Purification and characterization of laccase from the white rot fungus Trametes versicolor.
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Laccase is one of the ligninolytic enzymes of white rot fungus Trametes versicolor 951022, a strain first isolated in Korea. This laccase was purified 209-fold from culture fluid with a yield of 6.2% using ethanol precipitation, DEAE-Sepharose, Phenyl-Sepharose, and Sephadex G-100 chromatography. T. versicolor 951022 excretes a single monomeric laccase showing a high specific activity of 91,443 U/mg for 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) as a substrate. The enzyme has a molecular mass of approximately 97 kDa as determined by SDS-PAGE, which is larger than those of other laccases reported. It exhibits high enzyme activity over broad pH and temperature ranges with optimum activity at pH 3.0 and a temperature of 50 degrees C. The Km value of the enzyme for substrate ABTS is 12.8 micrometer and its corresponding Vmax value is 8125.4 U/mg. The specific activity and substrate affinity of this laccase are higher than those of other white rot fungi, therefore, it may be potentially useful for industrial purposes. (+info)
Cloning of a manganese peroxidase cDNA gene repressed by manganese in Trametes versicolor.
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White-rot fungi have the following enzyme systems for lignin degradation: laccase, lignin peroxidase and manganese peroxidase. There are other types of peroxidases related to lignin degradation, one of which we have cloned a cDNA gene of manganese-repressed peroxidase (MrP) in Trametes versicolor isolated in South Korea. The mrp transcript level has been decreased by 1 micrometer of Mn(2+). (+info)
Evaluation of the monophyly of Fomitopsis using parsimony and MCMC methods.
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To evaluate the monophyly of Fomitopsis and elucidate phylogenetic relationships of its members, partial nuclear large subunit (partial 28S) ribosomal RNA genes were sequenced from 10 species of Fomitopsis and 15 related species. Phylogenetic analyses indicated that Fomitopsis was phylogenetically heterogeneous and its members were divided into three subgroups. The constrained tree excluding F. palustris (the type species of Pilatoporus) from Fomitopsis core group was rejected, thus rejecting the taxonomic concept to segregate Pilatoporus from Fomitopsis. The monophyly of taxa belonging to F. rosea complex was rejected, thus rejecting the complex definition based on morphological similarities. The exclusion of Piptoporus betulinus (the type species of Piptoporus) from Fomitopsis core group was rejected and Piptoporus proved to be heterogeneous in both best MP and MAP trees. The monophyly of F. officinalis with Fomitopsis core group also was rejected. Fomitopsis officinalis was closely related to Antrodia xantha and formed an independent lineage from Fomitopsis core group at the basal position of brown rotting fungi comprising Antrodia, Daedalea, Fomitopsis, Piptoporus and Postia. The MAP tree topologyobtained from MCMC computation of Bayesian inference was similar to the one of the best MP tree based on the parsimony analysis but showed a higher likelihood score in the Kishino-Hasegawa test and reflected better evolutionary patterns for the phylogeny of Fomitopsis. (+info)
Identification of medicinal mushroom species based on nuclear large subunit rDNA sequences.
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The purpose of this study was to develop molecular identification method for medical mushrooms and their preparations based on the nucleotide sequences of nuclear large subunit (LSU) rDNA. Four specimens were collected of each of the three representative medicinal mushrooms used in Korea: Ganoderma lucidum, Coriolus versicolor, and Fomes fomentarius. Fungal material used in these experiments included two different mycelial cultures and two different fruiting bodies from wild or cultivated mushrooms. The genomic DNA of mushrooms were extracted and 3 nuclear LSU rDNA fragments were amplified: set 1 for the 1.1-kb DNA fragment in the upstream region, set 2 for the 1.2-kb fragment in the middle, and set 3 for the 1.3-kb fragment downstream. The amplified gene products of nuclear large subunit rDNA from 3 different mushrooms were cloned into E. coli vector and subjected to nucleotide sequence determination. The sequence thus determined revealed that the gene sequences of the same medicinal mushroom species were more than 99.48% homologous, and the consensus sequences of 3 different medicinal mushrooms were more than 97.80% homologous. Restriction analysis revealed no useful restriction sites for 6-bp recognition enzymes for distinguishing the 3 sequences from one another, but some distinctive restriction patterns were recognized by the 4-bp recognition enzymes AccII and HhaI. This analysis was also confirmed by PCR-RFLP experiments on medicinal mushrooms. (+info)
Protective effects of a neutral polysaccharide isolated from the mycelium of Antrodia cinnamomea on Propionibacterium acnes and lipopolysaccharide induced hepatic injury in mice.
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Mycelia of Antrodia cinnamomea were extracted with chloroform and hot water. A neutral polysaccharide named ACN2a separated from the water extract was purified using 10% CCl3COOH, and repeated column chromatography on HW-65 and DE-52 cellulose. Its structure was determined by chemical and spectroscopic analyses. ACN2a was composed of Gal, Glc, Fuc, Man and GalN (in the ratio 1:0.24:0.07:0.026:faint), in which an alpha-D-(1-->6)-Gal linkage accounted for 73% of all linkages. The ratio of branch points was about 16% of the total residual numbers, and branches were attached to C-2 of galactosyl residues of the main chain. ACN2a had an average molecular weight of 12.9x10(5) Daltons, [alpha]D25=+115 degrees (c=0.44, H2O); [eta]=0.0417dl.g-1, Cp=0.2663 cal/(g. degrees C). The hepatoprotective effect of ACN2a was evaluated using a mouse model of hepatic injury that was induced by Propionibacterium acnes (P. acnes) and lipopolysaccharide (LPS). The administration of ACN2a (0.4, 0.8 g/kg/d, p.o.), significantly prevented increases in serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) enzyme activities in mice treated with P. acnes-LPS, indicating hepatoprotective activity in vivo. (+info)
Filtrate of fermented mycelia from Antrodia camphorata reduces liver fibrosis induced by carbon tetrachloride in rats.
