Purificação parcial, propriedades e caracterização cinética de proteases tripsina-like do intestino médio da lagarta da soja, Anticarsia gemmatalis

• Autor(a) principal: Xavier, Luciana Pereira
• Data de Publicação: 2006
• Tipo de documento: Tese
• Idioma: por
• Título da fonte: LOCUS Repositório Institucional da UFV
• Texto Completo: http://locus.ufv.br/handle/123456789/304
• topic: Anticarsia gemmatalis; Interação inseto-planta; Serino-proteases; Tripsina-like; Anticarsia gemmatalis; Interaction insect-plant; Serine protease; Trypsin-like; CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA

The identification and characterization of proteases from the insect midgut are of great importance for research and development pest resistant cultivars of crop species. