Production, partial purification and characterization of ligninolytic enzymes from selected basidiomycetes mushroom fungi.

In recent years, many research on the quantity of lignocellulosic waste have been developed. The production, partial purification, and characterisation of ligninolytic enzymes from various fungi are described in this work. On the 21st day of incubation in Potato Dextrose (PD) broth, Hypsizygus ulmarius developed the most laccase (14.83 × 10-6 IU/ml) and manganese peroxidase (24.11 × 10-6 IU/ml), while Pleurotus florida produced the most lignin peroxidase (19.56 × -6 IU/ml). Laccase (Lac), lignin peroxidase (LiP), and manganese peroxidase (MnP), all generated by selected basidiomycetes mushroom fungi, were largely isolated using ammonium sulphate precipitation followed by dialysis. Laccase, lignin peroxidase, and manganese peroxidase purification findings indicated 1.83, 2.13, and 1.77 fold purity enhancements, respectively. Specific activity of purified laccase enzyme preparations ranged from 305.80 to 376.85 IU/mg, purified lignin peroxidase from 258.51 to 336.95 IU/mg, and purified manganese peroxidase from 253.45 to 529.34 IU/mg. H. ulmarius laccase (376.85 IU/mg) with 1.83 fold purification had the highest specific activity of all the ligninolytic enzymes studied, followed by 2.13 fold purification in lignin peroxidase (350.57 IU/mg) and manganese peroxidase (529.34 IU/mg) with 1.77-fold purification. Three notable bands with molecular weights ranging from 43 to 68 kDa and a single prominent band with a molecular weight of 97.4 kDa were identified on a Native PAGE gel from mycelial proteins of selected mushroom fungus. The SDS PAGE profiles of the mycelial proteins from the selected mushroom fungus were similar to the native PAGE. All three partially purified ligninolytic isozymes display three bands in native gel electrophoresis, with only one prominent band in enzyme activity staining. The 43 kDa, 55 kDa, and 68 kDa protein bands correspond to laccase, lignin peroxidase, and manganese peroxidase, respectively.

Evaluation of antidiabetic activity of Pleurotus pulmonarius against streptozotocin-nicotinamide induced diabetic wistar albino rats.

The current research aims to evaluate the antidiabetic properties of Pleurotus pulmonarius, an edible basidiomycetes mushroom fungi in diabetic induced wistar albino rats. Mycelial Hot Water Extracts (HWE) and Acetone Extracts (AE) of Pleurotus pulmonarius was orally administrated to STZ-NA induced (55 mg/kilogram body weight) diabetic wistar albino rats at a concentration of 200 and 400 mg/kg for 4 weeks. The outcomes revealed that the HWE of Pleurotus pulmonarius resulted in a significant (p < 0.001) reduction in blood glucose level. A noteworthy (p < 0.001) reduction in serum lipid profile and elevation in High-Density Lipoprotein Cholesterol (HDL-C) after administration with HWE, also demonstrating the protective effects of HWE in diabetes-related complications. Besides all antidiabetic parameters, pathological morphology of the pancreas, liver and kidney are regularised. This observation indicated that HWE of Pleurotus pulmonarius possessed higher antidiabetic activity than AE. Besides, HWE also promoted a significant control of alpha amylase enzyme in a concentration-dependent manner with a maximum activity of 99.23% inhibition at 1000 µg/ml. The outcomes of the present study indicated that the HWE possesses a potential antidiabetic activity both in vitro and in vivo. Thus, it can be used as a nontoxic complementary drug in the controlling of diabetes and related complications, thus providing scientific authentication of its use as an antidiabetic agent.

Characterization and biological activities of extracellular melanin produced by Schizophyllum commune (Fries).

