RESUMO
Manganese peroxidases (MnP) from the white-rot fungi Phanerochaete chrysosporium catalyse the oxidation of Mn2+ to Mn3+, a strong oxidizer able to oxidize a wide variety of organic compounds. Different approaches have been used to unravel the enzymatic properties and potential applications of MnP. However, these efforts have been hampered by the limited production of native MnP by fungi. Heterologous expression of MnP has been achieved in both eukaryotic and prokaryotic expression systems, although with limited production and many disadvantages in the process. Here we described a novel molecular approach for the expression and purification of manganese peroxidase isoform 1 (MnP1) from P. chrysosporium using an E. coli-expression system. The proposed strategy involved the codon optimization and chemical synthesis of the MnP1 gene for optimised expression in the E. coli T7 shuffle host. Recombinant MnP1 (rMnP1) was expressed as a fusion protein, which was recovered from solubilised inclusion bodies. rMnP1 was purified from the fusion protein using intein-based protein purification techniques and a one-step affinity chromatography. The designated strategy allowed production of an active enzyme able to oxidize guaiacol or Mn2+.
Assuntos
Escherichia coli/metabolismo , Expressão Gênica , Peroxidases/isolamento & purificação , Phanerochaete/enzimologia , Sequência de Aminoácidos , Ensaios Enzimáticos , Vetores Genéticos/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Padrões de Referência , SolubilidadeRESUMO
The glycoside hydrolase family 45 (GH45) of carbohydrate modifying enzymes is mostly comprised of ß-1,4-endoglucanases. Significant diversity between the GH45 members has prompted the division of this family into three subfamilies: A, B and C, which may differ in terms of the mechanism, general architecture, substrate binding and cleavage. Here, we use a combination of X-ray crystallography, bioinformatics, enzymatic assays, molecular dynamics simulations and site-directed mutagenesis experiments to characterize the structure, substrate binding and enzymatic specificity of the GH45 subfamily C endoglucanase from Phanerochaete chrysosporium (PcCel45A). We investigated the role played by different residues in the binding of the enzyme to cellulose oligomers of different lengths and examined the structural characteristics and dynamics of PcCel45A that make subfamily C so dissimilar to other members of the GH45 family. Due to the structural similarity shared between PcCel45A and domain I of expansins, comparative analysis of their substrate binding was also carried out. Our bioinformatics sequence analyses revealed that the hydrolysis mechanisms in GH45 subfamily C is not restricted to use of the imidic asparagine as a general base in the "Newton's cradle" catalytic mechanism recently proposed for this subfamily.
Assuntos
Celulase/química , Celulase/metabolismo , Phanerochaete/enzimologia , Catálise , Biologia Computacional , Cristalografia por Raios X , Ensaios Enzimáticos , Simulação de Dinâmica MolecularRESUMO
Ligninolytic enzyme production and lignin degradation are typically the rate-limiting steps in the biofuel industry. To improve the efficiency of simultaneous bio-delignification and enzyme production, Phanerochaete chrysosporium was transformed by shock wave-induced acoustic cavitation to co-overexpress 3 peroxidases and 1 laccase and test it on the degradation of sugarcane bagasse and wheat bran. Lignin depolymerization was enhanced by up to 25% in the presence of recombinant fungi in comparison with the wild-type strain. Sugar release on lignocellulose was 2- to 6-fold higher by recombinant fungi as compared with the control. Wheat bran ostensibly stimulated the production of ligninolytic enzymes. The highest peroxidase activity from the recombinant strains was 2.6-fold higher, whereas the increase in laccase activity was 4-fold higher in comparison to the control. The improvement of lignin degradation was directly proportional to the highest peroxidase and laccase activity. Because various phenolic compounds released during lignocellulose degradation have proven to be toxic to cells and to inhibit enzyme activity, a significant reduction (over 40%) of the total phenolic content in the samples treated with recombinant strains was observed. To our knowledge, this is the first report that engineering P. chrysosporium enhances biodegradation of lignocellulosic biomass.
