Xilanases produzidas por Aspergillus terreus : caracterização, degradação de biomassa lignocelulósica e efeito de compostos fenólicos

Tese (doutorado)—Universidade de Brasília, Instituto de Ciências Biológicas, Departamento de Biologia Celular Pós-Graduação em Biologia Molecular, 2013.Bagaço de cana de açúcar, piolho de algodão sujo e casca de soja são resíduos agroindustriais com elevado teor de holocelulose. O fungo filamentoso Aspergillus terreus fui cultivado por 6, 9, e 5 dias, nos meios contendo bagaço, piolho e casca de soja, respectivamente, como fontes de carbono. Quatro xilanases de baixas massas moleculares foram purificadas, com rendimentos variando entre 5,70 e 74,70% e maiores atividades no intervalo de pH 5,0-6,0 e temperaturas entre 45 e 60ºC. Os valores de Km para xilana de bétula solúvel variaram entre 0,42 e 15,33 mg/mL e para xilana insolúvel 0,47 e 10,90 mg/mL. As variações dos valores de Vmax para xilanas solúvel e insolúvel foram 0,15 e 5,37 UI/mL e 0,08 e 2,46 UI/mL, respectivamente. As 2+ 2+ +quatro enzimas foram ativadas pelo íon Mn (10 mM) e inibidas pelos íons Hg e K (1 e 10 mM). MFA mostrou uma distribuição bimodal de partículas globulares, indicando que Xyl T1 é maior que Xyl T2. Xyl T1 e Xyl T2 foram específicas para xilana como substrato. A espectrometria de massa dos digestos trípticos de Xyl T1 e Xyl T2 mostrou dois espectros diferentes. Xyl T1 e Xyl T3 foram inibidas em maior ou menor grau por todos os compostos fenólicos enquanto que Xyl T2 e Xyl T4 foram bastante resistentes a todos os compostos fenólicos testados. Para Xyl T1, houve um aumento ou diminuição do Km aparente com xilana de bétula, dependendo do tipo de composto fenólico utilizado, entretanto houve uma diminuição do Km aparente de Xyl T2 para xilana de bétula, após a incubação desta enzima com todos os compostos fenólicos. A análise do hidrolisado de três tipos de polpas kraft e de xilana de bétula solúvel e insolúvel por Xyl T1 e Xyl T2 mostrou uma predominância na liberação de xilobiose indicando um mecanismo de ação do tipo endo. A ausência de atividade celulolítica demonstra que essas enzimas têm potencial para o uso no branqueamento de papel. ______________________________________________________________________________ ABSTRACTThe filamentous fungus Aspergillus terreus was cultivated for 6, 9, and 5 days in media containing bagasse, dirty cotton residue and soybean residue, respectively, as the carbon sources. Low-molecular-weight xilanases, named Xyl T1, Xyl T2, Xyl T3 and Xyl T4, were purified with purification yields ranging between 5.70 and 74.70% and higher activities in the pH range 5.0-6.0 and temperatures between 45 and 60ºC. Km values for soluble and insoluble birchwood xylans were at the interval ranges of 0.42-15.33 mg/mL and 0.47-10.90 mg /mL, respectively. Vmax values for soluble and insoluble birchwood xylans ranged from 0.15 to 5.37 IU/mL and 0.08 to 2.46 IU/mL, 2+ respectively. All the above enzymes were activated by Mn (10 mM) and inhibited by 2+ +Hg and K (1 and 10 mM). AFM showed a bimodal distribution of globular particles, indicating that Xyl T1 is larger than Xyl T2. Xyl T1 and Xyl T2 were specific for xylan as substrate. Mass spectrometry showed two different fingerprinting spectra for Xyl T1 and Xyl T2, indicating that they are distinct enzymes. Xyl T1 and Xyl T3 were inhibited in a greater or lesser degree by all phenolic compounds, while Xyl T2 and Xyl T4 were very resistant to the inhibitory effect of all phenolic compounds tested. The apparent Km for Xyl T1 over birchwood xylan increased or decreased depending on the type of phenolic compound used, however, the apparent Km values of Xyl T2, using birchwood xylan as substrate, decreased in the presence of all phenolic compounds. The hydrolysis of cellulose pulps and soluble and insoluble birchwood xylan by Xyl T1 and Xyl T2 showed predominance in the release of xylobiose indicating an endo-type mechanism. The absence of cellulolytic activity indicates that these enzymes have a potential for use in the pulp bleaching process

