Enzimas degradadoras de parede celular de plantas produzidas por Rhizoctonia solani AG-1 IA: estudo da produção extracelular, purificação e caracterização
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Data
2016-11-18
Autores
Bistratini, Erica Aparecida Santos [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
O presente trabalho configura-se no estudo da produção de enzimas degradadoras de parede celular de plantas (EDPCP), produzida por Rhizoctonia solani AG-1 IA obtido de culturas de arroz, braquiária e soja. As enzimas inicialmente estudadas foram celulases, β-glucosidase, xilanase, pectinases, amilase, lípases e proteases excretadas extracelularmente para o meio de cultivo. Foram avaliados 87 isolados de Rhizoctonia solani AG-1 IA, e adotado o cultivo em estado sólido utilizando farelo de trigo como fonte de carbono inicial para indução da produção extracelular das enzimas e avaliar o potencial dos isolados. O estudo do efeito da fonte de carbono foi realizado com cinco isolados de cada tipo de cultura, os quais foram cultivados em farelo de trigo, braquiária triturada, palha de soja, palha de milho, sabugo de milho e palha de arroz. Os melhores resultados para atividade xilanase foi do isolado PA_B1F6 obtido de culturas de braquiária quando cultivado em farelo de trigo (15,82 U mL-1), para CMCase e Avicelase os melhores resultados foram obtidos com os isolados MA_217 e TO_064 obtidos de culturas de soja quando cultivado em palha de milho (3,24 U mL-1) e palha de soja (2,91 U mL-1), respectivamente, e para β glucosidase os melhores resultados foram obtidos com o isolado TO_022 (2,75 U mL-1) obtido de cultura de soja cultivado em braquiária. A atividade de amilase foi superior no cultivo em farelo de trigo para o isolado ROB4D7 (142,88 U mL-1) obtido da cultura de braquiária e a pectinase do isolado PA_B1F6 (17,69 U mL-1) obtido da cultura de braquiária quando cultivado em substrato braquiária. O isolado MT_S085 obtido da cultura de soja apresentou melhor atividade de protease (1154,52 UAP mL-1) no cultivo com farelo de trigo e melhor atividade lípase (30,57 U mL-1) quando cultivado em substrato de braquiária. Os cinco isolados de cada tipo de cultura foram cultivados nos substratos que indicaram melhor atividade xilanase com avaliação do perfil de atividade ao longo do tempo. Os isolados obtidos de cultura de braquiária foram os que exibiram maiores produções de atividade da enzima xilanase. Os cultivos utilizando braquiária triturada e farelo de trigo como substrato foram os que proporcionaram maior atividade xilanolítica, a qual, em geral, ocorreu com 120h. A xilanase bruta produzida pelos isolados obtidos de cultura de arroz (RR_A23), braquiária (PA_B1F6) e soja (MT_S085) mostrou temperatura ótima em 55 °C. O pH ótimo dessas xilanases foi pH 6,0 para o isolado RR_A23, pH 6,5 para PA_B1F6 e pH 7,5 para MT_S085. O pH de estabilidade ficou na faixa de pH 4,0 a 7,5. Foi possível verificar que as xilanases produzidas por esses isolados diferem com o tipo de substrato utilizado no cultivo verificado eletroforese em gel de poliacrilamida SDS-PAGE. Foram identificadas 35 sequências gênicas para a xilanase em Rhizoctonia solani, porém, não há até momento, sequência gênica descrita para xilanase produzida por Rhizoctonia solani AG-1 IA. Foram encontradas 4 sequências gênicas de Rhizoctonia solani AG-1 IA que apresentaram maior similaridade quando comparadas a sequências de xilanases descrita para outros grupos da mesma espécie. Desta forma foi realizada a purificação de uma xilanase produzida por Rhizoctonia solani AG-1 IA isolado da cultura de braquiária PA_B1F6. O cultivo em estado sólido foi realizado utilizando 5g de farelo de trigo com 0,5g de xilana e a purificação dessa xilanase necessitou de concentração por ultra filtração, precipitação com etanol e troca iônica em Hitrap SP Sepharose FF, para recuperar 4% da atividade inicial. A massa molecular foi estimada em 33,11 kDa. A xilanase purificada apresentou atividade ótima a 55 °C e maior atividade em pH 6,5. A enzima sofreu inibição por íons e agentes químicos e os parâmetros cinéticos para Km, Vmax, utilizando xilana de bétula (birchwood), como substrato foram 1,42 mg mL-1, 1415 mol L-1 mim-1, respectivamente.
