Extracellular peroxidase activity at the hyphal tips of the white-rot fungus Phanerochaete crassa WD1694

The white-rot fungus Phanerochaete crassa WD1694 was cultivated and peroxidase activity staining was performed to determine the sites at which the extracellular peroxidase reaction actually occurs in vivo. Although the ligninolytic peroxidases were found in the culture filtrates, the culture medium did not show a color reaction. However, a particularly strong color reaction was observed on the hyphal tips. Visible spectra and absorbance of the staining were analyzed by microspectrophotometry, and the catalytic rates of the peroxidase reaction at the hyphal tips were calculated. The estimated catalytic rate of the peroxidase reaction at the hyphal tips peaked at 794 μM/min, expressed as the consumption rate of H₂O₂, on day 3 of the cultivation. Analysis of the extracellular enzyme eluted with 0.1% Tween 80 from the mycelium revealed that manganese peroxidase accounted for 89% of all the peroxidase activity measured. The results clearly showed the existence of the concentrated manganese peroxidase reaction around the hyphal tips of the organism.     

Glyoxal oxidase supplies hydrogen peroxide at hyphal tips and on hyphal wall to manganese peroxidase of white-rot fungus Phanerochaete crassa WD1694

Peroxidase activity staining localized at hyphal tips of white-rot fungus Phanerochaete crassa WD1694 that was cultivated in a shaken liquid culture containing unbleached kraft pulp was investigated. Manganese peroxidase was detected in culture solution, washing solution of mycelium, and mycelial extract. Glyoxal oxidase was detected only in mycelial extract and was not detected in culture solution. Addition of hydrogen peroxide generated peroxidase activity staining in the culture solution. Addition of catalase resulted in no staining in the culture of P. crassa WD1694, and the addition of methylglyoxal resulted in marked peroxidase activity staining at hyphal tips and on hyphal wall. In an optimized culture, glyoxal oxidase was produced in culture solution. Although the production of glyoxal oxidase and manganese peroxidase had a positive correlation, the secretion and the peak of glyoxal oxidase was observed 3 and 2 days later than those of manganese peroxidase. The N-terminal sequence of purified glyoxal oxidase had very high homology with that of P. chrysosporium. These results elucidated the hydrogen peroxide supply system in lignin biodegradation by white-rot fungi, i.e., while remaining on the hyphal cell wall, glyoxal oxidase provides hydrogen peroxide to manganese peroxidase that had diffused into the culture solution beforehand.     

Extracellular peroxidase reaction at hyphal tips of white-rot fungus <Emphasis Type="Italic">Phanerochaete crassa</Emphasis> WD1694 and in fungal slime

The distribution of an extracellular peroxidase reaction by white-rot fungus Phanerochaete crassa WD1694 was visualized by peroxidase activity staining. The extracellular peroxidase reaction occurred at the hyphal tips and in the fungal slime filling the gaps between the hyphae. We investigated whether the peroxidase reaction occurred from the hyphal tips or in the slime. The hyphal tips were observed by phase-contrast microscopy, which showed that slime did not exist around the hyphal tips. Time-course observation of hyphal tips showed that peroxidase staining became thick and intense at the tips that did not have fungal slime. Daily observation of the peroxidase staining revealed that the staining was first observed at the hyphal tips. Furthermore, strongly stained hyphae were observed in the stained slime. These results suggested that an active species that oxidizes a peroxidase substrate is first produced at the tips of the hyphae, and then occurs in the slime via diffusion when slime exists around the hyphae. Our results show that the extracellular peroxidase reaction that is important to lignin biodegradation by white-rot fungi occurs directly at the tips of the hyphae and in the slime. Part of this report was presented at the 50th Lignin Symposium, October 19–20, 2005, Nagoya, Japan     

Selective staining and visualization of hyphal sheath of a white-rot fungus <Emphasis Type="Italic">Phanerochaete crassa</Emphasis> WD1694 with phloxine B

The hyphal sheath is a morphological feature of many kinds of fungi. Although the fine structures of the sheath have been studied in detail by a number of electron microscopy techniques, the function and physiology of the hyphal sheath are not yet clarified. One reason for this is that the hyphal sheath is a colorless, mucilaginous, and delicate material so that it is not easily identified. We developed a simple method to visualize and identify the hyphal sheath of the white-rot fungus Phanerochaete crassa WD1694. The small mycelial pellets in shaken liquid cultures of P. crassa WD1694 were stained directly with phloxine B. Both the hyphae and the hyphal sheath that filled the gaps between each of the hyphae were visualized and observed by light microscopy. The stained hyphae were further studied by transmission electron microscopy, atomic force microscopy, and fl uorescence microscopy. Based on these observations, we confirmed that the staining of the hyphae was also due to the presence of the hyphal sheath that closely covered the fungal cell wall. These results clearly showed that the hyphal sheath was selectively stained with phloxine B and could be observed and identified by conventional light microscopy. Part of this report was presented at the 50th Lignin Symposium, Nagoya, October 2005     

