Identification of redox activators for continuous reactivation of glyoxal oxidase from Trametes versicolor in a two-enzyme reaction cascade.

Glyoxal oxidases, belonging to the group of copper radical oxidases (CROs), oxidize aldehydes to carboxylic acids, while reducing O2 to H2O2. Their activity on furan derivatives like 5-hydroxymethylfurfural (HMF) makes these enzymes promising biocatalysts for the environmentally friendly synthesis of the bioplastics precursor 2,5-furandicarboxylic acid (FDCA). However, glyoxal oxidases suffer from inactivation, which requires the identification of suitable redox activators for efficient substrate conversion. Furthermore, only a few glyoxal oxidases have been expressed and characterized so far. Here, we report on a new glyoxal oxidase from Trametes versicolor (TvGLOX) that was expressed at high levels in Pichia pastoris (reclassified as Komagataella phaffii). TvGLOX was found to catalyze the oxidation of aldehyde groups in glyoxylic acid, methyl glyoxal, HMF, 2,5-diformylfuran (DFF) and 5-formyl-2-furancarboxylic acid (FFCA), but barely accepted alcohol groups as in 5-hydroxymethyl-2-furancarboxylic acid (HMFCA), preventing formation of FDCA from HMF. Various redox activators were tested for TvGLOX reactivation during catalyzed reactions. Among them, a combination of horseradish peroxidase and its substrate 2,2'-azino-di-(3-ethylbenzthiazoline sulfonic acid) (ABTS) most efficiently reactivated TvGLOX. Through continuous reactivation of TvGLOX in a two-enzyme system employing a recombinant Moesziomyces antarcticus aryl-alcohol oxidase (MaAAO) almost complete conversion of 8 mM HMF to FDCA was achieved within 24 h.

Enhanced laccase activity in Trametes versicolor (L.: Fr.) Pilát by host substrate and copper.

Laccases are appealing biocatalysts for various industrial utilizations. The fungus Trametes versicolor (L.: Fr.) Pilát causes white rot in wood and has been identified as an important fungal laccase producer. To investigate laccase production and activity in T. versicolor, the native isolate was collected from the host (Quercus castaneifolia) in the forests of Guilan province, northern Iran, and then purified and identified using the molecular marker. Its ability to produce laccase enzyme in the presence of different plant substrates including sawdust and wood chips of oak, poplar, and pine was evaluated. Also, the effect of copper as an enzyme inducer was investigated in vitro. The results showed that adding the wood to the culture medium increased laccase production, and among these, oak sawdust had the greatest effect, a 1.7-fold increase from that in the control (4.8 u/l vs. 2.8 u/l). Also, the enzyme extraction time effect on the optimal recovery yield showed that the 5-h enzyme extraction cycle resulted in the highest yield of the enzyme (18.97 u/l). Moreover, adding different concentrations of copper to the fungal culture medium increased the production of laccase, and the highest amount of enzyme (92.04 u/l) was obtained with 3.5 mM of CuSO4 along with oak sawdust. Based on the results, the addition of host wood sawdust ("oak" in this work) and copper particles together stimulates the fungal growth and the laccase production during submerged cultivation of T. versicolor. Therefore, it would be a safe and cheap strategy for the commercial production of laccase by filamentous fungi.

Arsenic(III)-induced oxidative defense and speciation changes in a wild Trametes versicolor strain.

Oxidative defense or arsenic(As) changes exhibited by Trametes versicolor in response to toxicity under As stress remain unclear. In this study, after internal transcribed spacer identification, a wild T. versicolor HN01 strain was cultivated under 40 and 80 mg/L of As III stress. The antioxidant contents by multifunctional microplate reader and the speciations of As by high performance liquid chromatography in conjunction with inductively coupled plasma mass spectrometry were examined to explore the detoxification mechanisms. The results demonstrated this strain could tolerate As concentration of 80 mg/L with a bio-enrichment coefficients of 11.25. Among the four antioxidants, the activities of catalase, superoxide dismutase, and glutathione in the As-stress group at 80 mg/L improved by 1.10, 1.09, and 20.47 times that of non-stress group, respectively. The speciation results indicated that AsV was the dominant species in the hyphae of T. versicolor regardless of no-stress or As-stress. The detoxification mechanisms of this strain were involved alleviating the toxicity by increasing the activities of antioxidants, especially glutathione, as well as by converting As III into As V and other less toxic As species. T. versicolor could be used as a bio-accumulator to deal with As exposure in contaminated environments based on its extraordinary As tolerance and accumulation capacities.