Assuntos
Biomassa , Lacase/biossíntese , Lacase/genética , Peroxidases/biossíntese , Peroxidases/genética , Phanerochaete/genética , Phanerochaete/metabolismo , Biodegradação Ambiental , Biocombustíveis , Celulose/metabolismo , Clonagem Molecular , Fibras na Dieta , Ergosterol , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Lignina/metabolismo , Engenharia Metabólica , Phanerochaete/enzimologia , Phanerochaete/crescimento & desenvolvimento , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharum , Transformação GenéticaRESUMO
For a long time, proteolytic enzymes have been employed as key tools of industrial processes, especially in the dairy industry. In the present work, we used Phanerochaete chrysosporium for biochemical characterization and analysis of catalytic specificity of an aspartic peptidase. Our results revealed an aspartic peptidase with molecular mass â¼38kDa, maximal activity at pH 4.5 and 50°C, and stability above 80% in the pH range of 3-8 and temperature up to 55°C for 1h. In a milk-clotting assay, this peptidase showed maximal milk clotting activity at 60-65°C and maintenance of enzymatic activity above 80% in the presence of 20mM CaCl2. In a specificity assay, we observed stronger restriction of catalysis at the S1 subsite, with a preference for lysine, arginine, leucine, tyrosine, and phenylalanine residues. The restricted proteolysis and milk-clotting potential are attractive properties for the use in cheese production.
Assuntos
Ácido Aspártico Proteases/metabolismo , Queijo/microbiologia , Indústria de Processamento de Alimentos , Leite/microbiologia , Phanerochaete/enzimologia , Animais , Proteínas Fúngicas/metabolismo , TemperaturaRESUMO
Peptidases are enzymes that catalyze the rupture of peptide bonds. Catalytic specificity studies of these enzymes have illuminated their modes of action and preferred hydrolysis targets. We describe the biochemical characteristics and catalytic specificity of a lysine-dependent peptidase secreted by the basidiomycete fungus Phanerochaete chrysosporium. We attained 5.7-fold purification of a â¼23-kDa neutral peptidase using size-exclusion (Sephadex G-50 resin) and ion-exchange (Source 15S resin) chromatography. Using the Fluorescence Resonance Energy Transfer substrate Abz-KLRSSKQ-EDDnp, we detected maximal activity at pH 7.0 and 45-55°C. The peptidase retained â¼80% of its enzymatic activity for a wide range of conditions (pH 4-9; temperatures up to 50°C for 1h). The peptidase activity was lowered by the ionic surfactants, sodium dodecyl sulfate and cetyltrimethylammonium bromide; the reducing agent, dithiothreitol; the chaotrope, guanidine; copper (II) ion; and the cysteine peptidase-specific inhibitors, iodoacetic acid and N-ethylmaleimide. The peptidase preferred the basic amino acids K and R and high selectivity on S'1 subsite, exhibiting a condition of lysine-dependence to catalysis on anchoring of this subsite.
Assuntos
Cisteína Proteases/química , Proteínas Fúngicas/química , Sequência de Aminoácidos , Biocatálise , Cisteína Proteases/isolamento & purificação , Inibidores de Cisteína Proteinase/química , Estabilidade Enzimática , Proteínas Fúngicas/isolamento & purificação , Concentração de Íons de Hidrogênio , Cinética , Lisina/química , Phanerochaete/enzimologia , Proteólise , Especificidade por SubstratoRESUMO
Phanerochaete chrysosporium belongs to a group of lignin-degrading fungi that secretes various oxidoreductive enzymes, including lignin peroxidase (LiP) and manganese peroxidase (MnP). Previously, we demonstrated that the heterologous expression of a versatile peroxidase (VP) in P. chrysosporium recombinant strains is possible. However, the production of laccases (Lac) in this fungus has not been completely demonstrated and remains controversial. In order to investigate if the co-expression of Lac and VP in P. chrysosporium would improve the degradation of phenolic and non-phenolic substrates, we tested the constitutive co-expression of the lacIIIb gene from Trametes versicolor and the vpl2 gene from Pleurotus eryngii, and also the endogenous genes mnp1 and lipH8 by shock wave mediated transformation. The co-overexpression of peroxidases and laccases was improved up to five-fold as compared with wild type species. Transformant strains showed a broad spectrum in phenolic/non-phenolic biotransformation and a high percentage in synthetic dye decolorization in comparison with the parental strain. Our results show that the four enzymes can be constitutively expressed in a single transformant of P. chrysosporium in minimal medium. These data offer new possibilities for an easy and efficient co-expression of laccases and peroxidases in suitable basidiomycete species.
