Evaluation of laccase production by Ganoderma lucidum in submerged and solid-state fermentation using different inducers.

Laccases are multicopper oxidases with high potential for industrial applications. Several basidiomycete fungi are natural producers of this enzyme; however, the optimization of production and selection of inducers for increased productivity coupled with low costs is necessary. Lignocellulosic residues are important lignin sources and potential inducers for laccase production. Pinus taeda, a dominant source of wood-based products, has not been investigated for this purpose yet. The aim of this study was to evaluate the production of laccase by the basidiomycete fungus Ganoderma lucidum in the presence of different inducers in submerged and solid-state fermentation. The results of submerged fermentation in presence of 5 µM CuSO 4 , 2 mM ferulic acid, 0.1 g/L P. taeda sawdust, or 0.05 g/L Kraft lignin indicated that although all the tested inducers promoted increase in laccase activity in specific periods of time, the presence of 2 mM ferulic acid resulted in the highest value of laccase activity (49 U/L). Considering the submerged fermentation, experimental design following the Plackett-Burman method showed that the concentrations of ferulic acid and P. taeda sawdust had a significant influence on the laccase activity. The highest value of 785 U/L of laccase activity on submerged fermentation was obtained on the seventh day of cultivation. Finally, solid-state fermentation cultures in P. taeda using ferulic acid or CuSO 4 as inducers resulted in enzymatic activities of 144.62 and 149.89 U/g, respectively, confirming the potential of this approach for laccase production by G. lucidum.

Conditions Affecting Lingzhi or Reishi Medicinal Mushroom Ganoderma lucidum (Agaricomycetes) Basidiome Quality, Morphogenesis, and Biodegradation of Wood By-products in Argentina.

Solid-state fermentation (SSF) with the medicinal higher Basidiomycete Ganoderma lucidum was studied as a strategy to use pine (Pinus radiata D. Don) and poplar (Populus nigra L.) wood chips and sawdust. Fruiting bodies were produced and the value of the biotransformed substrate was assessed. The highest mushroom yield (63 g dry weight per kilogram of dry substrate) was obtained with poplar sawdust and wood chips. Immersion of the bioreactors was a simple watering method that obtained suitable yields. Two morphological types were induced using 2 different incandescent light intensities. High light irradiation induced the highest valued mushroom morphology (as a whole product). Time course study of substrate biodegradation and mycelial growth dynamics indicated that the trophophase lasted 20 days and presented laccase activity of 0.01-0.03 units · g-1. The activity at idiophase was 10 times higher. Aqueous and alkali extracts, as well as carbohydrase enzyme profile activity, revealed differences in the properties of the residual substrate; some related to the substrate source are considered to be of concern for further use of this pretreated biomass. In view of the results obtained, we propose use of SSF of pine and poplar with G. lucidum to profitably recycle softwood by-products from the timber industry.

Evaluation of diuron tolerance and biotransformation by the white-rot fungus Ganoderma lucidum.

The white rot basidiomycete Ganoderma lucidum was evaluated for its capability to tolerate and to degrade the herbicide diuron. Diuron at a subtoxic concentration was added at the start of the cultivation in glucose liquid stationary cultures. Under this condition diuron was a laccase inducer. Almost 50% of the initially present diuron was removed after 15 d of cultivation. Two diuron metabolites were found N'-(3,4-dichlorophenyl)-N-methylurea (DCPMU) and 3,4-dichlorophenylurea (DCPU). The addition of the cytochrome P450 inhibitors 1-aminobenzotriazole and piperonyl butoxide reduced significantly the capability of the fungus in degrading diuron. The activities of superoxide dismutase and catalase were significantly increased in the mycelial extracts by the presence of diuron. On the other hand, diuron did not cause any significant alteration in the levels of reactive oxygen species. Additionally, laccase could also degrade diuron in vitro and this degradation was increased by the addition of synthetic mediators, 3-ethylbenzthiazoline-6-sulphonic acid and acetylacetone. Significant reduction in the toxicity, as evaluated by the Lactuca sativa bioassay, was observed after G. lucidum treatment. In conclusion, G. lucidum is able to metabolize diuron by intra- and extracellular mechanisms, without the accumulation of toxic products.