Transcriptomics and co-expression network analysis revealing candidate genes for the laccase activity of Trametes gibbosa.

BACKGROUND: Trametes gibbosa, which is a white-rot fungus of the Polyporaceae family found in the cold temperate zone, causes spongy white rot on wood. Laccase can oxidize benzene homologs and is one of the important oxidases for white rot fungi to degrade wood. However, the pathway of laccase synthesis in white rot fungi is unknown. RESULTS: The peak value of laccase activity reached 135.75 U/min/L on the 9th day. For laccase activity and RNA-seq data, gene expression was segmented into 24 modules. Turquoise and blue modules had greater associations with laccase activity (positively 0.94 and negatively -0.86, respectively). For biology function, these genes were concentrated on the cell cycle, citrate cycle, nicotinate, and nicotinamide metabolism, succinate dehydrogenase activity, flavin adenine dinucleotide binding, and oxidoreductase activity which are highly related to the laccase synthetic pathway. Among them, gene_8826 (MW199767), gene_7458 (MW199766), gene_61 (MW199765), gene_1741 (MH257605), and gene_11087 (MK805159) were identified as central genes. CONCLUSION: Laccase activity steadily increased in wood degradation. Laccase oxidation consumes oxygen to produce hydrogen ions and water during the degradation of wood. Some of the hydrogen ions produced can be combined by Flavin adenine dinucleotide (FAD) to form reduced Flavin dinucleotide (FADH2), which can be transmitted. Also, the fungus was starved of oxygen throughout fermentation, and the NADH and FADH2 are unable to transfer hydrogen under hypoxia, resulting in the inability of NAD and FAD to regenerate and inhibit the tricarboxylic acid cycle of cells. These key hub genes related to laccase activity play important roles in the molecular mechanisms of laccase synthesis for exploring industrial excellent strains.

ThhspA1 is involved in lacA transcriptional regulation of Trametes hirsuta AH28-2 exposed to o-toluidine.

White rot fungi, especially Trametes spp., respond to a wide range of aromatic compounds and dramatically enhance laccase activity, while the activation mechanisms remain to be elucidated. Here, we show that an Hsp70 homolog named ThhspA1 regulates the transcription of laccase LacA in Trametes hirsuta AH28-2 when confronted with o-toluidine. ThhspA1 is pulled down by lacA promoter sequence from the nuclear mixture extracted from T. hirsuta AH28-2 induced by 2 mM o-toluidine. Silencing of ThhspA1 results in a sharp decrease in lacA transcripts and laccase activity in vivo. By comparison, ThhspA1 overexpression does not affect lacA transcription, and laccase activity shows slight enhancement or remains unchanged upon induction with o-toluidine. Electrophoretic mobility shift assays suggest a direct interaction between ThhspA1 and the lacA promoter region. Further investigation shows that the integrity of ThhspA1 is critical since its substrate binding domain (SBD) and nucleotide-binding domain (NBD) are both necessary for DNA binding, with a higher affinity of SBD than NBD based on fluorescence polarization assay. Our results demonstrate that ThhspA1 functions as an aromatic-stress-related DNA binding transcriptional factor required for LacA expression.

Polysaccharides Produced by the Mushroom Trametes robiniophila Murr Boosts the Sensitivity of Hepatoma Cells to Oxaliplatin via the miR-224-5p/ABCB1/P-gp Axis.