Assuntos
Lacase/metabolismo , Peroxidases/metabolismo , Phanerochaete/genética , Clonagem Molecular , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Lacase/genética , Engenharia Metabólica/métodos , Peroxidases/genética , Phanerochaete/enzimologia , Phanerochaete/metabolismo , Fenóis/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transformação GenéticaRESUMO
The white-rot fungus Phanerochaete chrysosporium was investigated for its capacity to degrade the herbicide diuron in liquid stationary cultures. The presence of diuron increased the production of lignin peroxidase in relation to control cultures but only barely affected the production of manganese peroxidase. The herbicide at the concentration of 7 µ g/mL did not cause any reduction in the biomass production and it was almost completely removed after 10 days. Concomitantly with the removal of diuron, two metabolites, DCPMU [1-(3,4-dichlorophenyl)-3-methylurea] and DCPU [(3,4-dichlorophenyl)urea], were detected in the culture medium at the concentrations of 0.74 µ g/mL and 0.06 µ g/mL, respectively. Crude extracellular ligninolytic enzymes were not efficient in the in vitro degradation of diuron. In addition, 1-aminobenzotriazole (ABT), a cytochrome P450 inhibitor, significantly inhibited both diuron degradation and metabolites production. Significant reduction in the toxicity evaluated by the Lactuca sativa L. bioassay was observed in the cultures after 10 days of cultivation. In conclusion, P. chrysosporium can efficiently metabolize diuron without the accumulation of toxic products.
Assuntos
Biodegradação Ambiental , Sistema Enzimático do Citocromo P-450/metabolismo , Diurona/metabolismo , Phanerochaete/enzimologia , Humanos , Lignina/metabolismo , Oxirredução , Peroxidases/metabolismo , Phanerochaete/metabolismoRESUMO
White-rot fungi (WRF) are capable of degrading complex organic compounds such as lignin, and the enzymes that enable these processes can be used for the detoxification of recalcitrant organopollutants. The aim of this study is to evaluate a system based on the use of an in vitro ligninolytic enzyme for the detoxification of recalcitrant dye pollutants. The dyes selected for investigation were the anionic and cationic commercial azo dyes, basic blue 41 (BB41), acid black 1 (AB1), and reactive black 5 (RB5). A supernatant, cell-free culture of WRF with manganese peroxidase activity was used to investigate its degradative capacity under various conditions, and concentrations of cofactors, H(2)O(2) and Mn(2+). The assays were carried out using a 2(2) experimental designs whose variables were concentration of Mn(2+) (33 and 1,000 µM) and semicontinuous dosage of the H(2)O(2) (0.02 and 0.10 µmol) added at a frequency of 0.2 min(-1). The response variables analyzed were the efficiency and the initial rate of the decolorization process. The dye concentrations considered ranged from 10 to 200 mg L(-1). AB1 and RB5 were decolorized over the entire interval of concentrations studied; reaching efficiencies between 15 and 95%. Decolorization of up to 100 mg L(-1), BB41 had less than 30% efficiency. The decay of the concentration of AB1 was interpreted by two-stage kinetics model, with the exception of the condition of 33 µM Mn(2+)-0.02 µmol of H(2)O(2) in which only one stage was observed. For all assays performed with 33 µM Mn(2+), the initial rate of the decolorization process was found to be dependent on the dosage of H(2)O(2). The results of this study can be applied to the development bioreactors for the degradation of recalcitrant pollutants from the textile industry and may be used as a model for expanding the use of extracellular enzyme supernatants in bioremediation.