Production of Basidiomata and Ligninolytic Enzymes by the Lingzhi or Reishi Medicinal Mushroom, Ganoderma lucidum (Agaricomycetes), in Licuri (Syagrus coronata) Wastes in Brazil.

Ganoderma lucidum is a medicinal mushroom with different forms of bioactivity that has been used in popular medicine for centuries. This study aimed to test the application of agricultural wastes (fruit shells, leaves, and bracts) from the endemic Brazilian palm tree Syagrus coronata (licuri) as substrates for the production of G. lucidum basidiomata and ligninolytic enzymes via solid-state fermentation. The best culture conditions were the same for all substrates (pH 6.5, carbon-to-nitrogen ratio = 40, and temperature 30°C) and were established from preliminary assays. The yield was not significantly different for bracts (33.53 g/kg) and leaves (37.48 g/kg), nor for the biological efficiency in these same substrates: bracts, 3.35%; leaves, 3.75%. The highest laccase (13.80 U/L) and manganese peroxidase (14.92 U/L) activities were achieved after 14 and 28 days of incubation, respectively, using bracts as the substrate. Licuri residues are then potential substrates to be used in the bioconversion process for mycelia, basidiomata, and ligninolytic enzyme production by G. lucidum.

The Effect of Agaricus brasiliensis and Ganoderma lucidum Medicinal Mushroom Administration on the L-arginine/Nitric Oxide System and Rat Leukocyte Apoptosis in Experimental Type 1 Diabetes Mellitus.

Oxidative-nitrative stress develops as a result of hyperglycemia under diabetes mellitus. Formation of excessive reactive oxygen species and reactive nitrogen species leads to different cytotoxic effects and ultimately to increased cell death by apoptosis of immune-competent blood cells. This study showed the influence of medicinal mushroom (MM) administration on the L-arginine/nitric oxide (NO) system and rat leukocyte apoptosis under normal and experimental diabetic conditions. Animals were divided into 6 groups: (1) control, (2) control animals treated with Agaricus brasiliensis, (3) control animals treated with Ganoderma lucidum, (4) animals with experimental diabetes (EDM), (5) diabetic animals treated with A. brasiliensis, and (6) diabetic animals treated with G. lucidum. Control and diabetic animals were fed powdered mushrooms at a dose of 1 g/kg body weight. Administration of MMs to animals with diabetes caused a decrease in the activity of the NO synthase enzyme, as well as in the content of stable end products of NO metabolism-nitrates and nitrites-at the control level. The normalizing effect of mushrooms on the percentage of leukocytes that contain pro- (p53) and antiapoptotic (Bcl-2) proteins compared with the EDM group was shown by immunocytochemical analysis. Thus the administration of MMs under EDM showed a positive corrective action on the L-arginine/NO system and the ratio between p53 and Bcl-2 proteins in white blood cells, as well as on apoptotic index reduction.

Computational analysis and low-scale constitutive expression of laccases synthetic genes GlLCC1 from Ganoderma lucidum and POXA 1B from Pleurotus ostreatus in Pichia pastoris.