AIM: To investigate the mechanisms employed by PS-T (polysaccharides of Trametes, PS-T), the main active ingredient of Huaier granules, to improve the susceptibility of hepatoma cells to oxaliplatin (OXA). METHODS: Cell proliferation in response to PS-T was determined both in vitro and in vivo. The effects of PS-T on miRNAs were analyzed with the use of a microarray. MiRNAs were screened under specific conditions (P < .05, logFoldChange > ABS [1.5]) and further silenced or overexpressed by liposome transfection. Levels of ABCB1 mRNA and P-gp were detected by qRT-PCR and western blot analysis, respectively. A dual fluorescence assay was performed to determine whether miRNA directly targets ABCB1. RESULTS: PS-T enhanced the inhibitory effect of OXA in human hepatoma cells and xenografts. Among 5 up-regulated miRNAs, overexpression of only miR-224-5p inhibited the expression of ABCB1 mRNA and P-gp, while silencing of miR-224-5p had an opposite effect. Moreover, miR-224-5p can directly target the 3'-UTR of ABCB1. CONCLUSION: PS-T increases the sensitivity of human hepatoma cells to OXA via the miR-224-5p/ABCB1/P-gp axis.

Functional annotation and comparative modeling of ligninolytic enzymes from Trametes villosa (SW.) Kreisel for biotechnological applications.

Functional annotation of Trametes villosa genome was performed to search Class II peroxidase proteins in this white-rot fungus, which can be valuable for several biotechnological processes. After sequence identification and manual curation, five proteins were selected to build 3 D models by comparative modeling. Analysis of sequential and structural sequences from selected targets revealed the presence of two putative Lignin Peroxidase and three putative Manganese Peroxidase on this fungal genome. All 3 D models had a similar folding pattern from selected 3 D structure templates. After minimization and validation steps, the best 3 D models were subjected to docking studies and molecular dynamics to identify structural requirements and the interactions required for molecular recognition. Two reliable 3 D models of Class II peroxidases, with typical catalytic site and architecture, and its protein sequences are indicated to recombinant production in biotechnological applications, such as bioenergy.Communicated by Ramaswamy H. Sarma.

The mechanism of degradation of alizarin red by a white-rot fungus Trametes gibbosa.

BACKGROUND: Alizarin red (AR) is a typical anthraquinone dye, and the resulting wastewater is toxic and difficult to remove. A study showed that the white rot fungus Trametes gibbosa (T. gibbosa) can degrade dye wastewater by decolorization and has its own enzyme-producing traits. METHODS: In this study, transcriptome sequencing was performed after alizarin red treatment for 0, 3, 7, 10, and 14 h. The key pathways and key enzymes involved in alizarin red degradation were found to be through the analysis of KEGG and GO. The Glutathione S-transferase (GST), manganese peroxidase (MnP) and laccase activities of T. gibbosa treated with alizarin red for 0-14 h were detected. LC-MS and GC-MS analyses of alizarin red decomposition products after 7 h and 14 h were performed. RESULTS: The glutathione metabolic pathway ko00480, and the key enzymes GST, MnP, laccase and CYP450 were selected. Most of the genes encoding these enzymes were upregulated under alizarin red conditions. The GST activity increased 1.8 times from 117.55 U/mg prot at 0 h to 217.03 U/mg prot at 14 h. The MnP activity increased 2.9 times from 6.45 to 18.55 U/L. The laccase activity increased 3.7 times from 7.22 to 27.28 U/L. Analysis of the alizarin red decolourization rate showed that the decolourization rate at 14 h reached 20.21%. The main degradation intermediates were found to be 1,4-butene diacid, phthalic acid, 1,1-diphenylethylene, 9,10-dihydroanthracene, 1,2-naphthalene dicarboxylic acid, bisphenol, benzophenol-5,2-butene, acrylaldehyde, and 1-butylene, and the degradation process of AR was inferred. Overall, 1,4-butene diacid is the most important intermediate product produced by AR degradation. CONCLUSIONS: The glutathione metabolic pathway was the key pathway for AR degradation. GST, MnP, laccase and CYP450 were the key enzymes for AR degradation. 1,4-butene diacid is the most important intermediate product. This study explored the process of AR biodegradation at the molecular and biochemical levels and provided a theoretical basis for its application in practical production.

