Purificação de xilanase do fungo Myceliophthora heterothallica F.2.1.4 e aplicação de enzimas nativa purificada e recombinante para produção de xilo-oligossacarídeos
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Data
2019-02-28
Autores
Simões, Lorena Caixeta de Oliveira
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Universidade Estadual Paulista (Unesp)
Resumo
Xilanases são enzimas que agem na despolimerização da cadeia de xilana, principal componente da hemicelulose. Elas possuem diversas aplicações industriais tais como, na indústria de alimentos, do papel e de bioenergia. Neste contexto, há destaque para a produção de xilo-oligossacarídeos (XOS), que são considerados prebióticos. Estes possibilitam que probióticos sejam capazes de modular positivamente a microbiota intestinal, causando benefícios ao indivíduo hospedeiro. Nesse sentido, a busca de xilanases microbianas destaca-se como uma estratégia sustentável para a produção de prebióticos. Portanto, o objetivo deste trabalho foi purificar e caracterizar uma xilanase do fungo Myceliophthora heterothallica F.2.1.4 e aplicar esta e duas xilanases recombinantes GH11 do referido fungo para obtenção de XOS. A enzima nativa purificada, com massa molecular estimada em 27 kDa, apresentou atividade máxima em pH 4,5 e 65 °C, e manteve média maior que 90% de sua atividade residual quando exposta a temperaturas entre 30 e 60 °C por 1 hora. Essa enzima pode ser caracterizada como uma endoxilanase pertencente à família GH11. Foram produzidos XOS do bagaço pré-tratado com ozonólise seguida de extração alcalina, xilana extraída do bagaço in natura e xilana beechwood. Os produtos da hidrólise enzimática dos diferentes substratos foram identificados por eletroforese capilar e/ou HPAE-PAD, sendo que o hidrolisado de xilana beechwood pela xilanase nativa purificada, após 12 horas de hidrólise, apresentou 234,2 mg de XOS/g de xilana. As enzimas avaliadas possuem potencial para aplicação na produção de xilo-oligossacarídeos para uso como prebióticos.
Xylanases are enzymes that act in depolymerization of the xylan chain, the main component of hemicellulose. They have various industrial applications such as, food, paper and bioenergy industries. In this context, highlight the production of xylooligosaccharides (XOS), which are considered prebiotics. The XOS can be used with probiotic microorganisms to be able to positively modulate an intestinal microbiota, presenting benefits to the individual host. In this sense, a search for microbial xylanases stands out as a sustainable strategy for a production of prebiotics. Therefore, the objective of this work was to purify and characterize an xylanase of the fungus Myceliophthora heterothallica F.2.1.4 and to apply two recombinant enzymes of the same fungus to produce XOS. The enzyme, with the molecular weight estimated at 27 kDa, is at the maximum temperature of 4.5 and 65 °C, and continues more than 90% of its residual activity when exposed to temperatures between 30 and 60 °C for 1 hour. The purified native enzyme is presented as an endoxylanase for the GH11 family. XOS was produced from pretreated bagasse with ozonolysis followed by alkaline extraction, xylan extracted from the bagasse in natura and beeechwood xylan. The products of the enzymatic hydrolysis of the different substrates were identified by capillary electrophoresis and/or HPAE-PAD, being that the beechwood Xylan hydrolysate by xylanase purified native, after 12 hours of hydrolysis, showed 234.2 mg/g of xylan. Evaluated enzymes have potential for applications in the production of xylooligosaccharides for use as prebiotics.
Xylanases are enzymes that act in depolymerization of the xylan chain, the main component of hemicellulose. They have various industrial applications such as, food, paper and bioenergy industries. In this context, highlight the production of xylooligosaccharides (XOS), which are considered prebiotics. The XOS can be used with probiotic microorganisms to be able to positively modulate an intestinal microbiota, presenting benefits to the individual host. In this sense, a search for microbial xylanases stands out as a sustainable strategy for a production of prebiotics. Therefore, the objective of this work was to purify and characterize an xylanase of the fungus Myceliophthora heterothallica F.2.1.4 and to apply two recombinant enzymes of the same fungus to produce XOS. The enzyme, with the molecular weight estimated at 27 kDa, is at the maximum temperature of 4.5 and 65 °C, and continues more than 90% of its residual activity when exposed to temperatures between 30 and 60 °C for 1 hour. The purified native enzyme is presented as an endoxylanase for the GH11 family. XOS was produced from pretreated bagasse with ozonolysis followed by alkaline extraction, xylan extracted from the bagasse in natura and beeechwood xylan. The products of the enzymatic hydrolysis of the different substrates were identified by capillary electrophoresis and/or HPAE-PAD, being that the beechwood Xylan hydrolysate by xylanase purified native, after 12 hours of hydrolysis, showed 234.2 mg/g of xylan. Evaluated enzymes have potential for applications in the production of xylooligosaccharides for use as prebiotics.
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Palavras-chave
enzima fúngica, xilanase, purificação, xilo-oligossacarídeos, Fungal enzyme, Xylanase, purification, Xylooligosaccharides