Purificação e caracterização de proteases digestivas tripsina-like do intestino de lagarta da soja, envolvidas no mecanismo de interação planta-inseto

• Autor(a) principal: Reis, Denise Torres da Cruz
• Data de Publicação: 2009
• Tipo de documento: Tese
• Idioma: por
• Título da fonte: LOCUS Repositório Institucional da UFV
• Texto Completo: http://locus.ufv.br/handle/123456789/293
• topic: Tripsina-like; Planta-inseto; Lagarta da soja; Trypsin-like; Plant-insect; Velvetbean caterpillar; CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA::ENZIMOLOGIA

Anticarsia gemmatalis (Lepidoptera), the velvetbean caterpillar, is considered the main pest of soybean culture, causing important yield losses due to herbivorous attack. Taking into account the damage caused by the caterpillar and the economic relevance of soybean culture to Brazil, the search for alternative control methods is encouraged. As the digestive proteases play fundamental roles in the larvae physiology, the study of protease inhibitors as agents for pest control is receiving continuous attention. The decrease in proteolytic activity due to the ingestion of proteases inhibitors, by means of the incorporation in the diet or by the use of transgenic plants, impairs the digestion with consequences not only to the larvae growth and development but also to the adult fertility and fecundity. However, some insects develop resistance mechanisms to inhibitors involving their own digestive proteases. It turns out that the success of the strategies for insect control, acting through the gut, depends on a case by case analysis, considering the basic digestive process of the target insect and the enzymes that compose its proteolytic repertoire. Therefore, the selection or design of more specific and efficient inhibitors can be achieved. In this context, this work aimed to purify and characterize trypisin-like digestive proteases from Anticarsia gemmatalis. Soluble and non-soluble extracts were obtained from the gut of Anticarsia gemmatalis fifth instar larvae, reared in artificial diet. After being concentrated by ultrafiltration, the extracts were submitted to an affinity chromatrography on a p-aminobenzamidine column followed by an anionic chromatography. The enzymes were obtained after elution with a saline gradient (NaCl 0 to 1 mol.L-1). The yield of the soluble trypsin-like enzyme was 52% with a final specific activity of 26.75 μmol.L-1.min-1/mg protein. The enzyme present on the non-soluble fraction (membrane bound trypsin-like enzyme) was solubilized with detergent (CHAPS). After the purification steps the yield of this enzyme was 11%, with a final specific activity of 18.61 μmol.L-1.min-1/mg. The molecular mass determined for the soluble enzyme and for the membrane-bound enzyme were 24.9 kDa (SDS-PAGE) and 28.63 Da (MALDI-TOF), respectively. Both enzymes showed proteolytic activity in substrate gel containing casein. Due to the low yield and specific activity of the membrane bound trypsin-like enzyme, no further kinetic characterization was attempted. The maximum activity of the soluble trypsin-like enzyme was detected at pH 9.0 and 35°C with L-BApNA as substrate and at pH 8.0 and 25°C with L-TAME. The KM values obtained for LBApNA and L-TAME were 120 μmol.L-1 and 49 μmol.L-1, respectively. The soluble trypsin-like enzyme activity was inhibited by PMSF, TLCK and benzamidine, but not by TPCK, reinforcing its trypsin-like character. Additionaly, the activity of this enzyme was not affected by calcium ions (0 to 20 mmol.L-1). The soybean proteinaceous inhibitors SBTI and SBBI, at their highest concentration tested, 0.1 μmol.L-1 and 0.5 μmol.L-1, respectively, decreased the soluble trypsin-like enzyme activity at almost 80%, becoming candidates for in vivo inhibition assays. The soluble trypsin-like enzyme identity could not be established by the MASCOT search for homology using the tryptic peptides. However, the highest score was obtained for a trypsin-like enzyme from another Lepidoptera insect. The similarity of eletrophoretic and chromatographic profiles from soluble and membrane bound tripsin-like enzymes supports the hypothesis that the membrane bound enzyme can a precursor of the soluble enzyme, as described for other insects. The knowledge gained by this work brings perspectives for complementary studies which can further contribute to the development of strategies for pest control based on digestive proteases inhibition.