In vitro reduction of manganese dioxide by a ferrireductase system from the white-rot fungus <Emphasis Type="Italic">Phanerochaete sordida</Emphasis> YK-624

Reduction of manganese dioxide is demonstrated for an in vitro ferrireductase system that includes NADPH-dependent ferrireductase and the iron-binding compound (IBC) isolated from the white-rot fungus Phanerochaete sordida YK-624. The Fe(II)–IBC complex was more effective in reducing manganese dioxide to Mn(II) than were complexes of Fe(II) and organic acids of low molecular weight such as nitrilotriacetate, although IBC also reduced manganese dioxide to Mn(II) in the absence of Fe(II). The generated Fe(III)–IBC complex was a better substrate for NADPH-dependent ferrireductase than were other ferric chelates, suggesting that the Fe(III)–IBC complex is reduced to an Fe(II) complex by NADPH-dependent ferrireductase. Moreover, production of the Fe(III)–IBC complex by the reduction of manganese dioxide in a reaction system containing Fe(II) and IBC was observed to be coupled to reduction of the Fe(III)–IBC complex by NADPH-dependent ferrireductase. These results indicate that the ferrireductase system of P. sordida YK-624 plays an important role in the reduction of manganese dioxide, which is necessary for the production and function of manganese peroxidase.     

Screening of wood-rotting fungi for kraft pulp bleaching by the Poly R decolorization test and biobleaching of hardwood kraft pulp byPhanerochaete crassa WD1694

From among 419 wood-rotting fungi 10 were selected by the Poly R decolorization test, and their ability to bleach hardwood kraft pulp was assayed. Of the 10 selected, 6 fungi (i.e.,Phanerochaete crassa WD1694 and F150;Pleurotus pulmonarius PSC-H, PSC-M, and PSC-T;and Pleurotus species A119) showed much higher bleaching ability thanPhanerochaete chrysosponum BKMF1767 orTrametes versicolor WD1670, both of which are well-known high ligninolytic fungi.P. crassa WD1694 had the highest bleaching ability among the selected strains, and it increased the pulp brightness from 28 to 54, with a corresponding decrease in kappa number from 16 to 6 after 10 days of cultivation and alkali extraction. MnP was a predominant ligninolytic enzyme ofP. crassa WD1694 during the biobleaching.This research was presented in part at the 46th Annual Meeting of the Japan Wood Research Society, Kumamoto, April 1996     

Isolation of cDNA and genomic fragments encoding the major manganese peroxidase isozyme from the white rot basidiomycetePleurotus ostreatus

We have isolated the cDNA and genomic sequences encoding the major isozyme of manganese peroxidase, MnP3, from the white rot basidiomycetePleurotus ostreatus strain IS1. The genemnp3 is interrupted by 10 introns and encodes a mature protein of 357 amino acid residues with a 26-amino-acid signal peptide. The amino acid residues known to be involved in peroxidase function and those that form the Mn-binding site in thePanerochaete chrysosporium MnP isozyme are conserved in MnP3. Comparison of the deduced primary structure of MnP3 with those of other peroxidases from various white rot fungi suggested that MnPs fromP. ostreatus andTrametes versicolor belong to a subgroup that is more similar to the lignin peroxidases than MnPs fromP. chrysosporium orCeriporiopsis subvermispora.     

Studies on hypersaline-tolerant white-rot fungi III: biobleaching of unbleached kraft pulp by hypersaline-tolerant manganese peroxidase from a marine white rot isolate, <Emphasis Type="Italic">Phlebia </Emphasis>sp. MG-60

 A marine white rot isolate, Phlebia sp. MG-60, secreted lignin peroxidase (LiP), manganese peroxidase (MnP), and laccase under different sea salt incubation conditions. Its MnP production was strongly enhanced by adding 3% sea salts, and the MnP showed high tolerance to sea salts and NaCl. The crude enzyme secreted at 3% sea salt concentration by Phlebia sp. MG-60, in which the main component was MnP (cMnP), was then used to bleach unbleached hardwood kraft pulp (UKP) in vitro. The pulp was brightened 11 points by 4 U of cMnP, and the kappa number was decreased 6 points when only 0.5 mM H₂O₂ was added continuously. When 0.5 mM H₂O₂ (1.22 mg H₂O₂ /g pulp) was added at the initial bleaching, the pulp brightness increased 6 points with a dosage of 4 U of cMnP. When crude MnPs were employed to bleach UKP with organic-free model white-water instead of the Milli Q water usually used, the pulp was brightened 10 and 13 points by 4 and 20 U of cMnP, respectively, and 5 and 6 points by 4 and 20 U of MnP, respectively, of Phanerochaete chrysosporium. Received: September 28, 2001 / Accepted: March 15, 2002 Correspondence to:R. Kondo     

