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1.
Ecotoxicol Environ Saf ; 165: 278-283, 2018 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-30205329

RESUMO

Laccases are used in decolorization and biodegradation of synthetic dyes, bioremediation of industrial wastewaters and delignification of lignocellulosic compounds. The aims of the present study were the optimization of a recombinant laccase production in Yarrowia lipolytica yeast using sucrose as a main carbon source, and the application of the resulting enzyme to decolorization of synthetic dyes, which are problematic environmental pollutants. Taguchi's experimental design method was employed to optimize medium compounds. Recombinant laccase production by Y. lipolytica YL4 strain increased to 900 U L-1 after optimization of sucrose, ammonium chloride, yeast extract and thiamine levels in the modified PPB medium. Furthermore, the production rate reached 6760 U L-1 in a 5 L bioreactor which represents 4.5- and 33.5-fold increases compared to cultures that were in shake-flask with optimized and primary media, respectively. The supernatant containing secreted recombinant laccase was applied for decolorization of seven dyes. The effects of pH, the amount of enzyme and incubation period were verified. The effect of incubation time on dye decolorization by recombinant laccase was important, which has an influence of greater extent than 90% after 48 h for all dyes. The Trametes versicolor laccase can be efficiently produced in Y. lipolytica and the recombinant enzyme has a considerable potential in the decolorization of pollutant synthetic dyes.


Assuntos
Corantes/química , Poluentes Ambientais/química , Lacase/biossíntese , Yarrowia , Biodegradação Ambiental , Reatores Biológicos , Lacase/química , Lacase/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Sacarose/metabolismo , Fatores de Tempo , Trametes/genética , Águas Residuárias , Yarrowia/genética
2.
Molecules ; 23(9)2018 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-30200573

RESUMO

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.


Assuntos
Técnicas de Transferência de Genes , Terapia Genética , Compostos Fitoquímicos/química , Proteoglicanas/química , DNA/química , DNA/uso terapêutico , Humanos , Compostos Fitoquímicos/genética , Polímeros/química , Proteoglicanas/genética , Trametes/química , Trametes/genética , Transfecção
3.
FEBS Lett ; 592(18): 3163-3172, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30112765

RESUMO

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.


Assuntos
Cisteína/metabolismo , Proteínas Fúngicas/metabolismo , Glutationa Transferase/metabolismo , Trametes/enzimologia , Sequência de Aminoácidos , Biocatálise , Cristalografia por Raios X , Cisteína/química , Cisteína/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Glutationa/análogos & derivados , Glutationa/química , Glutationa/metabolismo , Glutationa Transferase/classificação , Glutationa Transferase/genética , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Modelos Moleculares , Filogenia , Ligação Proteica , Domínios Proteicos , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Trametes/genética
4.
J Biosci Bioeng ; 126(5): 559-566, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-29903678

RESUMO

The site-specific cross-linking of functional proteins creates macromolecular assemblies that exhibit unique biochemical and/or physicochemical properties. Herein, we explored the potential of laccase as a biocatalyst for the site-specific cross-linking of tyrosine-tagged proteins. Trametes sp. laccase (TL) was selected as the cross-linking catalyst, and Escherichia coli alkaline phosphatase (BAP) and antibody-binding proteins (pG2pAs) were employed as model proteins. The protein models were genetically fused to a peptide tag containing a tyrosine residue (Y-tag) at the N- and/or C-termini. Proteins without Y-tags were used as controls. The Y-tagged proteins could be recognized by TL as macromolecular substrates, leading to the oxidative formation of protein polymers, whereas no polymerization was observed with intact BAP or pG2pA. The TL-catalyzed cross-linking of Y-tagged proteins proceeded at a relatively high pH in comparison with that of small phenolic substrates. Co-polymers of BAP and pG2pA were able to be prepared by mixing the aqueous solution of each component in the presence of TL. A combination of bis-Y-tagged pG2pA (Y-pG2pA-Y) and Y-tagged BAP (BAP-Y) yielded functional co-polymers compatible with enzyme-linked immunosorbent assay (ELISA). The detection limit of the ELISA of ovalbumin with anti-OVA IgG depended on the molar ratio of BAP-Y and Y-pG2pA-Y in the TL-catalyzed cross-linking reaction. A high molar ratio of BAP-Y to Y-pG2pA-Y (75:1) resulted in the highest absorbance in the ELISA. The results suggested that the formation of a bifunctional protein polymer with a high molar ratio of signaling unit to antibody-binding unit gave better performance in antigen detection than using lower ratios.


