Isolamento de fungo produtor de enzimas xilanolíticas: produção e caracterização de xilanase
Carregando...
Data
2009-06-26
Autores
Benedetti, Ana Cláudia Elias Pião [UNESP]
Título da Revista
ISSN da Revista
Título de Volume
Editor
Universidade Estadual Paulista (Unesp)
Resumo
A descoberta de resíduos agrícolas, como fonte de energia renovável tem colaborado para o desenvolvimento industrial e preservação do meio ambiente. Desta maneira, a degradação da parede celular destes resíduos tem grande importância na fabricação de pães, alimentos, bebidas, têxtil, papel e celulose entre outros produtos. A degradação enzimática destes polímeros esta se tornando uma alternativa mais atraente do que a utilização de substâncias químicas e processos mecânicos. Alguns fungos termófilos participam desta degradação enzimática, como Humicola grisea var. thermoidea, isolado do solo, é conhecido como bom produtor do complexo xilanolítico. Além deste, um fungo termófilo isolado da polpa do fruto cupuaçuzeiro em decomposição foi estudado e produziu xilanase. Esses fungos foram mantidos em meios sólidos contendo ágar e farinha de aveia. O fungo isolado da polpa do fruto cresceu em meio liquido empregando diferentes resíduos agrícolas como fonte de carbono. Dentre os vários resíduos empregados para se otimizar a produção de xilanase, o melhor foi o sabugo de milho. Esse foi utilizado nos experimentos na proporção de 1,0% (m/v), em meio líquido contendo 0,1% CaCO3, 0,5% NaCl, 0,1% NH4Cl; 0,5% água de maceração de milho. O pH ótimo foi 5,0 e a temperatura ótima de 60ºC. A xilanase presente no extrato clarificado em caulim apresentou cinética Michaeliana com Km de 10,41 ± 0,282 mgmL-1 e Vmax 3,32 ± 0,053 U (mg protein)-1. Para acompanhar o processo de clarificação da enzima solúvel realizou-se SDS-PAGE verificando a presença de uma banda protéica com atividade xilanolítica de massa molecular de aproximadamente 30kDa e a Cromatografia Sílica Gel P60 para verificar os produtos da hidrólise da xilana em função do tempo e da associação de xilosidase do fungo termófilo Humicola com a xilanase do fungo isolado do fruto...
The researches of agricultural residues as renewable energy sources have been contributing for the industrial development and the environment conservation. Thus, the degradation of these residues’ cell walls has great importance in fabrication of bread, food, drinks, textile, paper, cellulose, and other products. The enzymatic degradation of such polymers is becoming an attractive option when compared to the use of chemical substances and mechanical processes. Some thermophilic fungi take role in this enzymatic degradation, such as Humicola grisea var. thermoidea, isolated from soil, and is known to be a good producer of the xylanolytic complex. Besides it, another thermophilic fungus, isolated from the Theobroma grandiflorum decomposing fruit pulp, was studied and also produced xylanases. These fungi were kept in solid media containing agar and oat bran. The fungus isolated from the fruit pulp grew in liquid media in wich different kinds of agricultural residues were employed as carbon sources. Among the various types of residues that were used to optimize the xylanase production, corn cobs proved to be the best one. It was used in assays in a proportion of 1,0% (m/v), in liquid media containing 0.1% of CaCO3, 0.5% of NaCl, 0.1% of NH4Cl, and 0.5% of corn steep liquor. The pH optima was 5.0 and the temperature optima was 60ºC. The xylanase present in the kaolin clarified extract showed a Km of 10.41 ± 0.282 mg.mL-1, and Vmax 3.32 ± 0.053 U (mg.protein)-1. In order to follow the soluble enzyme clarification process, SDS-PAGE was run, verifying the presence of a proteic band with xylanolytic activity of, approximately, 30kDa of molecular mass. Furthermore, Silica Gel 60 Thin Layer Chromatography (TLC) was used to verify the xylan hydrolysis products in function of time and the association of Humicola sp. thermophylic fungus xylosidase with the xylanase produced by the fruit pulp... (Complete abstract click electronic access below)
The researches of agricultural residues as renewable energy sources have been contributing for the industrial development and the environment conservation. Thus, the degradation of these residues’ cell walls has great importance in fabrication of bread, food, drinks, textile, paper, cellulose, and other products. The enzymatic degradation of such polymers is becoming an attractive option when compared to the use of chemical substances and mechanical processes. Some thermophilic fungi take role in this enzymatic degradation, such as Humicola grisea var. thermoidea, isolated from soil, and is known to be a good producer of the xylanolytic complex. Besides it, another thermophilic fungus, isolated from the Theobroma grandiflorum decomposing fruit pulp, was studied and also produced xylanases. These fungi were kept in solid media containing agar and oat bran. The fungus isolated from the fruit pulp grew in liquid media in wich different kinds of agricultural residues were employed as carbon sources. Among the various types of residues that were used to optimize the xylanase production, corn cobs proved to be the best one. It was used in assays in a proportion of 1,0% (m/v), in liquid media containing 0.1% of CaCO3, 0.5% of NaCl, 0.1% of NH4Cl, and 0.5% of corn steep liquor. The pH optima was 5.0 and the temperature optima was 60ºC. The xylanase present in the kaolin clarified extract showed a Km of 10.41 ± 0.282 mg.mL-1, and Vmax 3.32 ± 0.053 U (mg.protein)-1. In order to follow the soluble enzyme clarification process, SDS-PAGE was run, verifying the presence of a proteic band with xylanolytic activity of, approximately, 30kDa of molecular mass. Furthermore, Silica Gel 60 Thin Layer Chromatography (TLC) was used to verify the xylan hydrolysis products in function of time and the association of Humicola sp. thermophylic fungus xylosidase with the xylanase produced by the fruit pulp... (Complete abstract click electronic access below)
Descrição
Palavras-chave
Xilanases, Residuos agricolas, Caracterização física-cinética da enzima, Fungo termófilo, Xylanase, Thermophilic fungus, Xylanolytic complex
Como citar
BENEDETTI, Ana Cláudia Elias Pião. Isolamento de fungo produtor de enzimas xilanolíticas: produção e caracterização de xilanase. 2009. 108 f. Tese (doutorado) - Universidade Estadual Paulista, Faculdade de Ciências Farmacêuticas, 2009.