The hydrolysis of agro-industrial residues by holocellulose-degrading enzymes

Holocellulose structures from agro-industrial residues rely on main and side chain attacking enzymes with different specificities for complete hydrolysis. Combinations of crude enzymatic extracts from different fungal species, including Aspergillus terreus, Aspergillus oryzae, Aspergillus niger and Trichoderma longibrachiatum, were applied to sugar cane bagasse, banana stem and dirty cotton residue to investigate the hydrolysis of holocellulose structures. A. terreus and A. oryzae were the best producers of FPase and xylanase activities. A combination of A. terreus and A. oryzae extracts in a 50% proportion provided optimal hydrolysis of dirty cotton residue and banana stem. For the hydrolysis of sugar cane bagasse, the best results were obtained with samples only containing A. terreus crude extract

Avaliação da genotoxicidade do peróxido de hidrogênio em associação com o polimorfismo da haptoglobina

Dissertação (mestrado)—Universidade de Brasília, Faculdade de Medicina, 2007.Organismos aeróbicos sofrem ação constante dos radicais livres gerados · nesse processo. A reação de Fenton é a maior fonte de formação de OH in vivo, ++ uma vez que o Fe é o metal mais abundante do organismo e está mais capacitado a catalisar reações de oxidação em biomoléculas. É bem conhecido · que o H O causa quebrasnas fitasdo DNA por meio da geração de OH próximos 2 2 à molécula pela reação de Fenton. Dentre as enzimas séricas conhecidas, Hp tem ação antioxidante. Hp pode ++ ligar-se à hemoglobina livre no sangue, prevenindo a perda de Fe o que reduz os danos oxidativos ao DNA. O polimorfismo de Hp apresenta três fenótipos principais Hp 1-1, Hp 2-1 e Hp 2-2 sendo que o fenótipo Hp 1-1 confere maior atividade enzimática e conseqüentemente maior capacidade antioxidante, pois sendo uma molécula menor, liga-se mais efetivamente a Hb livre. Este estudo procurou avaliar os danos do DNA em leucócitos humanos de sangue periférico, coletados de doadores saudáveis, induzidos por exposição ao O e buscar possíveis associações entre o ID com os fenótipos de Hp. Para avaliação do ID ao DNA foi utilizado o teste do cometa. Utilizou-se a técnica de PCR e o gel de poliacrilamida para a genotipagem da Hp. Os resultados dos testes do cometa e das PCRs mostram uma diferença significante entre os IDs observados entre os indivíduos com o fenótipo Hp 1-1 e o fenótipo Hp 2-1. __________________________________________________________________________________________ ABSTRACTAerobic organisms, suffers constant action of free radicals produced in this · process. Fenton reaction is the major source of formation of OH hydroxyl in vivo, once iron is the most abundant metal in organism and is more able to catalyse oxidation reactions in biomolecules. It is well-known that H O causes strand 2 2 - breakes in DNA by the generation of OH near to DNA molecule by Fenton reaction. Among seric enzymes known, Hp acts as antioxidant. Hp can bind free hemoglobin in blood, preventing iron loss what reduces the oxidative damage in DNA. Hp polymorphism has three major phenotypes: Hp 1-1, Hp 2-1 and Hp 2-2. The phenotype Hp 1-1 has the highest enzymatic activity, because it codify a smaller molecule than that codified by the phenotypes Hp 1-2 and Hp 2-2, which binds more effectively to free hemoglobin. This study aimed to evaluate DNA damage in human leukocytes of peripheral O blood stream, collected from healthful donors, induced by the exposition to H 2 2 and then to find possible associations between DNA damage and haptoglobin phenotypes. Comet assay was carried out to evaluate DNA damage. PCR and polyacrylamide gel electrophoresys was used for genotyping of Hp. The results of comet assay nd PCRs shows a statistical difference between the DNA damage observed in individuals who carries phenotype Hp 1-1 and the individuals carring phenotype Hp 2-1

Produção e caracterização de um complexo enzimático de uma nova linhagem de Clostridium thermocellum com enfase em sua atividade de xilanase