Assuntos
Escherichia coli/metabolismo , Lacase/metabolismo , Oligopeptídeos/metabolismo , Proteínas Recombinantes/metabolismo , Trametes/enzimologia , Tirosina/metabolismo , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Catálise , Escherichia coli/genética , Lacase/química , Lacase/genética , Oligopeptídeos/genética , Organismos Geneticamente Modificados , Oxirredução , Trametes/genética , Tirosina/química
5.
Int J Med Mushrooms ; 20(1): 1-11, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29604909

RESUMO

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.


Assuntos
Agaricales/química , Agaricales/fisiologia , Madeira/metabolismo , Agaricales/classificação , Agaricales/genética , Linhagem Celular Tumoral , Proliferação de Células , Celulose/metabolismo , DNA Espaçador Ribossômico , Carpóforos/química , Carpóforos/isolamento & purificação , Células HEK293 , Células HeLa , Humanos , Dose Letal Mediana , Lignanas/metabolismo , Filogenia , Estudos Prospectivos , Federação Russa , Trametes/química , Trametes/genética , Trametes/isolamento & purificação
6.
Fungal Biol ; 122(5): 353-362, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29665961

RESUMO

Laccase (benzenediol:oxygen oxidoreductase, EC 1.10.3.2) is an enzyme that has been studied for over 100 y and is present in virtually all fungi. As increasing numbers of fungal genomes have been sequenced, it has become apparent that the laccase genes in white-rot fungi commonly form multigene families consisting of many nonallelic genes. Although a number of reports focussing on laccase gene expression in different fungal species were published over the decades, the fundamental questions of why fungi need such a redundant array of genes and how they manage this array to perform biological function(s) remain far from answered. In this article, we present a comprehensive study of the transcription of the whole Trametes hirsuta laccase multigene family under different conditions, including exposure to different nutritional factors such as nitrogen sources (organic and inorganic) and concentrations of nitrogen and carbon in the culture medium; in different growth phases (lag phase and stationary phase); and in the presence of different inducer agents (water-soluble lignin, bromocresol green dye, p-coumaric acid, ferulic acid, guaiacol, vanillin, veratryl alcohol, vanillic acid and syringic acid). Our findings are discussed in the context of the evolution of the laccase multigene family, and the presence of transcription-level subfunctionalization is highlighted.


Assuntos
Regulação Fúngica da Expressão Gênica , Lacase/biossíntese , Trametes/enzimologia , Trametes/genética , Transcrição Genética , Carbono/metabolismo , Meios de Cultura/química , Ativadores de Enzimas/metabolismo , Lacase/genética , Família Multigênica , Compostos de Nitrogênio/metabolismo , Trametes/crescimento & desenvolvimento , Trametes/metabolismo
7.
Appl Environ Microbiol ; 84(8)2018 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-29453263

