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Master's Dissertation
DOI
10.11606/D.97.2013.tde-02122013-170850
Document
Author
Full name
Fernanda de Lima Valadares
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
Lorena, 2013
Supervisor
Committee
Ferraz, André Luís (President)
Milagres, Adriane Maria Ferreira
Squina, Fabio Marcio
Title in Portuguese
Produção e uso de enzimas derivadas do fungo Pleurotus ostreatus na hidrólise de bagaço de cana pré-tratado por processo quimiotermomecânico
Keywords in Portuguese
Bagaço
Cana de açúcar
Celulases
Hidrólise enzimática
Pleurotus ostreatus
Pré-tratamento
Abstract in Portuguese
Fungos de decomposição branca atuam eficientemente na biodegradação de substratos altamente lignificados, como a madeira. Tal característica permite supor que esses organismos apresentem um sistema celulolítico com atividade diferenciada em substratos ricos em lignina. O presente trabalho avaliou o efeito da adição de enzimas derivadas do fungo de decomposição branca Pleurotus ostreatus em preparações de celulases comerciais durante a hidrólise enzimática do bagaço de cana previamente submetido a tratamento quimiotermomecânico com sulfito alcalino. Duas cargas de sulfito alcalino foram empregadas nos pré-tratamentos: uma mais elevada de 10 g de Na2SO3 e 5 g de NaOH para cada 100g de bagaço, que gerou um substrato de baixa recalcitrância; e uma carga diminuída à metade da anterior, que originou um substrato de elevada recalcitrância. Primeiramente, a produção de endoglucanases (EG) em cultivos submersos de P.ostreatus foi avaliada em diferentes fontes de carbono, sendo a maior produção de EG (342 UI L-1) verificada após 20 dias de cultivo em meio contendo bagaço de cana moído e carboximetilcelulose (CMC). Contudo, devido a CMC ser considerada um interferente nos ensaios de hidrólise do bagaço, optou-se por utilizar enzimas derivadas dos cultivos que empregaram somente bagaço de cana como fonte de carbono. Os experimentos de hidrólise empregaram cargas de enzimas correspondentes a 10FPU (carga alta) e 5FPU (carga média) de celulases derivadas de Trichoderma reesei ATCC 26921, misturadas com uma carga de 15 UI.g-1 de ?-glicosidase (BGL) derivadas de Aspergillus niger, para cada grama de bagaço. Para os experimentos de hidrólise que empregaram enzimas derivadas de P. ostreatus ajustou-se a carga de endoglucanase para que 50% da atividade fosse derivada de T. reesei, e 50% proveniente de P. ostreatus. A suplementação com enzimas de P. ostreatus causou uma alteração no teor das demais enzimas hidrolíticas, verificando-se valores de atividades de xilanases e celulases, com exceção das celobiohidrolases, superiores aos observados com o emprego da carga alta de enzimas comerciais. A conversão da celulose obtida durante a hidrólise dos bagaços pré-tratados mostraram que as enzimas de P. ostreatus proporcionaram valores de velocidade inicial de hidrólise equivalentes aos obtidos nos ensaios com carga alta de enzimas comerciais. Esse resultado foi atingido mesmo com uma carga de celobiohidrolases duas vezes inferior a existente nos ensaios com alta carga de enzimas comerciais, o que levou a considerar que as enzimas derivadas de P. ostreatus possam apresentar atividade celulolítica diferenciada. Além disso, o maior teor de enzimas xilanolíticas nos extratos de P. ostreatus resultou em maiores valores de conversão da xilana. A maior remoção de xilana também pode ter favorecido a maior conversão de celulose obtida mesmo com baixa carga de celobiohidrolases nas misturas reacionais, visto que a remoção da xilana associada à celulose aumentaria a disponibilidade do substrato às celulases. Contudo, a conversão de celulose a partir de 8-24h de hidrólise suplementada com enzimas de P. ostreatus foi ligeiramente inferior ao obtido na hidrólise com carga alta de celulases de T. reesei.
Title in English
Production and use of enzymes derived from the fungus Pleurotus ostreatus in the hydrolysis of sugarcane bagasse pretreated by chemithermomechanical process
Keywords in English
Cellulases
Enzymatic hydrolysis
Pleurotus ostreatus
Pretreatment
Sugarcane bagasse
Abstract in English
White-rot fungi are able to degrade highly lignified substrates, such as wood. This characteristic allows us to assume that these organisms possess a cellulolytic system with differentiated activity on lignin-rich substrates. This study evaluates how cellulolytic enzymes produced by the white-rot fungus Pleurotus ostreatus perform in the hydrolysis of pretreated sugarcane bagasse. The sugar cane bagasse was initially pretreated with two chemical loadings of alkaline sulphite: 10 g of Na2SO3 and 5 g of NaOH per 100g of pulp (high chemical load), generating a substrate with low recalcitrance; and a load decreased to half of the previous one, which gave a more recalcitrant substrate. The production of endoglucanases (EG) in submerged cultures of P.ostreatus was evaluated using different carbon sources in the culture media. The highest EG production (342 IU L-1) was observed after fungal growth for 20 days in the culture medium that contained sugarcane bagasse and carboxymethylcellulose (CMC) as carbon sources. However, residual CMC present in the culture extracts was considered to interfere in subsequent hydrolysis assays and we decided to use enzymes derived from the cultures that used only sugarcane bagasse as carbon source. The reference hydrolysis experiments were performed with enzyme loadings of 10 FPU (high loading) and 5 FPU (medium loading) from cellulases derived from Trichoderma reesei ATCC 26921 mixed with 15 UI of ?-glucosidase (BGL) from Aspergillus niger (enzyme loadings expressed in units per gram of pretreated bagasse). For the hydrolysis experiments that used enzymes from P. ostreatus, the enzyme loading was adjusted in order to have 50% of original endoglucanase activity from T. reesei enzymes replaced by enzymes from P. ostreatus enzymes. The addition of P. ostreatus enzymes caused a change in the overall levels of hydrolytic enzymes present in the reaction medium. Xylanase and beta-glucosidase activities were higher than those observed in the commercial enzymes mixture. However, the cellobiohydrolase levels were the half of the original values from the commercial enzymes. The cellulose conversion during the hydrolysis of pretreated bagasses showed that the enzymes from P. ostreatus provided initial hydrolysis rate values similar to those obtained in tests with the high loading of commercial enzymes. This result was achieved even with a cellobiohydrolase loading twice lower than in the assays with high loading of commercial enzymes, which led to the conclusion that the enzymes derived from P. ostreatus can show differentiated cellulolytic activity. In addition, the higher content of xylanolytic enzymes in P. ostreatus extracts resulted in higher xylan conversion. The higher removal of xylan may have also resulted in the higher conversion of cellulose, even with low cellobiohydrolases in the reaction mixtures, since removal of xylan increases the accessibility of the cellulases to the substrate. However, the cellulose conversion after 8-24h hydrolysis supplemented with enzymes from P. ostreatus was slightly lower than that obtained in the hydrolysis with high loading of cellulases from T. reesei.
 
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Publishing Date
2013-12-02
 
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