Lacasses are multicopper oxidases that can catalyze aromatic and non-aromatic compounds concomitantly with reduction of molecular oxygen to water. Fungal laccases have generated a growing interest due to their biotechnological potential applications, such as lignocellulosic material delignification, biopulping and biobleaching, wastewater treatment, and transformation of toxic organic pollutants. In this work we selected fungal genes encoding for laccase enzymes GlLCC1 in Ganoderma lucidum and POXA 1B in Pleurotus ostreatus. These genes were optimized for codon use, GC content, and regions generating secondary structures. Laccase proposed computational models, and their interaction with ABTS [2, 2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)] substrate was evaluated by molecular docking. Synthetic genes were cloned under the control of Pichia pastoris glyceraldehyde-3-phosphate dehydrogenase (GAP) constitutive promoter. P. pastoris X-33 was transformed with pGAPZαA-LaccGluc-Stop and pGAPZαA-LaccPost-Stop constructs. Optimization reduced GC content by 47 and 49% for LaccGluc-Stop and LaccPost-Stop genes, respectively. A codon adaptation index of 0.84 was obtained for both genes. 3D structure analysis using SuperPose revealed LaccGluc-Stop is similar to the laccase crystallographic structure 1GYC of Trametes versicolor. Interaction analysis of the 3D models validated through ABTS, demonstrated higher substrate affinity for LaccPost-Stop, in agreement with our experimental results with enzymatic activities of 451.08 ± 6.46 UL-1 compared to activities of 0.13 ± 0.028 UL-1 for LaccGluc-Stop. This study demonstrated that G. lucidum GlLCC1 and P. ostreatus POXA 1B gene optimization resulted in constitutive gene expression under GAP promoter and α-factor leader in P. pastoris. These are important findings in light of recombinant enzyme expression system utility for environmentally friendly designed expression systems, because of the wide range of substrates that laccases can transform. This contributes to a great gamut of products in diverse settings: industry, clinical and chemical use, and environmental applications.

Optimization of the Yield of Lingzhi or Reishi Medicinal Mushroom, Ganoderma lucidum (Higher Basidiomycetes), Cultivated on a Sunflower Seed Hull Substrate Produced in Argentina: Effect of Olive Oil and Copper.

Sunflower seed hulls were used as the main component of a solid substrate for the cultivation of the lingzhi or reishi medicinal mushroom Ganoderma lucidum. This study evaluated the effects of supplementing the substrate with olive oil and copper (II) on the mushroom production (MP) parameters and fruiting body total triterpenoid content. The addition of 1.5% olive oil increased total MP by 21.7% (dry basis) in 3 flushes. Copper (60 ppm) increased the daily productivity of the first flush (MP per day) by both reducing the time needed to harvest the crop and increasing the MP. However, the MP at the second and third flushes was reduced. When both supplements were combined, the MP at the first flush was 43% higher than with control treatment. No significant change in mushroom total triterpenoid content was observed by the addition of supplements to the substrate. An increase of 145-155% in the mushroom copper content was obtained by the addition of 60 ppm copper to the substrate. It is thus recommended to use substrate formulations containing both olive oil and copper (II) and harvest just the first flush.

Improved 1-Deoxynojirimycin (DNJ) production in mulberry leaves fermented by microorganism.

DNJ, an inhibitor of α-glucosidase, is used to suppress the elevation of postprandial hyperglycemia. In this study, we focus on screening an appropriate microorganism for performing fermentation to improve DNJ content in mulberry leaf. Results showed that Ganoderma lucidum was selected from 8 species and shown to be the most effective in improvement of DNJ production from mulberry leaves through fermentation. Based on single factor and three factor influence level tests by following the Plackett-Burman design, the optimum extraction yield was analyzed by response surface methodology (RSM). The extracted DNJ was determined by reverse-phase high performance liquid chromatograph equipped with fluorescence detector (HPLC-FD). The results of RSM showed that the optimal condition for mulberry fermentation was defined as pH 6.97, potassium nitrate content 0.81% and inoculums volume 2 mL. The extraction efficiency reached to 0.548% in maximum which is 2.74 fold of those in mulberry leaf.

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.

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.

Improved 1-Deoxynojirimycin (DNJ) production in mulberry leaves fermented by microorganism

DNJ, an inhibitor of α-glucosidase, is used to suppress the elevation of postprandial hyperglycemia. In this study, we focus on screening an appropriate microorganism for performing fermentation to improve DNJ content in mulberry leaf. Results showed that Ganoderma lucidum was selected from 8 species and shown to be the most effective in improvement of DNJ production from mulberry leaves through fermentation. Based on single factor and three factor influence level tests by following the Plackett-Burman design, the optimum extraction yield was analyzed by response surface methodology (RSM). The extracted DNJ was determined by reverse-phase high performance liquid chromatograph equipped with fluorescence detector (HPLC-FD). The results of RSM showed that the optimal condition for mulberry fermentation was defined as pH 6.97, potassium nitrate content 0.81% and inoculums volume 2 mL. The extraction efficiency reached to 0.548% in maximum which is 2.74 fold of those in mulberry leaf.