RESUMO

Trametesversicolor is a wood-inhabiting agaricomycete known for its ability to cause strong white-rot decay on hardwood and for its high tolerance of phenolic compounds. The goal of the present work was to gain insights into the molecular biology and biochemistry of the heme-including class II and dye-decolorizing peroxidases secreted by this fungus. Proteomic analysis of the secretome of T. versicolor BRFM 1218 grown on oak wood revealed a set of 200 secreted proteins, among which were the dye-decolorizing peroxidase TvDyP1 and the versatile peroxidase TvVP2. Both peroxidases were heterologously produced in Escherichia coli, biochemically characterized, and tested for the ability to oxidize complex substrates. Both peroxidases were found to be active against several substrates under acidic conditions, and TvDyP1 was very stable over a relatively large pH range of 2.0 to 6.0, while TvVP2 was more stable at pH 5.0 to 6.0 only. The thermostability of both enzymes was also tested, and TvDyP1 was globally found to be more stable than TvVP2. After 180 min of incubation at temperatures ranging from 30 to 50°C, the activity of TvVP2 drastically decreased, with 10 to 30% of the initial activity retained. Under the same conditions, TvDyP1 retained 20 to 80% of its enzyme activity. The two proteins were catalytically characterized, and TvVP2 was shown to accept a wider range of reducing substrates than TvDyP1. Furthermore, both enzymes were found to be active against two flavonoids, quercetin and catechin, found in oak wood, with TvVP2 displaying more rapid oxidation of the two compounds. They were tested for the ability to decolorize five industrial dyes, and TvVP2 presented a greater ability to oxidize and decolorize the dye substrates than TvDyP1.IMPORTANCETrametesversicolor is a wood-inhabiting agaricomycete known for its ability to cause strong white-rot decay on hardwood and for its high tolerance of phenolic compounds. Among white-rot fungi, the basidiomycete T. versicolor has been extensively studied for its ability to degrade wood, specifically lignin, thanks to an extracellular oxidative enzymatic system. The corresponding oxidative system was previously studied in several works for classical lignin and manganese peroxidases, and in this study, two new components of the oxidative system of T. versicolor, one dye-decolorizing peroxidase and one versatile peroxidase, were biochemically characterized in depth and compared to other fungal peroxidases.


Assuntos
Corantes/metabolismo , Proteínas Fúngicas/genética , Peroxidases/genética , Trametes/genética , Poluentes Químicos da Água/metabolismo , Proteínas Fúngicas/metabolismo , Oxirredução , Peroxidases/metabolismo , Proteômica , Trametes/enzimologia
8.
Protein Expr Purif ; 141: 39-43, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-28918197

RESUMO

Laccase is used in various industrial fields, and it has been the subject of numerous studies. Trametes versicolor laccase has one of the highest redox potentials among the various forms of this enzyme. In this study, we optimized the expression of laccase in Saccharomyces cerevisiae. Optimizing the culture conditions resulted in an improvement in the expression level, and approximately 45 U/L of laccase was functionally secreted in the culture. The recombinant laccase was found to be a heavily hypermannosylated glycoprotein, and the molecular weight of the carbohydrate chain was approximately 60 kDa. These hypermannosylated glycans lowered the substrate affinity, but the optimum pH and thermo-stability were not changed by these hypermannosylated glycans. This functional expression system described here will aid in molecular evolutionary studies conducted to generate new variants of laccase.


Assuntos
Lacase/química , Lacase/metabolismo , Saccharomyces cerevisiae/genética , Trametes/enzimologia , Clonagem Molecular , Evolução Molecular Direcionada , Estabilidade Enzimática , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Lacase/genética , Lacase/isolamento & purificação , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Trametes/genética
9.
Biochimie ; 142: 183-190, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28943302

RESUMO

Utilization of laccases in biotechnology and bioremediation has created a strong demand for the characterization of new enzymes and an increase in production of known laccases. Thus, additional research into these enzymes is critically needed. In this study, we report a comparative study of the biochemical and transcriptional properties of two different laccase isozymes from Trametes hirsuta 072 - the constitutive and inducible forms. A recombinant LacC enzyme was expressed in Penicillium canescens to characterize its properties. LacC is single-purpose enzyme, unlike LacA, which can operate efficiently under a wide range of temperatures and pHs (55-70 °C and pH 3-5, respectively). LacC has a lower RedOx potential than LacA and does not oxidize substrates containing amine groups. Expression of the lacC gene was selective compared to that of the lacA gene and increased significantly in the presence of complex synthetic compounds such as dyes and xenobiotics. This study shows that laccases from the multigene families of basidiomycetes differ significantly in their properties, thus providing a complementary effect during lignin degradation.