Uma nova linhagem de bactéria (ISO II) foi isolada de esterco bovino e identificada como filogeneticamente próxima à bactéria termofílica Clostridium thermocellum. A nova linhagem produziu atividades de xilanase, mananase, pectinase e celulase quando cultivada em meio de cultura líquido contendo engaço de bananeira como fonte de carbono. O perfil de produção enzimática após crescimento em engaço de bananeira mostrou que as atividades de xilanase e celulase foram detectadas em diferentes períodos de incubação. Um complexo enzimático, contendo atividades de xilanase, celulase e mananase, foi isolado de amostras de sobrenadante do meio de cultura da linhagem ISO II crescida em engaço de bananeira. O complexo foi parcialmente purificado por ultrafiltração e cromatografia de filtração em gel em coluna de Sephacryl S-300. Análise de zimograma mostrou 05 sub-unidades com atividade de xilanase. A amostra enzimática apresentou bandas únicas de proteína e atividade de xilanase após eletroforese sob condições não-desnaturantes. A hidrólise de xilana foi ótima no intervalo de temperatura de 55-75ºC e pH 6,0. A xilanase foi estável a 65ºC, mantendo 80% de sua atividade original após 12 h de incubação. Os valores de Km aparente, usando arabinoxilanas insolúveis e solúveis como substratos, foram 1,54 and 11,53 mg/mL, respectivamente. A xilanase foi ativada por ditiotreitol, L-triptofano and L-cisteina e fortemente inibida por N-bromosuccinamida e CoCl2. A caracterização da mananase do complexo mostrou Km e temperatura ótima de 0,846 mg/mL e 65ºC, respectivamente e pH 8,0. Ao contrário da xilanase, a mananase foi menos estável a 65ºC com meia vida de 2,5 h e inibida por ditiotreitol e Ca2+.A new bacterial strain (ISO II) was isolated from manure cow and identified as phylogenetically close to the thermophilic cellulolytic bacterium Clostridium thermocellum. The new strain produced extracellular xylanase, pectinase, mannanase and cellulase activities when grown in liquid culture medium containing banana stem as carbon source. The enzyme production profile after growth on banana stem showed that xylanase and cellulase activities were detected in different incubation periods. An enzyme complex containing xylanase, cellulase and mannanase activities was isolated from culture supernatant samples of strainISO II. The complex was partially purified by ultrafiltration and gel filtration chromatography on Sephacryl S-300. Zymogram analysis after SDS-PAGE presented at least 05 subunits with xylanase activity. The enzyme showed single protein and xylanase activity bands after electrophoresis under non-denaturing conditions. The hydrolysis of xylan was optimal at temperature range of 55-75ºC and pH 6.0. Xylanase activity was quite stable at 65ºC, retaining 80% of its original activity after 12 h incubation. The apparent Km values, using insoluble and soluble arabinoxylans as substrates, were 1.54 and 11.53 mg/mL, respectively. Xylanase was activated by dithiothreitol, L-tryptophan and L-cysteine and strongly inhibited by N-bromosuccinimide and CoCl2. The characterization of mannanase showed Km and temperature optimum of 0.846 mg/mL and 65ºC, respectively and pH 8.0. By contrast to xylanase, it was less stable at 65ºC with half-life of 2.5 h and inhibited by dithiothreitol and Ca2+

Production and characterization of an enzyme complex from a new strain of Clostridium thermocellum with emphasis on its xylanase activity Produção e caracterização de um complexo enzimático de uma nova linhagem de Clostridium thermocellum com enfase em sua atividade de xilanase