The Present work covers the study of the production of degradative enzymes from plant cell wall (EDPCP) produced by Rhizoctonia solani AG-1 IA isolated from rice, signalgrass and soybean. The enzymes studied initially were cellulases, β-glucosidase, xylanase, pectinase, amylase, lipase and proteases extracellularly secreted into the culture medium. It was evaluated 87 isolates of Rhizoctonia solani AG-1 IA and adopted cultivation in the solid state using wheat bran as the initial carbon source for inducing the production of extracellular enzymes and assess the potential isolates. The study of the effect of carbon source was performed with five isolates of each type of culture which were cultured on wheat bran, comminuted signalgrass, soybean straw, corn stover, corn cobs and rice straw. The best result results for xylanase activity was the isolated PA_B1F6 from cultures of signalgrass grown in wheat bran (15.82 U mL-1) to Avicelase and CMCase and the best results were obtained for the isolated of soybean MA_217 and TO_064 when grown corn straw (3.24 U ml-1) and soybean straw (2.91 U ml-1), respectively. For β-glucosidase the best results were obtained for the isolated of soybean TO_022 (2.75 U ml-1) grown in signalgrass. The amylase activity was higher in culture with wheat bran for isolated ROB4D7 from the signalgrass culture (142.88 U mL-1). The best pectinase activity was by the PA_B1F6 isolated signalgrass culture (17.69 U mL-1) when grown in signalgrass as substrate. The MT_S085 isolated from soybean showed better protease activity (UAP 1154.52 ml-1) in culture with wheat bran. For this same isolated, but when grown in signalgrass substrate was observed the best lipase activity (30.57 U mL-1). The five isolates of each type were grown in culture substrates that indicated better xylanase activity to evaluate the activity profile over time. Isolates obtained from signalgrass culture were those who generally had higher levels of xylanase activity. Crops using signalgrass and wheat bran as substrate presented the best xylanase activity and, in general, the cultivation time with greater enzymatic activity in 120h. The crude xylanase produced by rice culture isolates (RR_A23), signalgrass (PA_B1F6) and soybean (MT_S085) showed optimum temperature at 55 ° C. The optimal pH of these xylanase was pH 6.0 for isolated RR_A23, pH 6.5 for PA_B1F6, and pH 7.5 for MT_S085. The pH was stable at pH 4.0 to 7.5. It was possible to verify that the xylanases produced by these isolates differ with the type of substrate used in cultivation when subjected to polyacrylamide gel electrophoresis SDS-PAGE. They were identified 35 gene sequences for the xylanase in Rhizoctonia solani, however, there is not until now, gene sequence described for xylanase produced by Rhizoctonia solani AG-1 IA. It was found 4 gene sequences of Rhizoctonia solani AG-1 IA that showed greater similarity when compared to sequences of xylanase described for other groups of the same species. Thus, it was performed the purification of a xylanase produced by Rhizoctonia solani AG-1 IA isolated PA_B1F6 from the signalgrass culture. The cultivation in the solid state was carried out using wheat bran with xylan. For the purification of this xylanase concentration was performed by ultrafiltration, ethanol precipitation and ion exchange in Hitrap SP Sepharose FF, which can recover 4% of the initial activity. The molecular mass was estimated as 33.11 kDa. The purified xylanase showed optimal activity at 55 ° C and higher activity at pH 6.5. The enzyme was inhibited by ions and modifying agents and the kinetic parameters Km, Vmax, using xylan birch (beerchwood) as substrate were 1.42 mg mL-1, 1415 mol L-1 mim-1, respectively.