Assuntos
Lacase/genética , Família Multigênica/genética , Trametes/enzimologia , Trametes/genética , Isoenzimas/genética
10.
Sci Rep ; 7(1): 3008, 2017 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-28592865

RESUMO

Several methods have been reported for drying mushroom specimens for population genetic, taxonomic, and phylogenetic studies. However, most methods have not been directly compared for their effectiveness in preserving mushroom DNA. In this study, we compared silica gel drying at ambient temperature and oven drying at seven different temperatures. Two mushroom species representing two types of fruiting bodies were examined: the fleshy button mushroom Agaricus bisporus and the leathery shelf fungus Trametes versicolor. For each species dried with the eight methods, we assessed the mushroom water loss rate, the quality and quantity of extracted DNA, and the effectiveness of using the extracted DNA as a template for PCR amplification of two DNA fragments (ITS and a single copy gene). Dried specimens from all tested methods yielded sufficient DNA for PCR amplification of the two genes in both species. However, differences among the methods for the two species were found in: (i) the time required by different drying methods for the fresh mushroom tissue to reach a stable weight; and (ii) the relative quality and quantity of the extracted genomic DNA. Among these methods, oven drying at 70 °C for 3-4 h seemed the most efficient for preserving field mushroom samples for subsequent molecular work.


Assuntos
Agaricus/química , DNA Fúngico/isolamento & purificação , DNA Espaçador Ribossômico/isolamento & purificação , Dessecação/métodos , Biologia Molecular/métodos , Trametes/química , Agaricus/genética , DNA Fúngico/genética , DNA Espaçador Ribossômico/genética , Reação em Cadeia da Polimerase , Temperatura , Trametes/genética
11.
Mycopathologia ; 182(7-8): 755-759, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28324243

RESUMO

PURPOSE: We report the first case of human infection and keratitis secondary to Trametes betulina, a rare filamentous fungus. METHODS: Clinical examination including external and slit-lamp examination and corneal scrapings with microbiologic evaluation were performed on a patient with chronic allergic conjunctivitis, entropion and a long-standing corneal ulcer resistant to treatment. RESULTS: The culture from the corneal scraping revealed a basidiomycetous fungus which was submitted for identification. DNA extraction with sequencing and analysis of the ITS and D1/D2 regions were performed on the isolate and demonstrated 100% similarity to Lenzites betulina/Trametes betulina. Susceptibility testing demonstrated potent in vitro activity of voriconazole (MIC < 0.03 µg/ml). The patient was treated with voriconazole, and the corneal ulcer and infiltrate resolved. The infection resulted in corneal thinning and a dense central corneal scar. Penetrating keratoplasty was performed 5 months after diagnosis and treatment and revealed stromal scarring without fungal elements. CONCLUSION: This is the first reported case of keratitis caused by Trametes betulina. This organism should be considered in the differential diagnosis for rare filamentous fungal keratitis and its treatment with voriconazole also noted.


Assuntos
Córnea/microbiologia , Ceratite/diagnóstico , Ceratite/patologia , Trametes/isolamento & purificação , Idoso , Antifúngicos/farmacologia , DNA Fúngico/química , DNA Fúngico/genética , DNA Ribossômico/química , DNA Ribossômico/genética , DNA Espaçador Ribossômico/química , DNA Espaçador Ribossômico/genética , Humanos , Ceratite/microbiologia , Ceratite/cirurgia , Ceratoplastia Penetrante , Masculino , Testes de Sensibilidade Microbiana , Técnicas Microbiológicas , Análise de Sequência de DNA , Trametes/classificação , Trametes/genética , Voriconazol/farmacologia
12.
PLoS One ; 12(3): e0173813, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28301519

