Produção, purificação, caracterização bioquímica e determinação do padrão de ação de protease do fungo termofílico Thermoascus aurantiacus
Carregando...
Data
2007-02-23
Autores
Merheb, Carolina Wingeter [UNESP]
Título da Revista
ISSN da Revista
Título de Volume
Editor
Universidade Estadual Paulista (Unesp)
Resumo
Proteases constituem um dos grupos mais importantes de enzimas industriais, sendo o setor alimentício um dos que mais as utilizam, pois atuam sobre proteínas. Este trabalho teve como objetivo produzir, purificar e caracterizar a protease do fungo termofílico Thermoascus aurantiacus. A produção foi em fermentação em estado sólido e o extrato enzimático bruto foi caracterizado. A atividade de protease, no extrato enzimático bruto, foi inibida por ácido etilenodiaminotetracético (EDTA) (60,61%) e por fluoreto fenimetilsulfonil (PMSF) (56,37%) e foi levemente estimulada por Ca2+. Seu padrão de ação hidrolítico sobre a caseína foi estudado por UREA-PAGE, mostrando diferença significativa em relação a uma renina microbiana recombinante. O extrato enzimático bruto foi purificado através de precipitação com álcool a 72% seguido de cromatografias em resinas Sephadex G75 e Sephacryl S100. Apresentou rendimento de 0,4% e um fator de purificação de 80 vezes. A massa molecular de 24,5 KDa foi estimada por SDS-PAGE. A protease pura apresentou inibição somente por EDTA (96,70%) e ativação por sulfato ferroso, revelando-se uma metaloprotease ativada por ferro. O pH ótimo foi 5,5; a temperatura ótima foi 75°C e foi termoestável a 65°C por 1 hora retendo mais de 70% de atividade original. Apresentou aumento de 151,0% de atividade na presença de Tween-20 e, através dos estudos de cinética enzimática, hidrolisou melhor a caseína do que a azocaseína.
Proteases are a very important group of industrial enzymes, and are most employed in the food segment due to their action on proteins. This work had the aim to produce, purify and characterize the protease from the thermophilic Thermoascus aurantiacus. The production was carried out in solid state fermentation and the crude enzymatic extract was characterized. The proteolytic activity, in the crude extract, was inhibited by ethylenediaminetetraacetic acid (EDTA) (60.61%) and by phenylmethylsulphonyl fluoride (PMSF) (56.37%) and was slightly stimulated by Ca2+. Its hydrolytic action profile on casein was studied by UREA-PAGE, showing significant difference when compared to a recombinant microbial rennet. The crude enzymatic extract was purified through precipitation with ethanol at 72% followed by cromatographies in columns of Sephadex G75 and Sephacryl S100. It was purified 80-fold and exhibited recovery of total activity of 0.4%. SDS-PAGE analysis indicated an estimated molecular mass of 24.5 KDa. The purified protease was only inhibited by EDTA (96.70%) and stimulated by Fe2+ revealing to be a metalloprotease activated by iron. The optimum pH was 5.5, optimum temperature was 75°C and it was thermostable at 65°C for 1 hour maintaining more then 70% of original activity. It showed increase of 151.0% in activity in the presence of Tween-20 and, through enzyme kinetics studies, better hydrolyzed casein than azocasein.
Proteases are a very important group of industrial enzymes, and are most employed in the food segment due to their action on proteins. This work had the aim to produce, purify and characterize the protease from the thermophilic Thermoascus aurantiacus. The production was carried out in solid state fermentation and the crude enzymatic extract was characterized. The proteolytic activity, in the crude extract, was inhibited by ethylenediaminetetraacetic acid (EDTA) (60.61%) and by phenylmethylsulphonyl fluoride (PMSF) (56.37%) and was slightly stimulated by Ca2+. Its hydrolytic action profile on casein was studied by UREA-PAGE, showing significant difference when compared to a recombinant microbial rennet. The crude enzymatic extract was purified through precipitation with ethanol at 72% followed by cromatographies in columns of Sephadex G75 and Sephacryl S100. It was purified 80-fold and exhibited recovery of total activity of 0.4%. SDS-PAGE analysis indicated an estimated molecular mass of 24.5 KDa. The purified protease was only inhibited by EDTA (96.70%) and stimulated by Fe2+ revealing to be a metalloprotease activated by iron. The optimum pH was 5.5, optimum temperature was 75°C and it was thermostable at 65°C for 1 hour maintaining more then 70% of original activity. It showed increase of 151.0% in activity in the presence of Tween-20 and, through enzyme kinetics studies, better hydrolyzed casein than azocasein.
Descrição
Palavras-chave
Microbiologia industrial, Enzimas de fungos - Aplicações industriais, Enzimas proteoliticas - Aplicações industriais, Protease - Purificação, Protease - Caracterização bioquímica
Como citar
MERHEB, Carolina Wingeter. Produção, purificação, caracterização bioquímica e determinação do padrão de ação de protease do fungo termofílico Thermoascus aurantiacus. 2007. 99 f. Dissertação (mestrado) - Universidade Estadual Paulista, Instituto de Biociências, Letras e Ciências Exatas, 2007.