Clonagem e expressão de serino-proteases de Anticarsia gemmatalis e caracterização cinético-enzimática de tripsinas-like purificadas, produzidas por sua microbiota intestinal
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2012-07-30
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Universidade Federal de Viçosa
Resumo
A lagarta da soja, Anticarsia gemmatalis, é considerada uma das principais pragas da cultura da soja. Os danos causados pelo ataque deste inseto associado à relevância econômica do cultivo da soja para o Brasil e para o mundo fomentam a busca por alternativas no controle deste inseto. Estratégias de controle de insetos-pragas baseadas no uso de inibidores de proteases têm sido estudadas e o conhecimento das enzimas digestivas tem se mostrado fundamental. Neste contexto, este trabalho teve como objetivo identificar genes de serino-proteases de A. gemmatalis e avaliar sua expressão diante de inibidores de serino-proteases bem como purificar e caracterizar serino-proteases produzidas pela microbiota intestinal da lagarta da soja. Foram isolados três genes distintos de serino proteases do genoma da lagarta da soja chamados de Agem 1, Agem 2 e Agem 3, indicando que os genes de serino-proteases de A. gemmatalis estão organizados em família multigênica. Os três genes mostraram alta identidade com tripsinas de outros insetos da ordem Lepidoptera. Foi verificado que a expressão do gene Agem 2 se sobressaiu em relação ao gene Agem 1 e Agem 3. As sequências descritas neste trabalho evidenciaram ser genes de tripsinas sensíveis e/ou insensíveis ao inibidor de serino-protease Benzamidina. O inibidor sintético Berenil foi potencialmente eficiente na supressão dos genes de tripsinas isolados neste estudo. Os genes de tripsinas isolados mostraram serem sensíveis aos inibidores proteicos da soja SKTI e SBBI, diminuindo sua expressão durante o tratamento. Esses estudos mostram que o significado da expressão diferencial de proteases digestivas diante de inibidores de proteases nunca pode ser subestimado. O processo de purificação das enzimas produzidas por Bacillus cereus, Staphylococcus xylosus, Enterococcus mundtii e Enterococcus gallinarum isoladas do trato intestinal de A. gemmatalis, foi realizado em cromatografia de afinidade (p-aminobenzamidina). As massas moleculares estimadas das enzimas foram de aproximadamente 25 kDa (SDS-PAGE). As enzimas apresentaram maior atividade em temperatura a 40ºC e em pH 7,5 para B. cereus, pH 10,0 para E.munditti, e pH 8,5 para S.xylosus e E.galinarum. Os íons cálcio não afetaram a atividade enzimática nas concentrações testadas. Os valores de KM da serino protease de E. gallinarum, B. cereus, S. xylosus e E. mundtii foram de 0,35mM, 0,18 mM, 0,21 mM e 0,22 mM, respectivamente. As enzimas foram sensíveis à inibição por inibidores típicos de serino- proteases e tripsina, como Aprotinina, Berenil e SKTI. Suas atividades não foram alteradas pelos inibidores TPCK de quimiotripsina, Pepistatina A de aspartil-proteases, E-64 de cisteíno-proteases e EDTA de metalo-proteases. Esses resultados em conjunto demonstram que as bactérias sintetizam e excretam no lúmen intestinal de A. gemmatalis enzimas tripsinas-like com características semelhantes às produzidas pelo próprio inseto. Através do conhecimento obtido neste trabalho surgem novas perspectivas para a realização de pesquisas complementares que poderão contribuir para o desenvolvimento de estratégias de controle de pragas baseadas na inibição de proteases digestivas, tanto produzidas pelo inseto quanto pela sua microbiota intestinal.