RESUMO

Ligninolytic heme peroxidases comprise an extensive family of enzymes, which production is characteristic for white-rot Basidiomycota. The majority of fungal heme peroxidases are encoded by multigene families that differentially express closely related proteins. Currently, there were very few attempts to characterize the complete multigene family of heme peroxidases in a single fungus. Here we are focusing on identification and characterization of peroxidase genes, which are transcribed and secreted by basidiomycete Trametes hirsuta 072, an efficient lignin degrader. The T. hirsuta genome contains 18 ligninolytic peroxidase genes encoding 9 putative lignin peroxidases (LiP), 7 putative short manganese peroxidases (MnP) and 2 putative versatile peroxidases (VP). Using ddPCR method we have quantified the absolute expression of the 18 peroxidase genes under different culture conditions and on different growth stages of basidiomycete. It was shown that only two genes (one MnP and one VP) were prevalently expressed as well as secreted into cultural broth under all conditions investigated. However their transcriptome and protein profiles differed in time depending on the effector used. The expression of other peroxidase genes revealed a significant variability, so one can propose the specific roles of these enzymes in fungal development and lifestyle.


Assuntos
Genes Fúngicos , Lignina/metabolismo , Família Multigênica , Peroxidases/metabolismo , Trametes/enzimologia , Peroxidases/classificação , Filogenia , Trametes/genética , Transcrição Genética
13.
J Gen Appl Microbiol ; 62(6): 303-312, 2017 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-27885193

RESUMO

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.


Assuntos
Hidrocarbonetos Policíclicos Aromáticos/metabolismo , Trametes/metabolismo , Biodegradação Ambiental , Carcinógenos/metabolismo , Carcinógenos/farmacologia , Meios de Cultura/química , DNA Espaçador Ribossômico , Fluorenos/metabolismo , Fluorenos/farmacologia , Glucose/farmacologia , Lacase/biossíntese , Peroxidases/biossíntese , Fenantrenos/metabolismo , Fenantrenos/farmacologia , Hidrocarbonetos Policíclicos Aromáticos/farmacologia , Pirenos/metabolismo , Pirenos/farmacologia , Temperatura , Tailândia , Trametes/genética , Trametes/crescimento & desenvolvimento , Trametes/isolamento & purificação
14.
Appl Biochem Biotechnol ; 181(3): 1228-1239, 2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-27744524

RESUMO

Laccases are used in numerous applications, from green degradation of various xenobiotic compounds, waste detoxification, textile dye bleaching, and delignification of lignocellulose materials to biofuel production. In this study, the recombinant Yarrowia lipolytica YL4 strain carrying the white-rot fungus Trametes versicolor laccase IIIb gene was used for laccase production from beet molasses as an agro-industrial residue. Response surface methodology was used to statistical optimization of the production of laccase by Y. lipolytica using an industrial medium containing molasses which allows a six times increase in laccase activity compared to primary medium contains glucose after 144 h. In bioreactor cultivation after 48 h, laccase production reached to 3.7- and 22.5-fold more than optimized and primary media in shake-flask cultures, respectively. Laccase productivity in bioreactor (0.0937 U/h) was higher than shake-flask culture (0.0084 U/h). The present study provides valuable information about statistical optimization of bioprocess development for cost-effective production of laccase and other heterologous proteins in Y. lipolytica from beet molasses as sole carbon source, thus allowing the valorization and decreasing environmental pollution of this agro-industrial waste.