Hybrid aspen with a transgene for fungal manganese peroxidase is a potential contributor to phytoremediation of the environment contaminated with bisphenol A

To assess the possible utility of a fungal gene for manganese-dependent peroxidase (MnP) produced by a transgenic plant in phytoremediation, we transformed hybrid aspen with a chimeric gene for MnP. Our gene construct allowed expression of the gene for MnP in plants and relatively high MnP activity was detected in the hydroponic medium in which roots of plants that expressed the transgene had been cultured. Some of our transgenic plants were able to remove bisphenol A from the medium more efficiently than wild-type plants. Our results demonstrate that, without any modification of the coding sequence, a chimeric gene for fungal MnP can be expressed in a woody plant, with secretion of active MnP from roots into the rhizosphere. Our strategy suggests new options using woody plants for phytoremediation.     

Isolation and sequence analysis of the promoter and an allelic sequence of the iron-sulfur protein subunit gene from the white-rot fungusPleurotus ostreatus

We have isolated a structural gene ofsdil, which encodes the iron-sulfur protein (Ip) subunit of succinate dehydrogenase (EC 1.3.99.1), from a white-rot basidiomycete,Pleurotus ostreatus. Here we report isolation of the promoter region ofsdil and an allelic sequence encoding the second-type cDNA fragment isolated in the former experiments. The nucleotide sequence analysis of the promoter region revealed the existence of putative CAAT and TATA boxes, which permits us to develop an expression system in this species. The Southern blot analysis and the restriction fragment length polymorphism assay using monokaryotic strains demonstrated that no family genes tosdil exist in the haploid genome ofP. ostreatus. Moreover, a genetic analysis to detect a linkage between thesdil genotypes and flutolanil resistance in the mutantP. ostreatus strains was also developed.     

Indole-3-carbaldehyde: a tyrosinase inhibitor from fungus YL185

Indole-3-carbaldehyde (1) was isolated as a tyrosinase inhibitor from the ethyl acetate-soluble fraction of extracellular fluids of unknown fungus YL185. The partial sequencing data of 18S ribosomal DNA (18S rDNA) indicate that this isolate belongs to the family Polyporaceae or Corticiaceae sensu lato. Indole-3-carbaldehyde inhibited the oxidation of l-3,4-dihydroxyphenylalanine (l-DOPA) by mushroom tyrosinase with a 50% inhibitory concentration (IC₅₀) of 1.3mM and showed inhibitory activity on melanin production in B16 melanoma cells. The aldehyde group of 1 plays an important role in eliciting tyrosinase inhibitory activity.     

Armillaramide, a new sphingolipid from the fungus Armillaria mellea.

A new C(18)-phytosphingosine ceramide containing non-hydroxy fatty acid, armillaramide (1), has been isolated together with ergosterol peroxide from the fruiting bodies of the basidiomycete Armillaria mellea. Its structure was established as (2S,3S,4R)-2-hexadecanoylamino-octadecane-1,3,4-triol by spectroscopic and chemical methods.     

Molecular cloning of a putative gene encoding isopentenyltransferase from pingyitiancha (Malus hupehensis) and characterization of its response to nitrate

A putative isopentenyltransferase (IPT) encoding gene was identified from a pingyitiancha (Malus hupehensis Rehd.) expressed sequence tag database, and the full-length gene was cloned by RACE. Based on expression profile and sequence alignment, the nucleotide sequence of the clone, named MhIPT3, was most similar to AtIPT3, an IPT gene in Arabidopsis. The full-length cDNA contained a 963-bp open reading frame encoding a protein of 321 amino acids with a molecular mass of 37.3 kDa. Sequence analysis of genomic DNA revealed the absence of introns in the frame. Quantitative real-time PCR analysis demonstrated that the gene was expressed in roots, stems and leaves. Application of nitrate to roots of nitrogen-deprived seedlings strongly induced expression of MhIPT3 and was accompanied by the accumulation of cytokinins, whereas MhIPT3 expression was little affected by ammonium application to roots of nitrogen-deprived seedlings. Application of nitrate to leaves also up-regulated the expression of MhIPT3 and corresponded closely with the accumulation of isopentyladenine and isopentyladenosine in leaves.     