A new bacterial strain (ISO II) was isolated from manure cow and identified as phylogenetically close to the thermophilic cellulolytic bacterium Clostridium thermocellum. The new strain produced extracellular xylanase, pectinase, mannanase and cellulase activities when grown in liquid culture medium containing banana stem as carbon source. The enzyme production profile after growth on banana stem showed that xylanase and cellulase activities were detected in different incubation periods. An enzyme complex containing xylanase, cellulase and mannanase activities was isolated from culture supernatant samples of strainISO II. The complex was partially purified by ultrafiltration and gel filtration chromatography on Sephacryl S-300. Zymogram analysis after SDS-PAGE presented at least 05 subunits with xylanase activity. The enzyme showed single protein and xylanase activity bands after electrophoresis under non-denaturing conditions. The hydrolysis of xylan was optimal at temperature range of 55-75ºC and pH 6.0. Xylanase activity was quite stable at 65ºC, retaining 80% of its original activity after 12 h incubation. The apparent Km values, using insoluble and soluble arabinoxylans as substrates, were 1.54 and 11.53 mg/mL, respectively. Xylanase was activated by dithiothreitol, L-tryptophan and L-cysteine and strongly inhibited by N-bromosuccinimide and CoCl2. The characterization of mannanase showed Km and temperature optimum of 0.846 mg/mL and 65ºC, respectively and pH 8.0. By contrast to xylanase, it was less stable at 65ºC with half-life of 2.5 h and inhibited by dithiothreitol and Ca2+.<br>Uma nova linhagem de bactéria (ISO II) foi isolada de esterco bovino e identificada como filogeneticamente próxima à bactéria termofílica Clostridium thermocellum. A nova linhagem produziu atividades de xilanase, mananase, pectinase e celulase quando cultivada em meio de cultura líquido contendo engaço de bananeira como fonte de carbono. O perfil de produção enzimática após crescimento em engaço de bananeira mostrou que as atividades de xilanase e celulase foram detectadas em diferentes períodos de incubação. Um complexo enzimático, contendo atividades de xilanase, celulase e mananase, foi isolado de amostras de sobrenadante do meio de cultura da linhagem ISO II crescida em engaço de bananeira. O complexo foi parcialmente purificado por ultrafiltração e cromatografia de filtração em gel em coluna de Sephacryl S-300. Análise de zimograma mostrou 05 sub-unidades com atividade de xilanase. A amostra enzimática apresentou bandas únicas de proteína e atividade de xilanase após eletroforese sob condições não-desnaturantes. A hidrólise de xilana foi ótima no intervalo de temperatura de 55-75ºC e pH 6,0. A xilanase foi estável a 65ºC, mantendo 80% de sua atividade original após 12 h de incubação. Os valores de Km aparente, usando arabinoxilanas insolúveis e solúveis como substratos, foram 1,54 and 11,53 mg/mL, respectivamente. A xilanase foi ativada por ditiotreitol, L-triptofano and L-cisteina e fortemente inibida por N-bromosuccinamida e CoCl2. A caracterização da mananase do complexo mostrou Km e temperatura ótima de 0,846 mg/mL e 65ºC, respectivamente e pH 8,0. Ao contrário da xilanase, a mananase foi menos estável a 65ºC com meia vida de 2,5 h e inibida por ditiotreitol e Ca2+

The enzyme interactome concept in filamentous fungi linked to biomass valorization

Biomass represents an abundant and inexpensive source of sugars and aromatic compounds that can be used as raw materials for conversion into value-added bioproducts. Filamentous fungi are sources of plant cell wall degrading enzymes in nature. Understanding the interactions between enzymes is crucial for optimizing biomass degradation processes. Herein, the concept of the interactome is presented as a holistic approach that depicts the interactions among enzymes, substrates, metabolites, and inhibitors. The interactome encompasses several stages of biomass degradation, starting with the sensing of the substrate and the subsequent synthesis of hydrolytic and oxidative enzymes (fungus-substrate interaction). Enzyme-enzyme interactions are exemplified in the complex processes of lignocellulosic biomass degradation. The enzyme-substrate-metabolite-inhibitor interaction also provides a better understanding of biomass conversion, allowing bioproduct production from recalcitrant agro-industrial residues, thus bringing greater value to residual biomass. Finally, technological applications are presented for optimizing the interactome at various levels

Use of Residual Biomass from the Textile Industry as Carbon Source for Production of a Low-Molecular-Weight Xylanase from Aspergillus oryzae

Pretreated dirty cotton residue (PDCR) from the textile industry was used as an alternative carbon source for the submerged cultivation of Aspergillus oryzae and the production of xylanases. The filtered culture supernatant was fractionated by ultrafiltration followed by three chromatographic steps, which resulted in the isolation of a homogeneous low-molecular-weight xylanase (Xyl-O1) with a mass of 21.5 kDa as determined by sodium dodecyl sulfate-polyacrilamide gel electrophoresis (SDS-PAGE) co-polymerized with 0.1% oat spelt xylan. Enzyme catalysis was the most efficient at 50 °C and pH 6.0. The Km values (mg·mL−1) for the soluble fraction of oat spelt and birchwood xylans were 10.05 and 3.34, respectively. Xyl-O1 was more stable in the presence of 5,5-dithio-bis-(2-nitrobenzoic acid) (DTNB), 1,4-dithiothreitol (DTT), l-cysteine or β-mercaptoethanol, which increased the rate of catalysis by 40%, 14%, 40% or 37%, respectively. The enzyme stability was improved at pH 7.0 in the presence of 20 mM l-cysteine, with the retention of nearly 100% of the activity after 6 h at 50 °C. Xyl-O1 catalyzed the cleavage of internal β-1,4 linkages of the soluble substrates containing d-xylose residues, with a maximum efficiency of 33% for the hydrolysis of birchwood xylan after 12 h of incubation. Identification of the hydrolysis products by high-performance anion exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD) indicated the predominance of the hydrolysis products X2-X6 during the first 12 h of incubation and the accumulation of higher xylooligomers after the elution of the last xylooligomer standard, xylohexaose