Assuntos
Reatores Biológicos , Proteínas Fúngicas/biossíntese , Lacase/biossíntese , Modelos Biológicos , Melaço , Trametes , Yarrowia/crescimento & desenvolvimento , Proteínas Fúngicas/genética , Lacase/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Trametes/enzimologia , Trametes/genética , Yarrowia/genética
15.
Fungal Biol ; 120(12): 1609-1622, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27890094

RESUMO

A Trametes versicolor laccase was functionally expressed on the membrane surface of Saccharomyces cerevisiae EBY100. Laccase expression was increased 6.57-fold by medium optimization and surpassed production by the native strain. Maximal laccase and biomass production reached 19 735 ± 1719 Ug-1 and 6.22 ± 0.53 gL-1 respectively, after 2 d of culture. Optimum oxidization of all substrates by laccase was observed at pH 3. Laccase showed high affinity towards substrates used with Km (mM) and Vmax (µmol min-1) values of 0.57 ± 0.0047 and 24.55 ± 0.64, 1.52 ± 0.52 and 9.25 ± 1.78, and 2.67 ± 0.12 and 11.26 ± 0.75, were reported for ABTS, 2, 6-DMP and GUA, respectively. EDTA and NaN3 displayed none competitive inhibition towards laccase activity. The optimum temperature for activity was 50 °C; however, the enzyme was stable over a wide range of temperatures (25-70 °C). The biologically immobilized laccase showed high reusability towards phenolic substrates and low reusability with non-phenolic substrates. High affinity for a diversity phenolic compounds and great ethanol tolerance substantiates this laccase/yeast biocatalyst potential for application in the production of bioethanol.


Assuntos
Técnicas de Visualização da Superfície Celular , Enzimas Imobilizadas/metabolismo , Expressão Gênica , Lacase/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/metabolismo , Meios de Cultura/química , Estabilidade Enzimática , Enzimas Imobilizadas/genética , Concentração de Íons de Hidrogênio , Lacase/química , Lacase/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Especificidade por Substrato , Temperatura , Trametes/enzimologia , Trametes/genética
16.
Sci Rep ; 6: 33237, 2016 09 12.
Artigo em Inglês | MEDLINE | ID: mdl-27616058

RESUMO

Transcriptomic analysis of cultured fungi suggests that many genes for secondary metabolite synthesis are presumably silent under standard laboratory condition. In order to investigate the expression of silent genes in symbiotic systems, 136 fungi-fungi symbiotic systems were built up by co-culturing seventeen basidiomycetes, among which the co-culture of Trametes versicolor and Ganoderma applanatum demonstrated the strongest coloration of confrontation zones. Metabolomics study of this co-culture discovered that sixty-two features were either newly synthesized or highly produced in the co-culture compared with individual cultures. Molecular network analysis highlighted a subnetwork including two novel xylosides (compounds 2 and 3). Compound 2 was further identified as N-(4-methoxyphenyl)formamide 2-O-ß-D-xyloside and was revealed to have the potential to enhance the cell viability of human immortalized bronchial epithelial cell line of Beas-2B. Moreover, bioinformatics and transcriptional analysis of T. versicolor revealed a potential candidate gene (GI: 636605689) encoding xylosyltransferases for xylosylation. Additionally, 3-phenyllactic acid and orsellinic acid were detected for the first time in G. applanatum, which may be ascribed to response against T.versicolor stress. In general, the described co-culture platform provides a powerful tool to discover novel metabolites and help gain insights into the mechanism of silent gene activation in fungal defense.


Assuntos
Ganoderma/metabolismo , Glicosídeos/metabolismo , Trametes/metabolismo , Sequência de Aminoácidos , Sobrevivência Celular/efeitos dos fármacos , Técnicas de Cocultura , Sequência Conservada , Avaliação Pré-Clínica de Medicamentos , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Ganoderma/genética , Glicosídeos/química , Glicosídeos/isolamento & purificação , Glicosídeos/farmacologia , Humanos , Metabolômica , Interações Microbianas , Pentosiltransferases/genética , Pentosiltransferases/metabolismo , Filogenia , Trametes/genética
17.
Artigo em Inglês | MEDLINE | ID: mdl-27490563