Assuntos
Compostos Azo/metabolismo , Corantes/metabolismo , Peróxido de Hidrogênio/análise , Peroxidases/metabolismo , Eliminação de Resíduos Líquidos/métodos , Biodegradação Ambiental , Reatores Biológicos/microbiologia , Resíduos Industriais , Lignina/metabolismo , Naftalenossulfonatos/metabolismo , Naftóis/metabolismo , Phanerochaete/enzimologia , Phanerochaete/crescimento & desenvolvimento , Poluentes Químicos da Água/metabolismoRESUMO
A strain of Aspergillus niger, previously isolated from sugarcane bagasse because of its capacity to degrade phenanthrene in soil by solid culture, was used to express a manganese peroxidase gene (mnp1) from Phanerochaete chrysosporium, aiming at increasing its polycyclic aromatic hydrocarbons degradation capacity. Transformants were selected based on their resistance to hygromycin B and the discoloration induced on Poly R-478 dye by the peroxidase activity. The recombinant A. niger SBC2-T3 strain developed MnP activity and was able to remove 95% of the initial phenanthrene (400 ppm) from a microcosm soil system after 17 days, whereas the wild strain removed 72% under the same conditions. Transformation success was confirmed by PCR amplification using gene-specific primers, and a single fragment (1,348 bp long, as expected) of the recombinant mnp1 was amplified in the DNA from transformants, which was absent from the parental strain.
Assuntos
Aspergillus niger/enzimologia , Aspergillus niger/metabolismo , Peroxidases/genética , Peroxidases/metabolismo , Fenantrenos/metabolismo , Poluentes do Solo/metabolismo , Aspergillus niger/genética , Biotransformação , Primers do DNA/genética , DNA Fúngico/genética , Phanerochaete/enzimologia , Phanerochaete/genética , Reação em Cadeia da Polimerase , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Seleção Genética , Fatores de Tempo , Transformação GenéticaRESUMO
This study provides a first attempt investigation of a serie of studies on the ability of Anthracophyllum discolor, a recently isolated white-rot fungus from forest of southern Chile, for the treatment of soil contaminated with pentachlorophenol (PCP) to future research on potential applications in bioremediation process. Bioremediation of soil contaminated with PCP (250 and 350 mg kg⻹ soil) was investigated with A. discolor and compared with the reference strain Phanerochaete chrysosporium. Both strains were incorporated as free and immobilized in wheat grains, a lignocellulosic material previously selected among wheat straw, wheat grains and wood chips through the growth and colonization of A. discolor. Wheat grains showed a higher growth and colonization of A. discolor, increasing the production of manganese peroxidase (MnP) activity. Moreover, the application of white-rot fungi immobilized in wheat grains to the contaminated soil favored the fungus spread. In turn, with both fungal strains and at the two PCP concentrations a high PCP removal (70-85%) occurred as respect to that measured with the fungus as free mycelium (30-45%). Additionally, the use of wheat grains in soil allowed the proliferation of microorganisms PCP decomposers, showing a synergistic effect with A. discolor and P. chrysosporium and increasing the PCP removal in the soil.
Assuntos
Agaricales/metabolismo , Pentaclorofenol/metabolismo , Doenças das Plantas/microbiologia , Poluentes do Solo/metabolismo , Triticum/microbiologia , Agaricales/enzimologia , Agaricales/crescimento & desenvolvimento , Agaricales/isolamento & purificação , Biodegradação Ambiental , Chile , Técnicas de Cultura , Proteínas Fúngicas/metabolismo , Peroxidases/metabolismo , Phanerochaete/enzimologia , Phanerochaete/crescimento & desenvolvimento , Phanerochaete/metabolismo , Microbiologia do SoloRESUMO
The fungus Phanerochaete flavido-alba is highly efficient in the oxidation of olive oil wastewater-derived polyphenols. This capability is largely due to the action of a multicopper-oxidase (MCO), encoded by the pfaL gene. We describe the sequence and organization of pfaL gene and the biochemical characterization and predicted 3D structural model of the encoded protein. pfaL gene organization and peptide sequence are highly similar to those of P. chrysosporium MCOs. However, PfaL is the first MCO in the Phanerochaete genus to show evident laccase activity. Phylogenetic analysis places PfaL in a differentiated sub-branch of ferroxidases. Protein structure analysis reveals close similarity of PfaL and ferroxidases and provides clues about the differences of activity between both types of enzymes. To summarize, P. flavido-alba laccase is the first enzyme in the novel and biochemically poorly defined group of "ferroxidases/laccases" that shows efficacious oxidation of laccase substrates, biotechnologically exploitable in bioremediation approaches.
