Isolation and characterization of midgut trypsin-like proteases from the Anticarsia gemmatalis
¹ Xavier, L.P; ¹ Oliveira, M.G.A.; ² Oliveira, J.S.; ² Santos, A.V.; ³ Guedes, R.N.C.; ² Santoro, M.M.
¹ Depto Bioquímica e Biologia Molecular – Universidade Federal de Viçosa/MG; ² Depto Bioquímica e Imunologia – Universidade Federal de Minas Gerais/MG; ³ Depto Biologia Animal – Universidade Federal de Viçosa/MG
The identification and characterization of proteases from the insect midgut is important for developing pest resistant cultivars of crop species. Isolation and characterization of proteases from larvae midguts of Anticarsia gemmatalis Hübner (Lepidoptera-Noctuidae) are underway. In this study, enzymatic extracts were obtained after successive cycles of freezing-and-thawing; the homogenates were centrifuged at 100,834 g for 1h10min and at 4°C, and the resulting supernatant was used as source of the soluble protein. The digestives trypsin-like proteases were obtained by a combination of p-aminobenzamidine agarose affinity chromatography and ion exchange chromatography, in a FPLC system. The purified sample showed different bands with estimated molecular weight values of 23 kDa, 26,3 kDa, 29 kDa and 34,3 kDa, determined by silver stained SDS-PAGE. Inhibition assays with TLCK, benzamidine and PMSF confirmed that the purified samples were indeed trypsin-like proteases. Glycosylation assay, in SDS-PAGE, showed positive results for bands in the range of 30 kDa. These proteases were prepared in an RP-HPLC C-18 chromatography and subsequently subjected to mass spectrometry (MALDI-TOF), were the value 7.997 Da of the molecular weight was obtained, a likely result of autolysis. Antibacterial assays carried out against Staphylococcus aureus and Escherichia coli showed negative results. In addition, other concentrations of purified samples will be tested to confirm these results. At this moment, these proteases are being subjected to ESI-Q-TOF and N-terminus sequence determination.Supported by: CAPES, FAPEMIG and CNPq.
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