The Present work covers the study of the production of degradative enzymes from plant cell wall (EDPCP) produced by Rhizoctonia solani AG-1 IA isolated from rice, signalgrass and soybean. The enzymes studied initially were cellulases, β-glucosidase, xylanase, pectinase, amylase, lipase and proteases extracellularly secreted into the culture medium. It was evaluated 87 isolates of Rhizoctonia solani AG-1 IA and adopted cultivation in the solid state using wheat bran as the initial carbon source for inducing the production of extracellular enzymes and assess the potential isolates. The study of the effect of carbon source was performed with five isolates of each type of culture which were cultured on wheat bran, comminuted signalgrass, soybean straw, corn stover, corn cobs and rice straw. The best result results for xylanase activity was the isolated PA_B1F6 from cultures of signalgrass grown in wheat bran (15.82 U mL-1) to Avicelase and CMCase and the best results were obtained for the isolated of soybean MA_217 and TO_064 when grown corn straw (3.24 U ml-1) and soybean straw (2.91 U ml-1), respectively. For β-glucosidase the best results were obtained for the isolated of soybean TO_022 (2.75 U ml-1) grown in signalgrass. The amylase activity was higher in culture with wheat bran for isolated ROB4D7 from the signalgrass culture (142.88 U mL-1). The best pectinase activity was by the PA_B1F6 isolated signalgrass culture (17.69 U mL-1) when grown in signalgrass as substrate. The MT_S085 isolated from soybean showed better protease activity (UAP 1154.52 ml-1) in culture with wheat bran. For this same isolated, but when grown in signalgrass substrate was observed the best lipase activity (30.57 U mL-1). The five isolates of each type were grown in culture substrates that indicated better xylanase activity to evaluate the activity profile over time. Isolates obtained from signalgrass culture were those who generally had higher levels of xylanase activity. Crops using signalgrass and wheat bran as substrate presented the best xylanase activity and, in general, the cultivation time with greater enzymatic activity in 120h. The crude xylanase produced by rice culture isolates (RR_A23), signalgrass (PA_B1F6) and soybean (MT_S085) showed optimum temperature at 55 ° C. The optimal pH of these xylanase was pH 6.0 for isolated RR_A23, pH 6.5 for PA_B1F6, and pH 7.5 for MT_S085. The pH was stable at pH 4.0 to 7.5. It was possible to verify that the xylanases produced by these isolates differ with the type of substrate used in cultivation when subjected to polyacrylamide gel electrophoresis SDS-PAGE. They were identified 35 gene sequences for the xylanase in Rhizoctonia solani, however, there is not until now, gene sequence described for xylanase produced by Rhizoctonia solani AG-1 IA. It was found 4 gene sequences of Rhizoctonia solani AG-1 IA that showed greater similarity when compared to sequences of xylanase described for other groups of the same species. Thus, it was performed the purification of a xylanase produced by Rhizoctonia solani AG-1 IA isolated PA_B1F6 from the signalgrass culture. The cultivation in the solid state was carried out using wheat bran with xylan. For the purification of this xylanase concentration was performed by ultrafiltration, ethanol precipitation and ion exchange in Hitrap SP Sepharose FF, which can recover 4% of the initial activity. The molecular mass was estimated as 33.11 kDa. The purified xylanase showed optimal activity at 55 ° C and higher activity at pH 6.5. The enzyme was inhibited by ions and modifying agents and the kinetic parameters Km, Vmax, using xylan birch (beerchwood) as substrate were 1.42 mg mL-1, 1415 mol L-1 mim-1, respectively.
Descrição
Palavras-chave
Fungo fitopatogênico, Enzimas microbianas, Xilanase, Sequências gênicas, Phytopathogenic fungus, Microbial enzymes, Xylanase, Genomic sequences