Assuntos
Ceruloplasmina/metabolismo , Proteínas Fúngicas/metabolismo , Lacase/metabolismo , Oxirredutases/genética , Phanerochaete/enzimologia , Sequência de Aminoácidos , Sequência de Bases , Biodegradação Ambiental , Ceruloplasmina/genética , Clonagem Molecular , Flavonoides/metabolismo , Proteínas Fúngicas/genética , Lacase/genética , Modelos Moleculares , Dados de Sequência Molecular , Phanerochaete/genética , Fenóis/metabolismo , Filogenia , Polifenóis , Alinhamento de Sequência , Especificidade por SubstratoRESUMO
Four white rot fungi (WRF) strains, Phanerochaete chrysosporium, Trametes versicolor, Coriolopsis polyzona and Pycnoporus coccineus, were tested for efficiency of treatment of Olive Oil mill wastewaters (OOMW) in relation with their cultivation mode, i.e. under the form of free mycelium, mycelium immobilized in alginate beads and solid state cultivation on Petri dishes. Study of biodegradation of phenolic compounds, chemical oxygen demand (COD) decrease and decolourisation of OOMW have shown that Coriolopsis polyzona and Pycnoporus coccineus degradation performances were apparently only slightly affected by the cell cultivation procedures experienced here. In contrast, Phanerochaete chrysosporium and Trametes versicolor showed respectively marked preferences for solid state and alginate immobilisation procedures. Both mono and polyphenolics were reduced to different extent during incubation depending on the strain, as shown by gel filtration analysis. Final pH obtained after fungal treatment of the OOMW based medium (initial pH of 5.0) was measured in order to evaluate the possibility of releasing friendly the treated wastewater in the environment. Laboratory studies as reported here may be useful for orienting the choice of a strain for treating pollution by OOMW in a particular real situation.
Assuntos
Basidiomycota/enzimologia , Fungos/enzimologia , Phanerochaete/enzimologia , Purificação da Água/métodos , Alginatos , /métodos , Peroxidases , Gorduras VegetaisRESUMO
We have previously identified and functionally characterized the transcription factor ACE1 (Pc-ACE1) from Phanerochaete chrysosporium. In Saccharomyces cerevisiae, ACE1 activates the copper-dependent transcription of target genes through a DNA sequence element named ACE. However, the possible target gene(s) of Pc-ACE1 were unknown. An in silico search led to the identification of putative ACE elements in the promoter region of mco1, one of the four clustered genes encoding multicopper oxidases (MCOs) in P. chrysosporium. Since copper exerts an effect at the transcriptional level in MCOs from several organisms, in this work we analysed the effect of copper on transcript levels of the clustered MCO genes from P. chrysosporium, with the exception of the transcriptionally inactive mco3. Copper supplementation of cultures produced an increment in transcripts from genes mco1 and mco2, but not from mco4. Electrophoretic mobility-shift assays revealed that Pc-ACE1 binds specifically to a probe containing one of the putative ACE elements found in the promoter of mco1. In addition, using a cell-free transcription system, we showed that in the presence of cuprous ion, Pc-ACE1 induces activation of the promoter of mco1, but not that of mco2.
