Produção de β-galactosidase por fungos filamentosos: screening, purificação e caracterização bioquímica
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Data
2020-02-28
Autores
Jesus, Luiz Felipe de Morais Costa de
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Universidade Estadual Paulista (Unesp)
Resumo
Conhecida por sua importância na indústria de alimentos e no tratamento de pessoas intolerantes a lactose, a enzima β-galactosidase (EC 3.2.1.23) promove a hidrólise da lactose nos monossacarídeos galactose e glicose. Algumas β-galactosidases ainda catalisam reações de transgalactosilação produzindo galactooligossarídeos (GOS), que são prebióticos. Diante disto, este estudo visou à seleção de uma linhagem de fungo filamentoso produtora de β-galactosidase, seguida da caracterização e aplicação da enzima no tratamento de efluente da indústria láctea. Entre as 10 linhagens de fungos filamentosos inicialmente estudadas, Trichoderma sp. foi o melhor produtor de β-galactosidase, sendo cultivado em Fermentação Submersa (FSbm) e Fermentação em Estado Sólido (FES). A maior produção de β-galactosidase por este fungo em FSbm (28 U/L) foi obtida em meio Nakao-lactose modificado adicionado de 3% (v/v) de soro de leite, pH inicial ajustado para 6, mantido sob agitação (120 rpm) por 312 h, a 30 °C. Quando utilizada FES a maior produção enzimática, 2,67 U/g de substrato, foi observada na presença de farelo de trigo umedecido com água de torneira (1:1,65; m/v), mantido a 30 ºC por 187 h. A β-galactosidase produzida em FSbm apresentou temperatura ótima de atividade de 55 °C e tempo de meia vida (T50) de 90 min de 35-45 °C. O pH ótimo aparente de atividade foi 4,2 e T50 de 180 min quando mantida em pH 4. A atividade enzimática foi aumentada na presença de Mn2+ e inibida na presença de íons ferro e de SDS. A β-galactosidase produzida em FES apresentou temperatura e pH ótimos de atividade de 55°C e 4,5, respectivamente. A atividade catalítica foi mantida por até 180 min quando incubada nas temperaturas de 35-45 °C, e acima de 50% quando mantida em pH ácidos e alcalinos por até 24 h. Ainda apresentou resistência a diversos compostos químicos. As β-galactosidases produzidas em FSbm e FES foram capazes de hidrolisar o soro de queijo oriundo da indústria láctea, reduzindo o teor de lactose. Além disto, a β-galactosidase produzida via FES catalisou reação de transgalactosilação, produzindo GOS. Deste modo, o fungo Trichoderma sp. mostrou-se como um interessante produtor de β-galactosidases com propriedades importantes para possíveis aplicações industriais.
The enzyme β-galactosidase (EC 3.2.1.23), known because its importance in the food industry and in the treatment of lactose intolerant people, promotes the hydrolysis of lactose in the monosaccharides galactose and glucose. Some βgalactosidases also catalyze transgalactosylation reactions producing galactooligosaccharides (GOS), which are prebiotics. In view of this, this study aimed the selection of a strain of filamentous fungus producing β-galactosidase, followed by characterization and application of the enzyme in the treatment of effluent from the dairy industry. Among the 10 strains of filamentous fungi initially studied, Trichoderma sp. was the best producer of β-galactosidase, being grown in Submerged Fermentation (SbmF) and Solid State Fermentation (SSF). The greatest production of β-galactosidase by this fungus in SbmF (28 U/L) was obtained in modified Nakao-lactose medium added with 3%(v/v) whey, initial pH adjusted to 6, kept under agitation (120 rpm) for 312 h at 30 °C. When SSF was used, the highest enzyme production, 2.67 U/g of substrate was observed in the presence of wheat bran moistened with tap water (1:1.65; w/v), maintained at 30 ºC for 187 h. The β-galactosidase produced in SbmF showed optimum activity at 55 °C and half-life (T50) of 90 min from 35-45 °C. The apparent optimum pH of activity was 4.2 and T50 of 180 min when maintained at pH 4. The enzymatic activity was increased by Mn2+ and inhibited by iron ions and SDS. The β-galactosidase produced in SSF had optima temperature and pH of activity at 55 °C and 4.5, respectively. The catalytic activity was maintained for up to 180 min when incubated from 35-45 °C, and above 50% when maintained at acidic and alkaline pH for up to 24 h. It also presented resistance to several chemical compounds. The β-galactosidases produced in SbmF and SSF were able to hydrolyze the cheese whey from the dairy industry, reducing its lactose content. In addition, β-galactosidase produced via FES catalyzed the transgalactosylation reaction, producing GOS. Thus, the fungus Trichoderma sp. was an interesting producer of β-galactosidases with important properties for possible industrial applications.
The enzyme β-galactosidase (EC 3.2.1.23), known because its importance in the food industry and in the treatment of lactose intolerant people, promotes the hydrolysis of lactose in the monosaccharides galactose and glucose. Some βgalactosidases also catalyze transgalactosylation reactions producing galactooligosaccharides (GOS), which are prebiotics. In view of this, this study aimed the selection of a strain of filamentous fungus producing β-galactosidase, followed by characterization and application of the enzyme in the treatment of effluent from the dairy industry. Among the 10 strains of filamentous fungi initially studied, Trichoderma sp. was the best producer of β-galactosidase, being grown in Submerged Fermentation (SbmF) and Solid State Fermentation (SSF). The greatest production of β-galactosidase by this fungus in SbmF (28 U/L) was obtained in modified Nakao-lactose medium added with 3%(v/v) whey, initial pH adjusted to 6, kept under agitation (120 rpm) for 312 h at 30 °C. When SSF was used, the highest enzyme production, 2.67 U/g of substrate was observed in the presence of wheat bran moistened with tap water (1:1.65; w/v), maintained at 30 ºC for 187 h. The β-galactosidase produced in SbmF showed optimum activity at 55 °C and half-life (T50) of 90 min from 35-45 °C. The apparent optimum pH of activity was 4.2 and T50 of 180 min when maintained at pH 4. The enzymatic activity was increased by Mn2+ and inhibited by iron ions and SDS. The β-galactosidase produced in SSF had optima temperature and pH of activity at 55 °C and 4.5, respectively. The catalytic activity was maintained for up to 180 min when incubated from 35-45 °C, and above 50% when maintained at acidic and alkaline pH for up to 24 h. It also presented resistance to several chemical compounds. The β-galactosidases produced in SbmF and SSF were able to hydrolyze the cheese whey from the dairy industry, reducing its lactose content. In addition, β-galactosidase produced via FES catalyzed the transgalactosylation reaction, producing GOS. Thus, the fungus Trichoderma sp. was an interesting producer of β-galactosidases with important properties for possible industrial applications.
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Palavras-chave
β-galactosidase, Trichoderma, GOS, Transgalactosilação