Produção de β-D-frutofuranosidases pelo fungo filamentoso Aspergillus niveus através de fermentação em estado sólido, purificação e caracterização bioquímica
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Data
2016-07-26
Autores
Fernandes, Maysa Lima Parente [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
As invertases ou β-D-frutofuranosidases (EC 3.2.1.26) são responsáveis pela produção da mistura equimolar de glicose e frutose, conhecida como açúcar invertido, através da hidrólise da ligação β 2-1 da molécula de sacarose. Essa mistura apresenta sabor mais doce que a sacarose e é destinada à diversos fins na indústria alimentícia (doces, xaropes, leite condensado e bebidas). É importante destacar que o produto obtido por hidrólise enzimática é bastante superior ao obtido por hidrólise ácida. A hidrólise catalisada pela enzima β-D-frutofuranosidase produz um xarope de alta qualidade com ausência de hidroximetilfurfural e sem desenvolvimento de cor. Diante do interesse industrial e biotecnológico, e das diversas aplicações dessa enzima, o objetivo deste trabalho foi investigar a produção de β-D-frutofuranosidase extracelular pelo fungo filamentoso Aspergillus niveus através de Fermentação em Estado Sólido (FES) purificando-a e caracterizando-a bioquimicamente. Entre os substratos testados em FES, a maior produção β-D-frutofuranosidásica foi obtida em casca de mandioca, com granulometria de 10 mesh, umidificada na proporção de 1:1 com água de torneira, a uma temperatura de 30ºC com umidade relativa de 30%, por um período de 9 dias de incubação. A β-D-frutofuranosidase extracelular obtida em FES foi purificada 6,53 vezes com recuperação de 5,27%, obtendo-se em SDS-PAGE 8% uma única banda protéica (37 kDa), e massa molecular nativa de 91,2 kDa estimada por Sepharose CL-6B. A temperatura e pH ótimos de atividade foram 55ºC e 4,5, respectivamente. A enzima apresentou boa estabilidade em 50ºC e 60ºC por uma hora, e em ampla faixa de pH com atividade residual de 80% em pH 8,0 por uma hora. A atividade β-D-frutofuranosidásica foi ativada principalmente na presença de Mn2+. A enzima foi resistente a maioria dos sais e solventes testados. Os valores de Km e Vmax utilizando a sacarose como substrato foram 22,98 mM e 120,48 U/mg de proteína, respectivamente. Na presença de Mn2+ os valores foram 16,31 mM e 0,30 U/mg de proteína, respectivamente. A enzima não foi capaz de hidrolisar a inulina, caracterizando uma β-D-frutofuranosidase verdadeira, e apenas glicose e frutose foram observadas como produtos de hidrólise da sacarose em alta concentração; não foi observada a produção de frutooligossacarídeos (FOS).
The invertases or β-D-fructofuranosidases (EC 3.2.1.26) are responsible for the production of an equimolar mixture of glucose and fructose, known as invert sugar, by hydrolysis of β 2-1 bond of the sucrose molecule. This mixture is sweeter than sucrose and is intended for various purposes in the food industry (sweets, syrup, condensed milk and drinks). It is important to note that the product obtained by enzymatic hydrolysis is much higher than that obtained by acid hydrolysis. The hydrolysis catalyzed by β-D-fructofuranosidase enzyme produces a high-quality syrup without hydroxymethylfurfural and color development. Before the industrial and biotechnological interest, and the various applications of this enzyme, the aim of this study was to investigate the production of the extracellular β-D-fructofuranosidase by filamentous fungus Aspergillus niveus under Solid Fermentation State (SSF) purifying it and characterizing it biochemically. Among the substrates tested in the SSF, the highest yield of β-D-fructofuranosidase was obtained in cassava hulls with a particle size of 10 mesh, humidified in the ratio 1:1 with tap water and maintained at 30°C with a relative humidity of 30%, for 9 days of incubation. The extracellular β-Dfructofuranosidase obtained in SSF was purified 6.53-folds with a recovery of 5.27%. A single protein band (37 kDa) was obtained in 8% SDS-PAGE and the native molecular mass was estimated as 91.2 kDa by Sepharose CL-6B. The optimum temperature and pH for activity was 55°C and 4.5, respectively. The enzyme showed good stability at 50°C and 60°C for one hour and 80% of activity was maintained at pH 8.0 for one hour. The β-D-fructofuranosidase activity was primarily activated by Mn2+ . In addition, the enzyme was resistant to most solvents and salts tested. The Km and Vmax values using sucrose as substrate were 22.98 mM and 120.48 U/mg, respectively. In the presence of Mn2+ the values were 16.31 mM and 0.30 U/mg respectively. The enzyme was not able to hydrolize inulin, characterizing a true invertase, and only glucose and fructose were observed as hydrolysis products at high sucrose concentration; production of fructooligosaccharides (FOS) was not observed.
The invertases or β-D-fructofuranosidases (EC 3.2.1.26) are responsible for the production of an equimolar mixture of glucose and fructose, known as invert sugar, by hydrolysis of β 2-1 bond of the sucrose molecule. This mixture is sweeter than sucrose and is intended for various purposes in the food industry (sweets, syrup, condensed milk and drinks). It is important to note that the product obtained by enzymatic hydrolysis is much higher than that obtained by acid hydrolysis. The hydrolysis catalyzed by β-D-fructofuranosidase enzyme produces a high-quality syrup without hydroxymethylfurfural and color development. Before the industrial and biotechnological interest, and the various applications of this enzyme, the aim of this study was to investigate the production of the extracellular β-D-fructofuranosidase by filamentous fungus Aspergillus niveus under Solid Fermentation State (SSF) purifying it and characterizing it biochemically. Among the substrates tested in the SSF, the highest yield of β-D-fructofuranosidase was obtained in cassava hulls with a particle size of 10 mesh, humidified in the ratio 1:1 with tap water and maintained at 30°C with a relative humidity of 30%, for 9 days of incubation. The extracellular β-Dfructofuranosidase obtained in SSF was purified 6.53-folds with a recovery of 5.27%. A single protein band (37 kDa) was obtained in 8% SDS-PAGE and the native molecular mass was estimated as 91.2 kDa by Sepharose CL-6B. The optimum temperature and pH for activity was 55°C and 4.5, respectively. The enzyme showed good stability at 50°C and 60°C for one hour and 80% of activity was maintained at pH 8.0 for one hour. The β-D-fructofuranosidase activity was primarily activated by Mn2+ . In addition, the enzyme was resistant to most solvents and salts tested. The Km and Vmax values using sucrose as substrate were 22.98 mM and 120.48 U/mg, respectively. In the presence of Mn2+ the values were 16.31 mM and 0.30 U/mg respectively. The enzyme was not able to hydrolize inulin, characterizing a true invertase, and only glucose and fructose were observed as hydrolysis products at high sucrose concentration; production of fructooligosaccharides (FOS) was not observed.
Descrição
Palavras-chave
β-D-frutofuranosidases, Aspergillus niveus, Fermentação em estado sólido, β-D-fructofuranosidase, Solid-state fermentation