Melanins are enigmatic pigments produced by a wide variety of microorganisms including bacteria and fungi. Here, we have isolated and characterized extracellular melanin from mushroom fungus, Schizophyllum commune. The extracellular dark pigment produced by the broth culture of S. commune, after 21 days of incubation was recovered by hot acid-alkali treatment. The melanin nature of the pigment was characterized by biochemical tests and further, confirmed by UV, IR, EPR, NMR and MALDI-TOF Mass Spectra. Extracellular melanin, at 100 µg/ml, showed significant antibacterial activity against Escherichia coli, Bacillus subtilis, Klebsiella pneumoniae and Pseudomonas fluorescens and antifungal activity against Trichophyton simii and T. rubrum. At a concentration of 50 µg/ml, melanin showed high free radical scavenging activity of DPPH (2,2-diphenyl-1-picrylhydrazyl) indicating its antioxidant potential. It showed concentration dependent inhibition of cell proliferation of Human Epidermoid Larynx Carcinoma Cell Line (HEP-2). This study has demonstrated characterization of melanin from basidiomycetes mushroom fungus, Schizophyllum commune and its applications.

Comparative studies on lignin and polycyclic aromatic hydrocarbons degradation by basidiomycetes fungi.

A total of 130 wild basidiomycetes fungi were collected and identified. The polycyclic aromatic hydrocarbons (PAHs) degradation by the potential Phellinus sp., Polyporus sulphureus (in liquid state fermentation (LSF), solid state fermentation (SSF), in soil) and lignin biodegradation were compared with those of a bacterial isolate and their corresponding cocultures. The PAHs degradation was higher in LSF and the efficiency of the organisms declined in SSF and in soil treatment. Phellinus sp. showed better degradation in SSF and in soil. Bacillus pumilus showed higher degradation in LSF. B. pumilus was seen to have lower lignin degradation than the fungal cultures and the cocultures could not enhance the degradation. Phellinus sp. which had higher PAHs and lignin degradation showed higher biosurfactant production than other organism. Manganese peroxidase (MnP) was the predominant enzyme in Phellinus sp. while lignin peroxidase (Lip) was predominant in P. sulphureus.

Microbial production of poly-beta-hydroxybutyrate by marine microbes isolated from various marine environments.

Considering the industrial interest of Poly-beta-hydroxybutyrate (PHB), bacteria isolated from the various marine arenas were screened for their ability to accumulate PHB and were compared with Wausteria eutropha (MTCC-1285). Among the 42 isolates, four strains showed the accumulation of PHB. The maximum PHB producer Vibrio sp. (MK4) was further studied in detail. To increase the productivity, steps were taken to evaluate the effect of carbon sources, nitrogen sources, pH and sodium chloride concentration on PHB productivity by MK4. The optimized conditions were further used for the batch fermentation over a period of 72 h. Significantly higher maximum biomass of 9.1g/L with a PHB content of 4.223 g/L was obtained in a laboratory-scale bioreactor at 64 h, thus giving a productivity of 0.065 g/L/h. The extracted polymer was compared with the authentic PHB and was confirmed to be PHB using FTIR analysis and (1)H NMR analysis. Thus, the study highlights the potential of the use of Vibrio sp (MK4) in the commercial production of PHB.

Polycyclic aromatic hydrocarbons (PAHs) biodegradation by basidiomycetes fungi, Pseudomonas isolate, and their cocultures: comparative in vivo and in silico approach.

The polycyclic aromatic hydrocarbons (PAHs) biodegradation potential of the five basidiomycetes' fungal monocultures and their cocultures was compared with that of a Pseudomonas isolate recovered from oil-spilled soil. As utilization of hydrocarbons by the microorganisms is associated with biosurfactant production, the level of biosurfactant production and its composition by the selected microorganisms was also investigated. The Pseudomonas isolate showed higher ability to degrade three of the five PAHs but the isolate did not produce biosurfactant higher than C. versicolor and P. ostreatus. Among the PAHs, the most effective biodegradation of PAH--pyrene (42%)--was obtained with the fungus C. versicolor. Cocultures involving the fungi and Pseudomonas could not significantly degrade the selected PAHs compounds above that degraded by the most efficient monoculture. A slight increase in pyrene degradation was observed in cocultures of C. versicolor and F. palustris (93.7% pyrene). The crude biosurfactant was biochemically characterized as a multicomponent surfactant consisting of protein and polysaccharides. The PAH biodegradation potential of the basidiomycetes fungi positively correlated with their potential to express ligninolytic enzymes such as lignin peroxidase (Lip), manganese peroxidase (Mnp), and laccase. The present study utilized in silico method such as protein-ligand docking using the FRED in Open Eye software as a tool to assess the level of ligninolytic enzymes and PAHs interactions. The in silico analysis using FRED revealed that of the five PAHs, maximum interaction occurred between pyrene and all the three ligninolytic enzymes. The results of the in silico analysis corroborated with our experimental results showing that pyrene was degraded to the maximum extent by species such as C. versicolor and P. ostreatus.