Comparative mitochondrial genome analysis reveals intron dynamics and gene rearrangements in two Trametes species.

Trametes species are efficient wood decomposers that are widespread throughout the world. Mitogenomes have been widely used to understand the phylogeny and evolution of fungi. Up to now, two mitogenomes from the Trametes genus have been revealed. In the present study, the complete mitogenomes of two novel Trametes species, Trametes versicolor and T. coccinea, were assembled and compared with other Polyporales mitogenomes. Both species contained circular DNA molecules, with sizes of 67,318 bp and 99,976 bp, respectively. Comparative mitogenomic analysis indicated that the gene number, length and base composition varied between the four Trametes mitogenomes we tested. In addition, all of the core protein coding genes in Trametes species were identified and subjected to purifying selection. The mitogenome of T. coccinea contained the largest number of introns among the four Trametes species tested, and introns were considered the main factors contributing to size variations of Polyporales. Several novel introns were detected in the Trametes species we assembled, and introns identified in Polyporales were found to undergo frequent loss/gain events. Large-scale gene rearrangements were detected between closely related Trametes species, including gene inversions, insertions, and migrations. A well-supported phylogenetic tree for 77 Basidiomycetes was obtained based on the combined mitochondrial gene set using 2 phylogenetic inference methods. The results showed that mitochondrial genes are effective molecular markers for understanding the phylogeny of Basidiomycetes. This study is the first to report the mitogenome rearrangement and intron dynamics of Trametes species, which shed light on the evolution of Trametes and other related species.

Decolorization of Textile Effluent by Trametes hirsuta Bm-2 and lac-T as Possible Main Laccase-Contributing Gene.

The decolorization of dye and textile effluent by Trametes hirsuta was studied in both induced and non-induced media. A removal of 70-100% of the color was achieved through adsorption and the action of laccases. Laccase activity was increased significantly with the addition of grapefruit peel (4000 U/mL) and effluent with grapefruit peel (16,000 U/mL) in comparison with the basal medium (50 U/mL). Analysis of the expression of laccase isoenzymes lac-B and lac-T revealed clear differences in the expression of these genes. The low levels of expression of lac-B in all media suggest a basal or constitutive gene expression, whereas lac-T was over-expressed in the media with effluent, and showed an up/down regulation depending on culture conditions and time. The results obtained suggest that the lac-T gene of T. hirsuta is involved in the decolorization of dyes.

A Phytochemical-Based Copolymer Derived from Coriolus versicolor Polysaccharopeptides for Gene Delivery.

Coriolus versicolor is an herb widely used for cancer treatment in traditional Chinese medicine. Its active ingredients, polysaccharopeptides (PSP), have been used for adjuvant therapies in cancer treatment. This study conjugates Coriolus versicolor PSP with poly(ethylenimine) (PEI) to generate a PSP-PEI copolymer for gene transfer. After PEI conjugation, both the pH buffering capacity and DNA compaction ability of PSP are significantly increased. Compared with that of PSP, the transfection efficiency of PSP-PEI is 10 to 20-fold higher in vitro. This is a proof-of-concept study reporting the direct use of bioactive phytochemicals from traditional Chinese medicine for gene vector development. The promising performance of PSP-PEI raises the possibility that bioactive herbal ingredients can be further developed as a multi-therapeutic gene carrier for tackling cancers.

Trametes versicolor glutathione transferase Xi 3, a dual Cys-GST with catalytic specificities of both Xi and Omega classes.