Assuntos
Cobre/metabolismo , Proteínas de Ligação a DNA/genética , Oxirredutases/genética , Phanerochaete/genética , Fatores de Transcrição/genética , Transcrição Gênica , Sequência de Bases , Sítios de Ligação/genética , Sequência Consenso/genética , Ensaio de Desvio de Mobilidade Eletroforética , Proteínas Fúngicas/genética , Regulação da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Phanerochaete/enzimologia , Phanerochaete/metabolismo , Regiões Promotoras Genéticas , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
We describe the structure, organization, and transcriptional impact of repetitive elements within the lignin-degrading basidiomycete, Phanerochaete chrysosporium. Searches of the P. chrysosporium genome revealed five copies of pce1, a ~1,750-nt non-autonomous, class II element. Alleles encoding a putative glucosyltransferase and a cytochrome P450 harbor pce insertions and produce incomplete transcripts. Class I elements included pcret1, an intact 8.14-kb gypsy-like retrotransposon inserted within a member of the multicopper oxidase gene family. Additionally, we describe a complex insertion of nested transposons within another putative cytochrome P450 gene. The disrupted allele lies within a cluster of >14 genes, all of which encode family 64 cytochrome P450s. Components of the insertion include a disjoint copia-like element, pcret3, the pol domain of a second retroelement, pcret2, and a duplication of an extended ORF of unknown function. As in the case of the pce elements, pcret1 and pcret2/3 insertions are confined to single alleles, transcripts of which are truncated. The corresponding wild-type alleles are apparently unaffected. In aggregate, P. chrysosporium harbors a complex array of repetitive elements, at least five of which directly influence expression of genes within families of structurally related sequences.
Assuntos
Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica/genética , Genes Fúngicos , Mutagênese Insercional , Phanerochaete/genética , Retroelementos/genética , Alelos , Sequência de Bases , Sistema Enzimático do Citocromo P-450/biossíntese , Sistema Enzimático do Citocromo P-450/genética , Proteínas Fúngicas/biossíntese , Regulação Enzimológica da Expressão Gênica/genética , Glucosiltransferases/biossíntese , Glucosiltransferases/genética , Lignina/metabolismo , Dados de Sequência Molecular , Fases de Leitura Aberta/genética , Phanerochaete/enzimologia , Transcrição GênicaRESUMO
Inspection of the genome of the ligninolytic basidiomycete Phanerochaete chrysosporium revealed an unusual peroxidase_like sequence. The corresponding full length cDNA was sequenced and an archetypal secretion signal predicted. The deduced mature protein (NoP, novel peroxidase) contains 295 aa residues and is therefore considerably shorter than other Class II (fungal) peroxidases, such as lignin peroxidases and manganese peroxidases. Comparative modeling of NoP was conducted using the crystal structures of Coprinus cinereus and Arthromyces ramosus peroxidases as templates. The model was validated by molecular dynamics and showed several novel structural features. In particular, NoP has only three disulfide bridges and tryptophan replaces the distal phenylalanine within the heme pocket.
Assuntos
Genes Fúngicos , Peroxidases/química , Peroxidases/genética , Phanerochaete/enzimologia , Phanerochaete/genética , Sequência de Aminoácidos , Cristalografia , Modelos Moleculares , Conformação ProteicaRESUMO
A cluster of multicopper oxidase genes (mco1, mco2, mco3, mco4) from the lignin-degrading basidiomycete Phanerochaete chrysosporium is described. The four genes share the same transcriptional orientation within a 25 kb region. mco1, mco2 and mco3 are tightly grouped, with intergenic regions of 2.3 and 0.8 kb, respectively, whereas mco4 is located 11 kb upstream of mco1. All are transcriptionally active, as shown by RT-PCR. Comparison of cDNAs and the corresponding genomic sequences identified 14-19 introns within each gene. Based on homology and intron composition, two subfamilies of mco sequences could be identified. The sequences have copper-binding motifs similar to ferroxidase proteins, but different from fungal laccases. Thus, these sequences constitute a novel branch of the multicopper oxidase family. Analysis of several cDNA clones obtained from poly(A) RNA revealed the presence of transcripts of various lengths. Splice variants from mco2, mco3 and mco4 were characterized. They generally exhibited the presence of one to five introns, whereas other transcripts lacked some exons. In all cases, the presence of introns leads to frame shifts that give rise to premature stop codons. In aggregate, these investigations show that P. chrysosporium possesses a novel family of multicopper oxidases which also feature clustering and incomplete processing of some of their transcripts, a phenomenon referred to in this paper as 'altered splicing'.