Enzyme activities and substrate degradation by fungal isolates on cassava waste during solid state fermentation.

The growth and bioconversion potential of selected strains growing on cassava waste substrate during solid state fermentation were assessed. Rhizopus stolonifer showed the highest and the fastest utilization of starch and cellulose in the cassava waste substrate. It showed 70% starch utilization and 81% cellulose utilization within eight days. The release of reducing sugars indicating the substrate saccharification or degradation potential of the organisms reached the highest value of 406.5 mg/g by R. stolonifer on cassava waste during the eighth day of fermentation. The protein content was gradually increased (89.4 mg/g) on the eighth day of fermentation in cassava waste by R. stolonifer. The cellulase and amylase activity is higher in R. stolonifer than A. niger and P. chrysosporium. The molecular mass of purified amylase and cellulase seemed to be 75 KDal, 85 KDal respectively.

Isolation, Regeneration and PEG-Induced Fusion of Protoplasts of Pleurotus pulmonarius and Pleurotus florida.

Inter-specific hybridization between Pleurotus pulmonarius and P. florida was attempted through PEG-induced protoplast fusion to select a fusant. The protocol for protoplast release, regeneration and fusion in these two Pleurotus species was standardized using the variables controlling the process. The mixture of mycolytic enzymes, i.e. commercial cellulase, crude chitinase and pectinase, KCl (0.6 M) as osmotic stabilizer, pH 6 of the phosphate buffer and an incubation time of 3 hours resulted in the maximum release of protoplasts from 3-day-old mycelia of P. florida (5.3~5.75 × 10(7) protoplasts/g) and P. pulmonarius (5.6~6 × 10(7) protoplasts/g). The isolated protoplasts of P. florida regenerated mycelium with 3.3% regeneration efficiency while P. pulmonarius showed 4.1% efficiency of regeneration. Polyethyleneglycol (PEG) - induced fusion of protoplasts of these two species resulted in 0.28% fusion frequency. The fusant produced fruiting bodies on paddy straw but required a lower temperature of crop running (24 ± 2℃) than its parents which could fruit at 28 ± 2℃. The stable fusant strain was selected by testing for the selected biochemical markers i.e. Carbendazim tolerance and utilization of the lignin degradation product, vanillin.

Raw starch degrading amylase production by various fungal cultures grown on cassava waste.

The solid waste of sago industry using cassava was fermented by Aspergillus niger, Aspergillus terreus and Rhizopus stolonifer in solid state fermentation. Cassava waste contained 52 per cent starch and 2.9 per cent protein by dry weight. The amylase activity was maintained at a high level and the highest amylase activity was observed on the 8(th) day in R. stolonifer mediated fermentation. R. stolonifer was more efficient than Aspergillus niger and Aspergillus terreus in bioconverting cassava waste into fungal protein (90.24 mg/g) by saccharifying 70% starch and releasing 44.5% reducing sugars in eight days of solid state fermentation.

Effect of 'Azotobacter' Bioinoculant on the Growth and Substrate Utilization Potential of Pleurotus eous Seed Spawn.

We investigated the effect of nitrogen fixing Azotobacter bioinoculant on the mycelial growth and the rate of substrate utilization by Pleurotus eous. The synergistic or antagonistic role of the microorganism during dual culturing with the mushroom or the competitor molds Trichoderma viride, and Trichoderma reesi was studied. Azotobacter was inhibitory to the molds, which are competitive to the mushroom in the seed spawn substrate, but was synergistic towards the mushroom. The growth, substrate utilization potential as total nitrogen content and cellulase enzyme activities of the mushroom in the seed spawn substrate were also enhanced in the presence of the bioinoculant at lower inoculum concentrations, upto 5 ml broth culture per spawn bottle.