Glutathione transferases (GSTs) from the Xi and Omega classes have a catalytic cysteine residue, which gives them reductase activities. Until now, they have been assigned distinct substrates. While Xi GSTs specifically reduce glutathionyl-(hydro)quinones, Omega GSTs are specialized in the reduction of glutathionyl-acetophenones. Here, we present the biochemical and structural analysis of TvGSTX1 and TvGSTX3 isoforms from the wood-degrading fungus Trametes versicolor. TvGSTX1 reduces GS-menadione as expected, while TvGSTX3 reduces both Xi and Omega substrates. An in-depth structural analysis indicates a broader active site for TvGSTX3 due to specific differences in the nature of the residues situated in the C-terminal helix α9. This feature could explain the catalytic duality of TvGSTX3. Based on phylogenetic analysis, we propose that this duality might exist in saprophytic fungi and ascomycetes.

Antiproliferative Activity and Cytotoxicity of Some Medicinal Wood-Destroying Mushrooms from Russia.

We analyzed the antiproliferative activity of 6 medicinal wood-destroying mushrooms (Fomes fomentarius, Fomitopsis pinicola, Trametes versicolor, Trichaptum biforme, Inonotus obliquus, and Coniophora puteana) that are common in deciduous and mixed coniferous forests in Central Russia. Morphological identification of strains collected from the wild was confirmed based on ribosomal DNA internal transcribed spacer phylogenetic analysis. We observed cytotoxic and cell growth-inhibitory effects of hot water extracts from mycelial biomass of 5 species-T. versicolor, C. puteana, F. fomentarius, F. pinicola, and I. obliquus-on leukemia cell lines (Jukart, K562, and THP-1); the effective extract concentrations were mostly less than 50 µg · mL-1. However, we observed no antiproliferative activity of dry biomass from methanol-chloroform (1:1) extracts of C. puteana and F. fomentarius. A chemosensitivity assay showed that the most effective polypore mushroom extract was the methanol extract of T. versicolor (strain It-1), which inhibited the growth of 6 various solid tumors (A-549 and SWi573 [lung], HBL-100 and T-47D [breast], HeLa [cervix], and WiDr [colon]) at concentrations below 45 µg · mL-1, with a concentration as low as 0.7-3.6 µg · mL-1 causing 50% reduction in the proliferation of cancer cells in lung and cervix tumors. Methanol extracts of F. pinicola and I. obliquus were less effective, with proliferation-inhibiting capacities at concentrations below 70 and 200 µg · mL-1, respectively. Thus, T. versicolor is a prospective candidate in the search for and production of new antiproliferative chemical compounds.

Biodegradation of polycyclic aromatic hydrocarbons by a thermotolerant white rot fungus Trametes polyzona RYNF13.

The biodegradation of three polycyclic aromatic hydrocarbons (PAHs), phenanthrene, fluorene, and pyrene, by a newly isolated thermotolerant white rot fungal strain RYNF13 from Thailand, was investigated. The strain RYNF13 was identified as Trametes polyzona, based on an analysis of its internal transcribed spacer sequence. The strain RYNF13 was superior to most white rot fungi. The fungus showed excellent removal of PAHs at a high concentration of 100 mg·L-1. Complete degradation of phenanthrene in a mineral salt glucose medium culture was observed within 18 days of incubation at 30°C, whereas 90% of fluorene and 52% of pyrene were degraded under the same conditions. At a high temperature of 42°C, the strain RYNF13 was still able to grow, and degraded approximately 68% of phenanthrene, whereas 48% of fluorene and 30% of pyrene were degraded within 32 days. Thus, the strain RYNF13 is a potential fungus for PAH bioremediation, especially in a tropical environment where the temperature can be higher than 40°C. The strain RYNF13 secreted three different ligninolytic enzymes, manganese peroxidase, laccase, and lignin peroxidase, during PAH biodegradation at 30°C. When the incubation temperature was increased from 30°C to 37°C and 42°C, only two ligninolytic enzymes, manganese peroxidase and laccase, were detectable during the biodegradation. Manganese peroxidase was the major enzyme produced by the fungus. In the culture containing phenanthrene, manganese peroxidase showed the highest enzymatic activity at 179 U·mL-1. T. polyzona RYNF13 was determined as a potential thermotolerant white rot fungus, and suitable for application in the treatment of PAH-containing contaminants.

