Produção, purificação, propriedades bioquímicas e aplicação de xilanases de Aspergillus hortae
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Data
2017-04-10
Autores
Nascimento, Juliana Montesino de Freitas [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
Xilanases são enzimas que possuem uma gama de aplicações biotecnológicas, como hidrólise enzimática da biomassa lignocelulósica para produção de etanol, branqueamento de polpa de papel Kraft, na ração animal, na indústria de alimentos como aditivo para melhorar a qualidade de pães, na clarificação de sucos, entre outras. Essa variedade de aplicações deve-se a atuação das xilanases sobre os resíduos de xilose da cadeia principal do xilano, o segundo principal componente da parede celular vegetal, liberando xilooligossacarídeos. A amplitude de aplicações confere às xilanases posição de destaque econômico e grande exploração científica, principalmente na busca por xilanases estáveis ou tolerantes a condições físico-químicas mais extremas de temperatura, pH e salinidade. Este estudo mostra, pela primeira vez, a produção, purificação e aplicação de xilanases de Aspergillus hortae, um fungo isolado de lagoas salinas da região do Pantanal do Mato Grosso do Sul, Brasil. A produção de xilanases foi realizada utilizando sabugo de milho como substrato. Após a realização de um planejamento experimental, as condições ótimas de cultura foram pH 5,36 e 35 ºC. As xilanases caracterizadas no filtrado de cultura apresentaram pH e temperatura ótimos de 6,5 e 55 ºC bem como estabilidade em pH 3,0 a 8,0 e cerca de 70% da atividade ainda estava presente após 5 h de incubação do filtrado a 50 ºC. A aplicação do filtrado de cultura na produção de pães, resultou na redução da firmeza do miolo, provocando um efeito anti-endurecimento, o que é um fator positivo quando se considera a vida de prateleira do pão. O filtrado de cultura foi submetido aos testes de purificação em trocador catiônico e aniônico bem como cromatografias de exclusão molecular, resultando na purificação de três xilanases, Xyl I, Xyl II e Xyl III, com massas moleculares de 33, 25 e 29 kDa, respectivamente. O pH ótimo das enzimas purificadas foi em torno de 6,0 a 6,5 e a temperatura ótima 55 ºC, exceto para Xyl III que foi a 65 ºC. As três enzimas apresentaram interessante tolerância e estabilidade ao NaCl, com destaque para Xyl I que foi ativada, especialmente a 4 M de NaCl, bem como permaneceram estáveis em 177%, 75% e 58%, respectivamente, quando incubadas a 2 M de NaCl por 3 h a 25 ºC. Os resultados deste trabalho indicam o potencial de aplicação destas enzimas em processos industriais como na panificação e também processos que requerem estabilidade em faixas de pH e salinidade mais extremas, como branqueamento de polpa de papel Kraft e preparação de alimentos derivados de frutos do mar, respectivamente.
Xylanases are enzymes that have a range of biotechnological applications, such as enzymatic hydrolysis of lignocellulosic biomass for ethanol production, bleaching of Kraft paper pulp, in animal feed, in the food industry as an additive to improve the quality of breads, clarification of juices, among others. This variety of applications is due to the action of xylanases on the xylose residues of the xylan main chain, the second main component of the plant cell wall, releasing xylooligosaccharides. The range of applications gives xylanases an outstanding economic position and great scientific exploration, mainly in the search for stable or tolerant xylanases to extreme physicochemical conditions, such as high temperature, pH or salinity. This study shows, for the first time, the production, purification and application of xylanases of Aspergillus hortae, a fungus isolated from saline lagoons in the Pantanal region of Mato Grosso do Sul, Brazil. The xylanases production was carried out using corn cobs as a substrate. After an experimental design, optimum culture conditions were pH 5.36 and 35 ºC. The xylanases characterized in the culture filtrate presented optimum pH and temperature of 6.5 and 55 ºC as well as stability at pH 3.0 to 8.0 and almost 70% of the activity was still present after 5 h incubation of the filtrate at 50 °C. The application of the culture filtrate in the bread production resulted in a reduction of the firmness of the crumb, causing an anti-stalling effect, which is a positive factor when considering the shelf life of the bread. The culture filtrate produced under the conditions described above was subjected to purification tests, which included chromatographic steps in cationic and anionic exchangers as well as exclusion chromatography, resulting in the purification of three xylanases, Xyl I, Xyl II and Xyl III, with molecular mass of 33, 25 and 29 kDa, respectively. The optimum pH of the purified enzymes was around 6.0-6.5 and the optimum temperature 55 °C, except for Xyl III which was at 65 °C. The three enzymes presented interesting tolerance and stability to NaCl, especially Xyl I, which was activated mainly at 4 M NaCl, as well as remained stable at 177%, 75% and 58%, respectively, when incubated at 2 M NaCl for 3 h at 25 ºC. The results of this work indicate the potential application of these enzymes in industrial processes such as baking and also processes that require stability in more extreme pH and salinity ranges, such as bleaching of Kraft paper pulp and preparation of foods derived from seafood, respectively.
Xylanases are enzymes that have a range of biotechnological applications, such as enzymatic hydrolysis of lignocellulosic biomass for ethanol production, bleaching of Kraft paper pulp, in animal feed, in the food industry as an additive to improve the quality of breads, clarification of juices, among others. This variety of applications is due to the action of xylanases on the xylose residues of the xylan main chain, the second main component of the plant cell wall, releasing xylooligosaccharides. The range of applications gives xylanases an outstanding economic position and great scientific exploration, mainly in the search for stable or tolerant xylanases to extreme physicochemical conditions, such as high temperature, pH or salinity. This study shows, for the first time, the production, purification and application of xylanases of Aspergillus hortae, a fungus isolated from saline lagoons in the Pantanal region of Mato Grosso do Sul, Brazil. The xylanases production was carried out using corn cobs as a substrate. After an experimental design, optimum culture conditions were pH 5.36 and 35 ºC. The xylanases characterized in the culture filtrate presented optimum pH and temperature of 6.5 and 55 ºC as well as stability at pH 3.0 to 8.0 and almost 70% of the activity was still present after 5 h incubation of the filtrate at 50 °C. The application of the culture filtrate in the bread production resulted in a reduction of the firmness of the crumb, causing an anti-stalling effect, which is a positive factor when considering the shelf life of the bread. The culture filtrate produced under the conditions described above was subjected to purification tests, which included chromatographic steps in cationic and anionic exchangers as well as exclusion chromatography, resulting in the purification of three xylanases, Xyl I, Xyl II and Xyl III, with molecular mass of 33, 25 and 29 kDa, respectively. The optimum pH of the purified enzymes was around 6.0-6.5 and the optimum temperature 55 °C, except for Xyl III which was at 65 °C. The three enzymes presented interesting tolerance and stability to NaCl, especially Xyl I, which was activated mainly at 4 M NaCl, as well as remained stable at 177%, 75% and 58%, respectively, when incubated at 2 M NaCl for 3 h at 25 ºC. The results of this work indicate the potential application of these enzymes in industrial processes such as baking and also processes that require stability in more extreme pH and salinity ranges, such as bleaching of Kraft paper pulp and preparation of foods derived from seafood, respectively.
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Palavras-chave
Aspergillus hortae, Produção de xilanases, Aplicação na panificação, Purificação e caracterização, Enzimas halotolerantes