Assuntos
Genes Fúngicos , Família Multigênica , Oxirredutases/genética , Phanerochaete/enzimologia , Phanerochaete/genética , Processamento Alternativo , Sequência de Aminoácidos , Sequência de Bases , DNA Complementar/genética , DNA Fúngico/genética , Dados de Sequência Molecular , RNA Fúngico/genética , RNA Mensageiro/genética , Homologia de Sequência de AminoácidosRESUMO
Lignin degradation by the white rot basidiomycete Phanerochaete chrysosporium involves various extracellular oxidative enzymes, including lignin peroxidase, manganese peroxidase, and a peroxide-generating enzyme, glyoxal oxidase. Recent studies have suggested that laccases also may be produced by this fungus, but these conclusions have been controversial. We identified four sequences related to laccases and ferroxidases (Fet3) in a search of the publicly available P. chrysosporium database. One gene, designated mco1, has a typical eukaryotic secretion signal and is transcribed in defined media and in colonized wood. Structural analysis and multiple alignments identified residues common to laccase and Fet3 sequences. A recombinant MCO1 (rMCO1) protein expressed in Aspergillus nidulans had a molecular mass of 78 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the copper I-type center was confirmed by the UV-visible spectrum. rMCO1 oxidized various compounds, including 2,2'-azino(bis-3-ethylbenzthiazoline-6-sulfonate) (ABTS) and aromatic amines, although phenolic compounds were poor substrates. The best substrate was Fe2+, with a Km close to 2 micro M. Collectively, these results suggest that the P. chrysosporium genome does not encode a typical laccase but rather encodes a unique extracellular multicopper oxidase with strong ferroxidase activity.
Assuntos
Ceruloplasmina/metabolismo , Oxirredutases , Phanerochaete/enzimologia , Sequência de Aminoácidos , Modelos Moleculares , Dados de Sequência Molecular , Oxirredutases/química , Oxirredutases/genética , Oxirredutases/metabolismo , Phanerochaete/genética , Alinhamento de Sequência , Análise de Sequência de DNA , Especificidade por SubstratoRESUMO
The possibility of using two by-products of the sugar cane industry, molasses and bagasse steam explosion liquor (SEL), for lignin peroxidase (LiP) production by Phanerochaete chrysosporium was investigated. For comparison, the fungus was initially cultivated in synthetic media containing either glucose, sucrose, xylose, or xylan as sole carbon sources. The effect of veratryl alcohol (VA) was also investigated in relation to the enzyme activity levels. Results showed that sucrose was not metabolized by this fungus, which precluded the use of molasses as a carbon source. Glucose, xylose, and xylan promoted equivalent cell growth. Enzyme levels in the absence of VA were lower than 28 UI/L and in the presence of VA reached 109 IU/L with glucose and 85 IU/L with xylose or xylan. SEL was adequate for P. chrysosporium LiP production as LiP activity reached 90 IU/L. When VA was added to this medium, enzyme concentration increased to 155 IU/L.
Assuntos
Celulose , Lignina/metabolismo , Peroxidases/metabolismo , Phanerochaete/enzimologia , Saccharum/química , Glucose/metabolismo , Cinética , Peroxidases/isolamento & purificação , Phanerochaete/crescimento & desenvolvimento , Vapor , Sacarose/metabolismo , Xilanos/metabolismo , Xilose/metabolismoRESUMO
The degradation potential of Phanerochaete sordida, Trametes trogii, Coprinus truncorum and Paecilomyces sp. upon yard wastes was evaluated. The species had been inoculated individually or in pairs formed by P. sordida and Paecilomyces sp., T. trogii and Paecilomyces sp., and C. truncorum and Paecilomyces sp. The highest level of endoxilanase activity was produced by P. sordida growing alone, during day 21 (1.09 U/g of dry material), but in P. sordida and Paecilomyces sp. cultures, the detected activity did not overcome 0.27 U/g of dry material during the whole experiment. T. trogii showed maximum activity on day 14 (0.78 U/g of dry material), but in T. trogii and Paecilomyces sp. cultures, the values increased until day 21 (1.07 U/g of dry material). P. sordida endocellulase activity reached its maximum on day 28 (0.08 U/g of dry material), but in P. sordida and Paecilomyces sp. cultures, this activity increased during the whole experiment (0.04 U/g of dry material). The major weight loss was found in P. sordida (27.6%). The possible beneficial effect of co-culture in yard wastes biodegradation is discussed.