Preliminary studies of new strains of Trametes sp. from Argentina for laccase production ability

Abstract Oxidative enzymes secreted by white rot fungi can be applied in several technological processes within the paper industry, biofuel production and bioremediation. The discovery of native strains from the biodiverse Misiones (Argentina) forest can provide useful enzymes for biotechnological purposes. In this work, we evaluated the laccase and manganese peroxidase secretion abilities of four newly discovered strains of Trametes sp. that are native to Misiones. In addition, the copper response and optimal pH and temperature for laccase activity in culture supernatants were determined.The selected strains produced variable amounts of laccase and MnP; when Cu2+ was added, both enzymes were significantly increased. Zymograms showed that two isoenzymes were increased in all strains in the presence of Cu2+. Strain B showed the greatest response to Cu2+ addition, whereas strain A was more stable at the optimal temperature and pH. Strain A showed interesting potential for future biotechnological approaches due to the superior thermo-stability of its secreted enzymes.

Optimal assembly strategies of transcriptome related to ploidies of eukaryotic organisms.

BACKGROUND: Several de novo transcriptome assemblers have been developed recently to assemble the short reads generated from the next-generation sequencing platforms and different strategies were employed for assembling transcriptomes of various eukaryotes without genome sequences. Though there are some comparisons among these de novo assembly tools for assembling transcriptomes of different eukaryotic organisms, there is no report about the relationship between assembly strategies and ploidies of the organisms. RESULTS: When we de novo assembled transcriptomes of sweet potato (hexaploid), Trametes gallica (a diploid fungus), Oryza meyeriana (a diploid wild rice), five assemblers, including Edena, Oases, Soaptrans, IDBA-tran and Trinity, were used in different strategies (Single-Assembler Single-Parameter, SASP; Single-Assembler Multiple-Parameters, SAMP; Combined De novo Transcriptome Assembly, CDTA, that is multiple assembler multiple parameter). It was found that CDTA strategy has the best performance compared with other two strategies for assembling transcriptome of the hexaploid sweet potato, whereas SAMP strategy with assembler Oases is better than other strategies for assembling transcriptomes of diploid fungus and the wild rice transcriptomes. CONCLUSION: Based on the results from ours and others, it is suggested that CDTA strategy is better used for transcriptome assembly of polyploidy organisms and SAMP strategy of Oases is outperformed for those diploid organisms without genome sequences.

Proteomics investigation reveals cell death-associated proteins of basidiomycete fungus Trametes versicolor treated with Ferruginol.

Ferruginol has antifungal activity against wood-rot fungi (basidiomycetes). However, specific research on the antifungal mechanisms of ferruginol is scarce. Two-dimensional gel electrophoresis and fluorescent image analysis were employed to evaluate the differential protein expression of wood-rot fungus Trametes versicolor treated with or without ferruginol. Results from protein identification of tryptic peptides via liquid chromatography­electrospray ionization tandem mass spectrometry (LC­ESI-MS/MS) analyses revealed 17 protein assignments with differential expression. Downregulation of cytoskeleton ß-tubulin 3 indicates that ferruginol has potential to be used as a microtubule-disrupting agent. Downregulation of major facilitator superfamily (MFS)­multiple drug resistance (MDR) transporter and peroxiredoxin TSA1 were observed, suggesting reduction in self-defensive capabilities of T. versicolor. In addition, the proteins involved in polypeptide sorting and DNA repair were also downregulated, while heat shock proteins and autophagy-related protein 7 were upregulated. These observations reveal that such cellular dysfunction and damage caused by ferruginol lead to growth inhibition and autophagic cell death of fungi.

Species delimitation in Trametes: a comparison of ITS, RPB1, RPB2 and TEF1 gene phylogenies.