The caterpillar, Anticarsia gemmatalis, is considered a major pest of soybean. The damage caused by this pest associated with the economic importance of soybean cultivation in Brazil and the world to promote the search for alternatives to control this insect. Strategies to control insect pests based on the use of protease inhibitors have been studied and knowledge of digestive enzymes has proved crucial. In this context, this study aimed to identify genes for serine proteases of A. gemmatalis and evaluate your expression face of inhibitors of serine proteases as well as purify and characterize serine proteases produced by the intestinal microbiota of the soybean caterpillar. Genes were isolated three distinct serine proteases of the genome of soybean caterpillar called Agem 1, Agem 2 and Agem 3, indicating that the genes of serine proteases of A. gemmatalis are organized into multigene family. The three genes showed high identity with trypsins from other insects of the order Lepidoptera. It was found that gene expression Agem 2 excelled compared to the genes Agem 1 and Agem 3. The sequences described herein have shown to be sensitive genes trypsins and/or insensitive to the serine protease inhibitor benzamidine. The synthetic inhibitor berenil was potentially effective in the suppression of genes trypsins isolated in this study. The genes of isolates showed trypsin to be sensitive to inhibitors of soybean protein SKTI and SBBI decreasing its expression throughout the treatment. These studies show that the significance of differential expression of digestive proteases before protease inhibitors can never be underestimated. The purification process of enzymes produced by Bacillus cereus, Staphylococcus xylosus, Enterococcus gallinarum, and Enterococcus mundtii isolated from the intestinal tract of A. gemmatalis was performed on affinity chromatography (p-aminobenzamidine). The molecular weights of the enzymes were estimated to approximately 25 kDa (SDS-PAGE). The enzymes were more active in temperature to 40 ° C and pH 7.5 for B. cereus, pH 10.0 for E.munditti and pH 8.5 for S.xylosus and E.galinarum. The calcium ions did not affect the enzyme activity at the concentrations tested. The values of KM serine protease of E. gallinarum, B. cereus, S. xylosus and E. mundtii were 0.35 mM, 0.18 mM, 0.21 mM and 0.22 mM, respectively. The enzymes were sensitive to inhibition by inhibitors of serine proteases typical and trypsin, as Aprotinin, Berenil and SKTI. His activities were not altered by inhibitors chymotrypsin of TPCK, Pepstatin A of aspartyl proteases, E-64 of cysteine proteases and EDTA of metalo-proteases. These results together demonstrate that the bacteria synthesize and secrete the intestinal lumen A. gemmatalis trypsin-like enzymes with similar characteristics to those produced by the insect. Through the knowledge gained in this work are new prospects for conducting additional research that will contribute to the development of pest control strategies based on the inhibition of digestive proteases, both produced by the insect as for their intestinal microbiota.
The caterpillar, Anticarsia gemmatalis, is considered a major pest of soybean. The damage caused by this pest associated with the economic importance of soybean cultivation in Brazil and the world to promote the search for alternatives to control this insect. Strategies to control insect pests based on the use of protease inhibitors have been studied and knowledge of digestive enzymes has proved crucial. In this context, this study aimed to identify genes for serine proteases of A. gemmatalis and evaluate your expression face of inhibitors of serine proteases as well as purify and characterize serine proteases produced by the intestinal microbiota of the soybean caterpillar. Genes were isolated three distinct serine proteases of the genome of soybean caterpillar called Agem 1, Agem 2 and Agem 3, indicating that the genes of serine proteases of A. gemmatalis are organized into multigene family. The three genes showed high identity with trypsins from other insects of the order Lepidoptera. It was found that gene expression Agem 2 excelled compared to the genes Agem 1 and Agem 3. The sequences described herein have shown to be sensitive genes trypsins and/or insensitive to the serine protease inhibitor benzamidine. The synthetic inhibitor berenil was potentially effective in the suppression of genes trypsins isolated in this study. The genes of isolates showed trypsin to be sensitive to inhibitors of soybean protein SKTI and SBBI decreasing its expression throughout the treatment. These studies show that the significance of differential expression of digestive proteases before protease inhibitors can never be underestimated. The purification process of enzymes produced by Bacillus cereus, Staphylococcus xylosus, Enterococcus gallinarum, and Enterococcus mundtii isolated from the intestinal tract of A. gemmatalis was performed on affinity chromatography (p-aminobenzamidine). The molecular weights of the enzymes were estimated to approximately 25 kDa (SDS-PAGE). The enzymes were more active in temperature to 40 ° C and pH 7.5 for B. cereus, pH 10.0 for E.munditti and pH 8.5 for S.xylosus and E.galinarum. The calcium ions did not affect the enzyme activity at the concentrations tested. The values of KM serine protease of E. gallinarum, B. cereus, S. xylosus and E. mundtii were 0.35 mM, 0.18 mM, 0.21 mM and 0.22 mM, respectively. The enzymes were sensitive to inhibition by inhibitors of serine proteases typical and trypsin, as Aprotinin, Berenil and SKTI. His activities were not altered by inhibitors chymotrypsin of TPCK, Pepstatin A of aspartyl proteases, E-64 of cysteine proteases and EDTA of metalo-proteases. These results together demonstrate that the bacteria synthesize and secrete the intestinal lumen A. gemmatalis trypsin-like enzymes with similar characteristics to those produced by the insect. Through the knowledge gained in this work are new prospects for conducting additional research that will contribute to the development of pest control strategies based on the inhibition of digestive proteases, both produced by the insect as for their intestinal microbiota.
Descrição
Palavras-chave
Serino-proteases, Anticarsia gemmatalis, Microbiota, Serine proteases, Anticarsia gemmatalis, Microbiota
Citação
PILON, Franciny Martins. Cloning and expression of serine proteases Anticarsia gemmatalis and kinetic characterization of enzyme-purified trypsin-like, produced by their intestinal microbiota. 2012. 131 f. Tese (Doutorado em Bioquímica e Biologia molecular de plantas; Bioquímica e Biologia molecular animal) - Universidade Federal de Viçosa, Viçosa, 2012.