Isolamento de fungos do bioma pantanal e estudos da produção de xilanase e celulase
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Data
2016-08-29
Autores
Santos, Wanderléia Rodrigues dos [UNESP]
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
Diante do recente interesse em valorizar os fungos degradadores da biomassa lignocelulósica oriunda das atividades agrícolas, agroindustriais e de florestas plantadas, torna-se necessário a prospecção de novos micro-organismos e a partir destes estudar seu potencial, bem como fatores que afetam o seu desenvolvimento, a produção de enzimas, e o rendimento como um todo. Após uma breve introdução e revisão de literatura, tem-se o Capítulo I expondo parte do projeto, onde o objetivo de estudo foi isolar e selecionar fungos da região do Pantanal Sul-mato-grossense, e testar sua eficiência na produção das enzimas xilanase, carboximetilcelulase (CMCase) e avicelase tendo 1% de palha de milho e papelão como substratos indutores no meio líquido de isolamento testados na temperatura de 35 e 45 °C. Neste processo, um total de 111 fungos foram obtidos, onde 80,7% dos isolados em meio contendo palha de milho, cresceram a temperatura de 35 °C e os isolados induzidos meio contendo papelão, 63,0% se manifestaram a 45 °C. O isolado WPPM 922 obteve as maiores atividades enzimáticas com 614,5 U g- 1, e 107,3 U g-1 e 5,37 U g- 1 para atividade de xilanase, CMCase e avicelase respectivamente, fermentado em farelo de trigo em 96 h a 35 °C. Oito isolados e o Trichoderma asperelloides F22, foram selecionados para avaliação do seu potencial na produção enzimática utilizando 6 tipos de resíduos agrícolas e agroindustriais. A casca de arroz foi o substrato que mais induziu as enzimas xilanase e CMCase pelos isolados testados. A maior produção de xilanase foi obtida pelo isolado WPPM 421A com 931,95 U g-1 em casca de arroz e por T. asperelloides F22 cerca de 876,48 U g -1 tendo o farelo de trigo como substrato. O isolado WPPM 622 apresentou a maior atividade da enzima CMCase, com atividade de 155,73 U g-1 na mistura bagaço de cana-de-açúcar, seguido pelo isolado WPPM 151 e o fungo T. asperelloides F22, com atividade de 60,52 U g-1 e 57,29 U g-1 respectivamente, fermentados em casca de arroz. No Capítulo II, descreve-se a importância das condições bioquímicas das enzimas xilanase e CMCase produzidas pelos isolados WPPM 421A e WPPM 622, e por T. asperelloides F22 quanto a temperatura e pH ótimos e estabilidade e a meia vida. O Trichoderma asperelloides F22 obteve seu pico de produção de xilanase em 72 h (606,23 U g-1), e o isolado WPPM 421A que obteve pico de produção de xilanase e CMCase em 96 h (547,27 U g-1 e 55,53 U g-1, respectivamente). O pH ótimo e estabilidade foi diferente para os isolados avaliados, no entanto, o isolado WPPM 421A, cultivado em farelo de trigo, demonstrou pH ótimo de 5,5, mas manteve sua atividade acima de 50% na faixa de pH 5-11. Quanto a termoestabilidade, o isolado WPPM 421A quando cultivado em sabugo de milho produziu xilanase mais termoestável, mantendo 67,4% da atividade a 90 °C. Essa xilanase conservou 60% de sua atividade após 12h de incubação a 60 °C, e manteve 50% de sua atividade após incubada 1 h a 70 °C. As propriedades estudadas colocam estas enzimas como promissoras para aplicações biotecnológicas.
