decolorization by selected white-rot fungi.

Five different strains of white-rot fungi have been tested for their ability to decolorize black liquor on plates and on solid-state fermentation using vermiculite as the solid inert support. Since the high salt concentration inhibited the growth of all fungi, the black liquor was dialyzed against distilled water prior to use. A preliminary step on plates was carried out to qualitatively determine the capacity of the fungal strains for black liquor decolorization. Out of the five fungi studied, Phanerochaete sordida, Pycnoporus sanguineus, and Trametes elegans exhibited the more conspicuous decolorization halos in malt extract medium, while the decolorization by all the strains was not evident when a defined culture medium was used. Cultures on solid-state fermentation using vermiculite as solid support were also tested, the liquid phase was malt extract or glucose-based medium and supplemented with different black liquor concentrations. Decolorization of black liquor was largely affected by the fungal strain, the concentration of black liquor, and the carbon source. The percentage of color removal ranged from 6.14% to 91.86% depending on the fungal strain and culture conditions. Maximal decolorization was observed in malt extract cultures after 60 cultivation days. Interestingly, decolorization in malt extract medium increased with increasing black liquor concentration. The highest decolorization value was achieved by Steccherinum sp. which reduced up to 91.86% the color of the black liquor in malt extract medium; this percentage is equivalent to 5.2 g L(-1) of decolorized black liquor, the highest value reported to date. Traditional technologies used for the treatment of black liquor are not always effective and may not to be an environmentally friendly process. Vermiculite-white-rot fungi systems are presented in this work as a promising efficient alternative for the treatment of black liquor.

Effects of nutrients, pH and water potential on exopolysaccharides production by a fungal strain belonging to Ganoderma lucidum complex.

Exopolysaccharide (EPS) production by Ganoderma lucidum in response to different culture conditions was studied. Cellulose and glucose, in defined media, resulted in the more efficient enhancers of EPS production among the carbon sources tested. In natural media cultures containing glucose and malt extract exhibited a marked increase (up to 29-fold) respect to defined media. Subsequently, high malt extract and glucose concentrations were tested. G. lucidum produced two fractions of EPS, water-soluble and water-insoluble under these culture conditions. The maximum value (15 g L(-1)) was reached at 21 days in the medium containing 60 g L(-1) malt extract and 40 g L(-1) glucose. The incomplete utilization of reducing sugars by the fungus in these media suggested that not only did high malt extract and glucose concentrations play a role in EPS production but also the water activity might be involved. A factorial uniform experimental design to test the effect of malt extract, polyethylene glycol (PEG, as water activity depressor), and initial pH on specific EPS production was applied. G. lucidum showed to be a more efficient specific EPS (mg EPS per g mycelium) producer at pH 3.5 in cultures containing the highest PEG and malt extract concentrations.

Geastrum episcopale: a new noticeable species with red-violet exoperidium.

A new species, Geastrum episcopale, is described from Argentina. Detailed descriptions and illustrations of fresh and dry basidiomata are provided along with photographs of microscopic elements, such as spores, capillitium, calcium oxalate crystals, and exo- and endoperidium surfaces. This fungus is typical of the genus in its morphology and ecology but clearly differs from every known species. This species can be distinguished easily by the red-violet (to magenta) exoperidium and the lilac endoperidium. Specimens were collected on wet soil composed of a superficial layer of ca. 5 cm leaf litter and 15 cm of particulate and melanized lignocellulosic material (pH 6.5) under Eugenia uruguayensis and Nectandra sp.

Influence of the carbon source on the growth and lignocellulolytic enzyme production by Morchella esculenta strains.

Growth and lignocellulolytic enzymes production by two Morchella esculenta strains (BAFC 1728 and BEL 124) growing in solid state fermentation using different lignocellulosic materials along 58 days was characterized. Both strains were able to grow on the three substrates: wheat bran, wheat bran plus corn starch, and rolled oat. The growth was characterized by measuring chitin content, reducing sugars, pH, dry weight loss, and extractable proteins, such parameters varied substantially with substrate and strain used. The maximum rate of growth in both strains was observed between 5 and 28 days. Regarding enzyme production, as a general trend strain BAFC 1728 produced the highest titres. The most evident difference was observed in laccase production by this strain on wheat bran, which exceeded that observed in strain BEL 124 by tenfold (7.45 U g(-1)).

Stabilization studies of Fomes sclerodermeus laccases.

Stability of laccase isoenzymes from a crude extract obtained from Fomes sclerodermeus grown on wheat bran medium was studied. The variables assessed were temperature, pH and additives. As revealed by PAGE, three bands of laccase, each with different thermal inactivation pattern, were detected in the crude extract: after 6h at 50 degrees C and pH 8, Lc2 was the most resistant, while the Lc1 and Lc3 bands were almost completely inactivated. This pattern of inactivation was observed at all temperatures and pH tested. Laccase activity was more stable in the 5-10 pH range when incubated at 40 and 50 degrees C; at 30 degrees C and 24h the enzyme remained fully active in the 3-11 pH range. The effect of additives (veratryl alcohol, trehalose, glycerol, mannitol, glutaraldehyde, CuSO(4) and 1-HBT) on laccase stability was tested. The stability was enhanced with CuSO(4) (1.25 mM), glycerol (0.2%) and mannitol (1%). The presence of both CuSO(4) and glycerol caused a 3-fold increase in the half-life values.

[Combined effect of copper and initial pH of the culture on production of laccase and manganese peroxidase by Stereum hirsutum (Willd) Pers]. / Efecto combinado del cobre y pH inicial del medio de cultivo sobre la producción de lacasa y manganeso peroxidasa por Stereum hirsutum (Willd) Pers.

Stereum hirsutum is a white-rot fungus which produces laccase and manganese peroxidase as part of its ligninolytic system. Maximal ligninases production (under the conditions studied) was obtained in the media containing 250 microM Cu++ along with a pH value of 5.5. The fitting of ligninase production to a linear equation showed negative interaction between increasing values of pH and concentration of copper.