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AIM: To investigate the effects of filtrate of fermented mycelia from Antrodia camphorata (FMAC) on liver fibrosis induced by carbon tetrachloride (CCl(4)) in rats. METHODS: Forty Wistar rats were divided randomly into control group and model group. All model rats were given 200 mL/L CCl(4) (2 mL/Kg, po) twice a week for 8 wk. Four weeks after CCl(4) treatment, thirty model rats were further divided randomly into 3 subgroups: CCl(4) and two FMAC subgroups. Rats in CCl(4) and 2 FMAC subgroups were treated with FMAC 0, 0.5 and 1.0 g/kg, daily via gastrogavage beginning at the fifth week and the end of the eighth week. Spleen weight, blood synthetic markers (albumin and prothrombin time) and hepatic malondialdehyde (MDA) and hydroxyproline (HP) concentrations were determined. Expression of collagen I, tissue inhibitor of metalloproteinases (TIMP)-1 and transforming growth factor beta1 (TGF-beta1) mRNA were detected by RT-PCR. Histochemical staining of Masson's trichrome was performed. RESULTS: CCl(4) caused liver fibrosis, featuring increased prothrombin time, hepatic MDA and HP contents, and spleen weight and decreased plasma albumin level. Compared with CCl(4) subgroup, FMAC subgroup (1 g/kg) significantly decreased the prothrombin time (36.7+/-7.2 and 25.1+/-10.2 in CCl(4) and FMAC groups, respectively, P<0.05) and increased plasma albumin concentration (22.7+/-1.0 and 30.7+/-2.5 in CCl(4) and FMAC groups, respectively, P<0.05). Spleen weight was significantly lower in rats treated with CCl(4) and FMAC (1 g/kg) compared to CCl(4) treated rats only (2.7+/-0.1 and 2.4+/-0.2 in CCl(4) and FMAC groups, respectively, P<0.05). The amounts of hepatic MDA and HP in CCl(4)+FAMC (1 g/kg) subgroup were also lower than those in CCl(4) subgroup (MDA: 3.9+/-0.1 and 2.4+/-0.6 in CCl(4) and CCl(4)+FMAC groups, respectively, P<0.01; HP: 1730.7+/-258.0 and 1311.5+/-238.8 in CCl(4) and CCl(4)+FMAC groups, respectively, P<0.01). Histologic examinations showed that CCl(4)+FMAC subgroups had thinner or less fibrotic septa than CCl(4) group. RT-PCR analysis indicated that FMAC (1 g/kg) reduced mRNA levels of collagen I, TIMP-1 and TGF-beta1 (collagen I: 5.63+/-2.08 and 1.78+/-0.48 in CCl(4) and CCl(4)+FMAC groups, respectively, P<0.01; TIMP-1: 1.70+/-0.82 and 0.34+/-0.02 in CCl(4) and CCl(4)+FMAC groups, respectively, P<0.01; TGF-beta1:38.03+/-11.9 and 4.26+/-2.17 in CCl(4) and CCl(4)+FMAC groups, respectively, P<0.01) in the CCl(4)-treated liver. CONCLUSION: It demonstrates that FMAC can retard the progression of liver fibrosis induced by CCl(4) in rats. (+info)
Termite-egg mimicry by a sclerotium-forming fungus.
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Mimicry has evolved in a wide range of organisms and encompasses diverse tactics for defence, foraging, pollination and social parasitism. Here, I report an extraordinary case of egg mimicry by a fungus, whereby the fungus gains competitor-free habitat in termite nests. Brown fungal balls, called 'termite balls', are frequently found in egg piles of Reticulitermes termites. Phylogenetic analysis illustrated that termite-ball fungi isolated from different hosts (Reticulitermes speratus, Reticulitermes flavipes and Reticulitermes virginicus) were all very similar, with no significant molecular differences among host species or geographical locations. I found no significant effect of termite balls on egg survivorship. The termite-ball fungus rarely kills termite eggs in natural colonies. Even a termite species (Reticulitermes okinawanus) with no natural association with the fungus tended termite balls along with its eggs when it was experimentally provided with termite balls. Dummy-egg bioassays using glass beads showed that both morphological and chemical camouflage were necessary to induce tending by termites. Termites almost exclusively tended termite balls with diameters that exactly matched their egg size. Moreover, scanning electron microscopic observations revealed sophisticated mimicry of the smooth surface texture of eggs. These results provide clear evidence that this interaction is beneficial only for the fungus, i.e. termite balls parasitically mimic termite eggs. (+info)
Purification and characterization of cellobiose dehydrogenase from white-rot basidiomycete Trametes hirsuta.
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In order to save energy during the pulp making process, we tried to use white-rot basidiomycete, Trametes hirsuta, which degrades lignin efficiently. But a decrease in paper strength caused by cellulolytic activity ruled this out for practical application. Since the cellulolytic activity of the fungus must be decreased, we purified and characterized a cellobiose dehydrogenase (CDH) that was reported to damage pulp fiber. The CDH in the culture filtrate of C. hirsutus was purified by freeze-thawing and chromatographic methods. The pI of the enzyme was 4.2 and its molecular weight was 92 kDa. The optimal temperature and pH of the enzyme were 60-70 degrees C and 5.0 respectively. Since the purified CDH decreased the viscosity of pulp in the presence of Fe(III) and cellobiose, it was shown that the suppression of CDH should be an effective way to reduce cellulose damage. (+info)