Improved 1-Deoxynojirimycin (DNJ) production in mulberry leaves fermented by microorganism

DNJ, an inhibitor of α-glucosidase, is used to suppress the elevation of postprandial hyperglycemia. In this study, we focus on screening an appropriate microorganism for performing fermentation to improve DNJ content in mulberry leaf. Results showed that Ganoderma lucidum was selected from 8 species and shown to be the most effective in improvement of DNJ production from mulberry leaves through fermentation. Based on single factor and three factor influence level tests by following the Plackett-Burman design, the optimum extraction yield was analyzed by response surface methodology (RSM). The extracted DNJ was determined by reverse-phase high performance liquid chromatograph equipped with fluorescence detector (HPLC-FD). The results of RSM showed that the optimal condition for mulberry fermentation was defined as pH 6.97, potassium nitrate content 0.81% and inoculums volume 2 mL. The extraction efficiency reached to 0.548% in maximum which is 2.74 fold of those in mulberry leaf.

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.

Hepatoprotective effects of mushrooms.

The particular characteristics of growth and development of mushrooms in nature result in the accumulation of a variety of secondary metabolites such as phenolic compounds, terpenes and steroids and essential cell wall components such as polysaccharides, b-glucans and proteins, several of them with biological activities. The present article outlines and discusses the available information about the protective effects of mushroom extracts against liver damage induced by exogenous compounds. Among mushrooms, Ganoderma lucidum is indubitably the most widely studied species. In this review, however, emphasis was given to studies using other mushrooms, especially those presenting efforts of attributing hepatoprotective activities to specific chemical components usually present in the mushroom extracts.

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.

Production of biomass, polysaccharides, and ganoderic acid using non-conventional carbon sources under submerged culture of the Lingzhi or Reishi medicinal mushroom, Ganoderma lucidum (W.Curt.:Fr.)P. Karst. (higher Basidiomycetes).

The effect of different non-conventional carbon sources was studied in the submerged culture of Lingzhi or Reishi medicinal mushroom, Ganoderma lucidum, for simultaneous production of mycelial biomass, bioactive ganoderic acid, and polysaccharides, in less time, using non-conventional carbon sources to minimize the high costs of current culture media. The optimal medium composition was defined as (g/L): 50 of barley flour, 0.2 of KH2PO4, 0.1 of MgSO4ⁱ7H2O, and 1 NH4Cl. Cultivated under this complex culture medium, the mycelial biomass production was 23.49 ± 0.37 g/L; the extracellular polysaccharides production was 2.72 ± 0.11 g/L; the intracellular polysaccharides production was 2.22 ± 0.06 g/L; the ganoderic acids production was 299.67 ± 11.63 mg/L. One liter of culture medium developed in this project was priced at USD $ 0.11 if barley flour is used as carbon source or $ 0.13 with oat flour in order to get a good amount of products of interest.

Comparative removal of bentazon by Ganoderma lucidum in liquid and solid state cultures.

Bentazon removal by Ganoderma lucidum cultured in liquid and solid state conditions was compared in this work. In solid state cultures, the fungus produced both ligninolytic enzymes, namely laccase and Mn peroxidase. In liquid cultures, the main ligninolytic enzyme produced was laccase. In both types of cultures bentazon improved the production of laccase without significant alteration in the production of Mn peroxidase. In solid state cultures, where high levels of both laccase and Mn peroxidase activities were found, the fungus was more resistant to the action of the herbicide (50 mM in solid state cultures against 20 mM in liquid cultures) and more efficient in removing bentazon (90% removal against 55% in liquid cultures after 10 days of cultivation). Furthermore, the solid state culture filtrates were more efficient in the in vitro degradation of bentazon than the liquid culture filtrates. These observations suggest that both enzymes, laccase and Mn peroxidase, are involved in bentazon degradation. The results further suggest that solid state cultures of Ganoderma lucidum could be useful in strategies designed to reduce environmental contamination by bentazon.

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.