RESUMO

Textile effluents are highly polluting and have variable and complex compositions. They can be extremely complex, with high salt concentrations and alkaline pHs. A fixed-bed bioreactor was used in the present study to simulate a textile effluent treatment, where the white-rot fungus, Trametes versicolor, efficiently decolourised the azo dye Reactive Black 5 over 28 days. This occurred under high alkaline conditions, which is unusual, but advantageous, for successful decolourisation processes. Active dye decolourisation was maintained by operation in continuous culture. Colour was eliminated during the course of operation and maximum laccase (Lcc) activity (80.2 U∙L(-1)) was detected after glycerol addition to the bioreactor. Lcc2 gene expression was evaluated with different carbon sources and pH values based on reverse transcriptase-PCR (polymerase chain reaction). Glycerol was shown to promote the highest lcc2 expression at pH 5.5, followed by sucrose and then glucose. The highest levels of expression occurred between three and four days, which corroborate the maximum Lcc activity observed for sucrose and glycerol on the bioreactor. These results give new insights into the use of T. versicolor in textile dye wastewater treatment with high pHs.


Assuntos
Reatores Biológicos , Carbono/metabolismo , Corantes/metabolismo , Concentração de Íons de Hidrogênio , Lacase/biossíntese , Indústria Têxtil , Trametes/metabolismo , Álcalis/metabolismo , Biodegradação Ambiental , Cor , Lacase/genética , Naftalenossulfonatos , Reação em Cadeia da Polimerase , Portugal , Trametes/genética , Poluentes Químicos da Água
18.
Braz. j. microbiol ; 47(2): 287-297, Apr.-June 2016. tab, graf
Artigo em Inglês | LILACS | ID: lil-780826

RESUMO

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.


Assuntos
Proteínas Fúngicas/metabolismo , Lacase/metabolismo , Trametes/enzimologia , Argentina , Temperatura , Estabilidade Enzimática , Proteínas Fúngicas/genética , Proteínas Fúngicas/química , Lacase/genética , Lacase/química , Trametes/isolamento & purificação , Trametes/genética
19.
Braz J Microbiol ; 47(2): 287-97, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26991301

RESUMO

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 Cu(2+) was added, both enzymes were significantly increased. Zymograms showed that two isoenzymes were increased in all strains in the presence of Cu(2+). Strain B showed the greatest response to Cu(2+) 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.


Assuntos
Proteínas Fúngicas/metabolismo , Lacase/metabolismo , Trametes/enzimologia , Argentina , Estabilidade Enzimática , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Lacase/química , Lacase/genética , Temperatura , Trametes/genética , Trametes/isolamento & purificação
20.
PLoS One ; 11(2): e0147997, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26849129

RESUMO

Fungal laccases are enzymes that have been studied because of their ability to decolorize and detoxify effluents; they are also used in paper bleaching, synthesis of polymers, bioremediation, etc. In this work we were able to express a laccase from Trametes (Pycnoporus) sanguineus in the filamentous fungus Trichoderma atroviride. For this purpose, a transformation vector was designed to integrate the gene of interest in an intergenic locus near the blu17 terminator region. Although monosporic selection was still necessary, stable integration at the desired locus was achieved. The native signal peptide from T. sanguineus laccase was successful to secrete the recombinant protein into the culture medium. The purified, heterologously expressed laccase maintained similar properties to those observed in the native enzyme (Km and kcat and kcat/km values for ABTS, thermostability, substrate range, pH optimum, etc). To determine the bioremediation potential of this modified strain, the laccase-overexpressing Trichoderma strain was used to remove xenobiotic compounds. Phenolic compounds present in industrial wastewater and bisphenol A (an endocrine disruptor) from the culture medium were more efficiently removed by this modified strain than with the wild type. In addition, the heterologously expressed laccase was able to decolorize different dyes as well as remove benzo[α]pyrene and phenanthrene in vitro, showing its potential for xenobiotic compound degradation.


Assuntos
Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Lacase/genética , Trametes/genética , Trichoderma/genética , Xenobióticos/farmacologia , Ativação Enzimática , Estabilidade Enzimática , Cinética , Proteínas Recombinantes , Especificidade por Substrato , Trametes/enzimologia , Trichoderma/enzimologia
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