Before the recent interest in valuing the degrading fungi lignocellulosic biomass derived from agricultural, agroindustrial and planted forests, it is necessary to prospect for new microorganisms and from these study their potential as well as factors affecting their development the production of enzymes, and performance as a whole. After a brief introduction and review of the literature, there is Chapter I exposing part of the project, where the objective of the study was to isolate and select fungi from the Sul-mato-grossese Pantanal region, and test their efficiency in the production of xylanase enzymes, carboxymethylcellulase (CMCase) and avicelase having 1% corn straw and cardboard as an inducer substrate in the liquid isolation medium tested at 35 and 45 °C. In this process, a total of 111 fungi were obtained where 80.7% of the isolates were grown in medium with corn stover at 35 °C and induced in medium containing isolated cardboard, they are expressed in greater numbers at 45 °C, about 63.0%. Isolated WPPM 922 had the highest enzymatic activities 614.5 U g-1, and 107.3 U g-1 and 5.37 U g-1 xylanase, CMCase and avicelase respectively, fermented wheat bran at 96 h at 35 °C. Also in this chapter, eight isolates and the Trichoderma asperelloides F22, were tested for their potential to hydrolyze 6 types of agricultural and agro-industrial waste. Rice husk was the substrate that more induced the xylanase enzymes and CMCase the isolates tested. The highest xylanase production was obtained with the isolated WPPM 421A activity 931.95 U g-1 in rice husk, and T. asperelloides F22 with 876.48 U g-1 with wheat bran as substrate. The isolate WPPM 622 was the fungus that showed the highest activity of CMCase enzyme with activity 155.73 Ug-1 in the mixture of sugar cane bagasse and wheat bran, followed by isolated WPPM 151 and the T. asperelloides F22 with activity 60.52 U g-1 and 57.29 U g-1 respectively, fermented rice husk. In Chapter II, reports the importance of the minimum conditions for the fungi to express their potential to secrete degradative enzymes of cell wall, it aimed to with this process to obtain a kinetic production of xylanase enzymes and CMCase by isolated WPPM 421A and WPPM 622, and T. asperelloides F22 and after characterize the xylanase of these materials with the optimum temperature and pH, thermal and pH stability and half life of the enzyme. O Trichoderma asperelloides F22 obtained its peak xylanase production at 72 h (606.23 U g-1) and isolated WPPM 421A that obtained peak xylanase production and CMCase at 96 h (547.27 U g-1 and 55.53 U g-1, respectively). There was a difference in optimum pH and stability of the tested isolates, however, isolated WPPM 421A, cultivated in wheat bran showed optimum pH of 5.5, but maintained its activity above 50% in the pH 5-11 range. The thermostability, isolated WPPM 421A when grown on corn cobs produced more thermostable xylanase activity keeping in 67.4% when the reaction occurred at 90 °C, and also retained its activity by 60%, 12 h incubation at 60 °C, and retained 50% of its activity after incubated 1h at 70 °C. The properties studied enzymes make them promising for biotechnological applications. The region of Sul-mato-grossense has shown promise for the study of fungal microbiota for the production of enzymes, where 99% demonstrated ability to express cellulases and hemicellulases, with activity up to 900 U g- 1.
Before the recent interest in valuing the degrading fungi lignocellulosic biomass derived from agricultural, agroindustrial and planted forests, it is necessary to prospect for new microorganisms and from these study their potential as well as factors affecting their development the production of enzymes, and performance as a whole. After a brief introduction and review of the literature, there is Chapter I exposing part of the project, where the objective of the study was to isolate and select fungi from the Sul-mato-grossese Pantanal region, and test their efficiency in the production of xylanase enzymes, carboxymethylcellulase (CMCase) and avicelase having 1% corn straw and cardboard as an inducer substrate in the liquid isolation medium tested at 35 and 45 °C. In this process, a total of 111 fungi were obtained where 80.7% of the isolates were grown in medium with corn stover at 35 °C and induced in medium containing isolated cardboard, they are expressed in greater numbers at 45 °C, about 63.0%. Isolated WPPM 922 had the highest enzymatic activities 614.5 U g-1, and 107.3 U g-1 and 5.37 U g-1 xylanase, CMCase and avicelase respectively, fermented wheat bran at 96 h at 35 °C. Also in this chapter, eight isolates and the Trichoderma asperelloides F22, were tested for their potential to hydrolyze 6 types of agricultural and agro-industrial waste. Rice husk was the substrate that more induced the xylanase enzymes and CMCase the isolates tested. The highest xylanase production was obtained with the isolated WPPM 421A activity 931.95 U g-1 in rice husk, and T. asperelloides F22 with 876.48 U g-1 with wheat bran as substrate. The isolate WPPM 622 was the fungus that showed the highest activity of CMCase enzyme with activity 155.73 Ug-1 in the mixture of sugar cane bagasse and wheat bran, followed by isolated WPPM 151 and the T. asperelloides F22 with activity 60.52 U g-1 and 57.29 U g-1 respectively, fermented rice husk. In Chapter II, reports the importance of the minimum conditions for the fungi to express their potential to secrete degradative enzymes of cell wall, it aimed to with this process to obtain a kinetic production of xylanase enzymes and CMCase by isolated WPPM 421A and WPPM 622, and T. asperelloides F22 and after characterize the xylanase of these materials with the optimum temperature and pH, thermal and pH stability and half life of the enzyme. O Trichoderma asperelloides F22 obtained its peak xylanase production at 72 h (606.23 U g-1) and isolated WPPM 421A that obtained peak xylanase production and CMCase at 96 h (547.27 U g-1 and 55.53 U g-1, respectively). There was a difference in optimum pH and stability of the tested isolates, however, isolated WPPM 421A, cultivated in wheat bran showed optimum pH of 5.5, but maintained its activity above 50% in the pH 5-11 range. The thermostability, isolated WPPM 421A when grown on corn cobs produced more thermostable xylanase activity keeping in 67.4% when the reaction occurred at 90 °C, and also retained its activity by 60%, 12 h incubation at 60 °C, and retained 50% of its activity after incubated 1h at 70 °C. The properties studied enzymes make them promising for biotechnological applications. The region of Sul-mato-grossense has shown promise for the study of fungal microbiota for the production of enzymes, where 99% demonstrated ability to express cellulases and hemicellulases, with activity up to 900 U g- 1.
Descrição
Palavras-chave
Xilanase, Endoglucanase, Fermentação sólida, Resíduos lignocelulósicos, Xylanase, Solid fermentation, Lignocellulosic waste