A novel extracellular peroxidase and nucleases from a milky sap of Chelidonium majus

Using affinity chromatography, SDS-PAGE, peroxidase activity assay and mass spectrometry data, a new extracellular peroxidase (CMP) from Chelidonium majus milky sap was isolated and characterized. The protein has a molecular weight of about 40 kDa and belongs to secretory class III plant peroxidases. The peroxidase activity is also accompanied by DN-ase activities. A novel CMP combined with other proteins is probably involved in development and differentiation of the plant and defence against different pathogens. It suggests that the biological activity of C. majus whole plants and extracts may depend not only on its alkaloidal content but also on the presence of biologically active proteins.     

Molecular cloning and expression analysis of a gene encoding a putative beta-ketoacyl-acyl carrier protein (ACP) synthase III (KAS III) from Jatropha curcas

A cDNA clone encoding a putative beta-ketoacyl-acyl carrier protein (ACP) synthase III (KAS III) was isolated from Jatropha curcas L., a woody oil plant. The cDNA clone (named JcKAS III) contained a 1203-bp open reading frame coding for 400 amino acids with a predicted molecular mass of about 42 kDa. The deduced amino acid sequence of the cDNA clone shares about 80% identity to KAS III from other plants, and contains a conserved Cys(176) in the active site and the amino acid motif G(355)NTSAAS(361) which is responsible for binding regulatory acyl-ACPs. Southern blotting analysis indicated that JcKAS III is a single copy gene in the J. curcas genome. Quantitative real-time PCR analysis showed that JcKAS III was expressed in all tissues examined with highest expression in roots, and that expression of JcKAS III increased as seeds developed.     

The genes encoding glycoside hydrolase family 6 and 7 cellulases from the brown-rot fungus <Emphasis Type="Italic">Coniophora puteana</Emphasis>

Four genes encoding glycoside hydrolase (GH) family 6 and 7 cellulases (cel6A, cel6B, cel7A, and cel7B) were obtained from the brown-rot fungus Coniophora puteana by genomic polymerase chain reaction (PCR) using consensus degenerate hybrid oligonucleotide primers (CODEHOPs) designated from the amino acid sequence of cellobiohydrolases (CBHs) from white-rot fungi. The nucleotide sequences of four genes showed high homology with basidiomycetes CBHs, suggesting the fi rst cloning of the genes encoding Cel6 and Cel7 from brown-rot fungi. PCR using CODEHOP pairs at the catalytic domain successfully amplifi ed both cel6A and cel6B, whereas only cel6A fragment was obtained using the primers including the carbohydrate-binding modules (CBMs), suggesting lack of CBM in Cel6B. Moreover, both cel7A and cel7B were amplified by the PCR using CODEHOP pairs at the catalytic domain, but not by those including CBM, suggesting the absence of Cel7 with CBM in the fungus. From these results, three of four cellulases from C. puteana may not carry CBM, which has an important role for the degradation of crystalline cellulose.     

Mutagenesis, heterogeneous gene expression, and purification and amino acid substitution analyses of plant peroxidase, PrxA3a

We introduced mutations into prxA3a, a peroxidase gene of hybrid aspen, Populus kitakamiensis, to substitute the amino acid residues at the surface of the protein, and analyzed substrate specificities. PrxA3a and mutated enzymes heterogeneously gene expressed in Saccharomyces cerevisiae were purified by Ni affinity chromatography, hydrolysis of sugar chain (Endoglycosidase H_f) and gel filtration. The substrate specificities were altered by substituted amino acid residues. PrxA3a F77Y A165W acquired the substrate specificity to m-chlorophenol. PrxA3a F77Y and PrxA3a F77YA165W could polymerize sinapyl alcohol. In addition, PrxA3a A165W, F77Y, and F77YA165W improved cytochrome c oxidizing activity. These substituted amino acid residues should function as a catalytic site outside of the heme pocket.     

Isolation and identification of a new anti-inflammatory cyathane diterpenoid from the medicinal fungus Cyathus hookeri Berk

A new cyathane diterpene, named as cyathin I (1), as well as two known cyathane diterpenes (12R)-11a,14a-epoxy-13a,14b,15-trihydroxycyath-3-ene (2) and erinacine I (3), were isolated from the liquid culture of Cyathus hookeri Berk. Their structures were elucidated on the basis of extensive spectroscopic analysis. Compounds 1–3 showed inhibition against nitric oxide production in macrophages with an IC₅₀ value of 15.5, 52.3, and 16.8 μM, respectively.