Clonagem e expressão de serino-proteases de Anticarsia gemmatalis e caracterização cinético-enzimática de tripsinas-like purificadas, produzidas por sua microbiota intestinal

• Autor(a) principal: Pilon, Franciny Martins
• Data de Publicação: 2012
• Tipo de documento: Tese
• Idioma: por
• Título da fonte: LOCUS Repositório Institucional da UFV
• Texto Completo: http://locus.ufv.br/handle/123456789/319
• topic: Serino-proteases; Anticarsia gemmatalis; Microbiota; Serine proteases, Anticarsia gemmatalis; Microbiota; CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA::ENZIMOLOGIA

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.