Trametes is a cosmopolitan genus of white rot polypores, including the "turkey tail" fungus, T. versicolor. Although Trametes is one of the most familiar genera of polypores, its species-level taxonomy is unsettled. The ITS region is the most commonly used molecular marker for species delimitation in fungi, but it has been shown to have a low molecular variation in Trametes resulting in poorly resolved phylogenies and unclear species boundaries, especially in the T. versicolor species complex (T. versicolor sensu stricto, T. ochracea, T. pubescens, T. ectypa). Here we evaluate the performance of three protein-coding genes (TEF1, RPB1, RPB2) for species delimitation and phylogenetic reconstruction in Trametes. We obtained 59 TEF1, 34 RPB1 and 55 RPB2 sequences from 69 individuals, focusing on the T. versicolor complex and performed phylogenetic analyses with maximum likelihood and parsimony methods. All three protein-coding genes outperformed ITS for separating species in the T. versicolor complex. The multigene phylogenetic analysis shows the highest amount of resolution and supported nodes separating T. ectypa, T. ochracea, T. pubescens and T. versicolor with strong support. In addition three slineages are resolved in the species complex of T. elegans. The T. elegans complex includes three species: T. elegans (based on material from Puerto Rico, Belize, the Philippines), T. aesculi (from North America) and T. repanda (from Papua New Guinea, the Philippines, Venezuela). The utility of gene markers varies, with TEF1 having the highest PCR and sequencing success rate and RPB1 offering the best backbone resolution for the genus.

Construction and direct electrochemistry of orientation controlled laccase electrode.

A laccase has multiple redox centres. Chemisorption of laccases on a gold electrode through a polypeptide tag introduced at the protein surface provides an isotropic orientation of laccases on the Au surface, which allows the orientation dependent study of the direct electrochemistry of laccase. In this paper, using genetic engineering technology, two forms of recombinant laccase which has Cys-6×His tag at the N or C terminus were generated. Via the Au-S linkage, the recombinant laccase was assembled orientationally on gold electrode. A direct electron transfer and a bioelectrocatalytic activity toward oxygen reduction were observed on the two orientation controlled laccase electrodes, but their electrochemical behaviors were found to be quite different. The orientation of laccase on the gold electrode affects both the electron transfer pathway and the electron transfer efficiency of O2 reduction. The present study is helpful not only to the in-depth understanding of the direct electrochemistry of laccase, but also to the development of laccase-based biofuel cells.

Trametes versicolor protein YZP activates regulatory B lymphocytes - gene identification through de novo assembly and function analysis in a murine acute colitis model.

BACKGROUND: Trametes versicolor (Yun-Zhi) is a medicinal fungus used as a chemotherapy co-treatment to enhance anti-tumor immunity. Although the efficacies of T. versicolor extracts have been documented, the active ingredients and mechanisms underlying the actions of these extracts remain uncharacterized. RESULTS: We purified a new protein, YZP, from the fruiting bodies of T. versicolor and identified the gene encoding YZP using RNA-seq and de novo assembly technologies. YZP is a 12-kDa non-glycosylated protein comprising 139 amino acids, including an 18-amino acids signal peptide. YZP induced a greater than 60-fold increase in IL-10 secretion in mice B lymphocytes; moreover, YZP specifically triggered the differentiation of CD1d(+) B cells into IL-10-producing regulatory B cells (Bregs) and enhanced the expression of CD1d. YZP-induced B cells suppressed approximately 40% of the LPS-activated macrophage production of inflammatory cytokines in a mixed leukocyte reaction and significantly alleviated the disease activity and colonic inflammation in a DSS-induced acute colitis murine model. Furthermore, YZP activated Breg function via interaction with TLR2 and TLR4 and up-regulation of the TLR-mediated signaling pathway. CONCLUSIONS: We purified a novel Breg-stimulating protein, YZP, from T. versicolor and developed an advanced approach combining RNA-seq and de novo assembly technologies.to clone its gene. We demonstrated that YZP activated CD1d(+) Breg differentiation through TLR2/4-mediated signaling pathway, and the YZP-stimulated B cells exhibited anti-inflammatory efficacies in vitro and in murine acute colitis models.