Produção de tanases por biofilmes de Aspergillus ochraceus e potencial de aplicação
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Data
2019-02-22
Autores
Aracri, Fernanda Mansano
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Universidade Estadual Paulista (Unesp)
Resumo
A utilização de biofilmes como forma inovadora para cultivar fungos filamentosos tem sido cada vez mais empregada na produção de enzimas. Quando o fungo se desenvolve aderido a uma superfície, formando então o biofilme, seu metabolismo é amplificado e consequentemente sua secreção enzimática. Deste modo, a fermentação por biofilme (FB) torna-se uma alternativa atrativa para a produção de enzimas, que são um dos principais produtos de interesse biotecnológico. Dentre as enzimas produzidas por fungos filamentosos, as tanases se destacam devido sua ampla gama de aplicações industriais, como na indústria de alimentos, bebidas, ração animal, farmacêutica e química. Estas enzimas atuam na hidrólise de ligações éster de taninos hidrolisáveis, como o ácido tânico, liberando glicose e ácido gálico, o qual é intermediário na síntese de compostos como o propil-galato (utilizado como aditivo na indústria de alimento), pirogalol (conservante) e trimetropina (antibacteriano). Desta forma, esta pesquisa visou a produção de tanases por biofilmes de Aspergillus ochraceus desenvolvidos em suporte inerte de polietileno. Em FB o fungo foi cultivado em condições ótimas em meio Khanna tendo 1,5% (m/v) de ácido tânico como fonte de carbono e 0,1% (m/v) de extrato de levedura como fonte adicional de nitrogênio, por 72 horas a 28ºC, 50 rpm e pH inicial 5,0. A tanase de A. ochraceus apresentou temperatura ótima aparente de 30ºC e pH ótimo aparente de 6,0. Mostrou também boa estabilidade na faixa de 30º-40ºC, apresentando ainda 50% de sua atividade inicial nos tempos de 100 e 180 minutos, respectivamente. Os valores de pH de 5,0-6,0 foram os mais estáveis. A atividade enzimática foi aumentada na presença de ureia, MnCl2 e NH4Cl, e inibida na presença de íons Ag+ e Ba2+ . A aplicação do extrato bruto para tratamento de efluentes de curtume do couro de bode reduziu em 83% o teor de taninos presentes após 2 horas de incubação. Também reduziu em 62% o teor de taninos presentes na farinha de sorgo após 5 dias de tratamento. A enzima contida no extrato foi capaz de produzir propil-galato, sendo a maior produção obtida em 96 horas de reação. Os biofilmes foram ainda reutilizados e um aumento de 20,5% e 30,5% foram observados na atividade enzimática, respectivamente, na primeira e segunda reutilização. Foi realizada a microscopia eletrônica de varredura dos biofilmes (MEV), na qual a formação de canais foi observada, o que permite a troca eficiente de nutrientes entre o biofilme fúngico e o ambiente. Deste modo, o fungo A. ochraceus em FB apresentou bom potencial na produção de tanase com propriedades importantes para aplicação industrial.
The use of biofilms as an innovative way to grow filamentous fungi has been increasingly employed in the production of enzymes. When the fungus develops adhered to a support, then forming the biofilm, its metabolism is amplified and consequently its enzymatic secretion. In this way, biofilm fermentation (BF) becomes an attractive alternative for the production of enzymes, which are one of the main products of biotechnological interest. Among the enzymes produced by filamentous fungi, the tannases stand out due to their wide range of industrial applications, such as the food, beverage, animal feed, pharmaceutical and chemical industries. These enzymes act on the hydrolysis of ester bonds of hydrolysable tannins, such as tannic acid, releasing glucose and gallic acid, which is intermediate in the synthesis of compounds such as propyl gallate (used as an additive in the food industry), pyrogallol (preservative) and trimethoprim (antibacterial). Thus, this research aimed at the production of tanases by Aspergillus ochraceus biofilms developed in inert polyethylene support. In BF the fungus was cultivated under optimum conditions in Khanna medium containing 1.5% (w/v) tannic acid as a carbon source, 0.1% (w/v) yeast extract as an additional source of nitrogen, for 72 hours at 28° C, 50 rpm and initial pH of 5.0. The A. ochraceus tannase had an optimum apparent temperature of 30ºC and an optimum apparent pH of 6.0. It also showed good stability in the 30-40 range presenting as well 50% of its activity on 100 and 180 minutes, respectively. The pH values of 5-6 were the most stable. The enzymatic activity was increased in the presence of urea, MnCl2 and NH4Cl, and inhibited in the presence of Ag+ and Ba2+ ions. The application of the crude extract to treat tannery effluents from goat leather reduced the content of tannins present after 2 hours of incubation by 83%. Also reduced by 62% the content of tannins present in sorghum flour after 5 days of treatment. The enzyme contained in the extract was also capable of producing propyl gallate, and the highest yield was obtained in 96 hours of reaction. Biofilms were also reused and an increase of 20.5% and 30.5% were observed in the enzymatic activity, in the first and second reuse respectively. Scanning electron microscopy of biofilms was performed, in which channel formation was observed, which allows the efficient exchange of nutrients between the fungi biofilm and the environment. Finally, the fungus A. ochraceus in BF presented good potential in the production of tanase with important properties for industrial application.
The use of biofilms as an innovative way to grow filamentous fungi has been increasingly employed in the production of enzymes. When the fungus develops adhered to a support, then forming the biofilm, its metabolism is amplified and consequently its enzymatic secretion. In this way, biofilm fermentation (BF) becomes an attractive alternative for the production of enzymes, which are one of the main products of biotechnological interest. Among the enzymes produced by filamentous fungi, the tannases stand out due to their wide range of industrial applications, such as the food, beverage, animal feed, pharmaceutical and chemical industries. These enzymes act on the hydrolysis of ester bonds of hydrolysable tannins, such as tannic acid, releasing glucose and gallic acid, which is intermediate in the synthesis of compounds such as propyl gallate (used as an additive in the food industry), pyrogallol (preservative) and trimethoprim (antibacterial). Thus, this research aimed at the production of tanases by Aspergillus ochraceus biofilms developed in inert polyethylene support. In BF the fungus was cultivated under optimum conditions in Khanna medium containing 1.5% (w/v) tannic acid as a carbon source, 0.1% (w/v) yeast extract as an additional source of nitrogen, for 72 hours at 28° C, 50 rpm and initial pH of 5.0. The A. ochraceus tannase had an optimum apparent temperature of 30ºC and an optimum apparent pH of 6.0. It also showed good stability in the 30-40 range presenting as well 50% of its activity on 100 and 180 minutes, respectively. The pH values of 5-6 were the most stable. The enzymatic activity was increased in the presence of urea, MnCl2 and NH4Cl, and inhibited in the presence of Ag+ and Ba2+ ions. The application of the crude extract to treat tannery effluents from goat leather reduced the content of tannins present after 2 hours of incubation by 83%. Also reduced by 62% the content of tannins present in sorghum flour after 5 days of treatment. The enzyme contained in the extract was also capable of producing propyl gallate, and the highest yield was obtained in 96 hours of reaction. Biofilms were also reused and an increase of 20.5% and 30.5% were observed in the enzymatic activity, in the first and second reuse respectively. Scanning electron microscopy of biofilms was performed, in which channel formation was observed, which allows the efficient exchange of nutrients between the fungi biofilm and the environment. Finally, the fungus A. ochraceus in BF presented good potential in the production of tanase with important properties for industrial application.
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Palavras-chave
Aspergillus ochraceus, Efluente, Fermentação por biofilme, Propil-galato, Tanino acil hidrolase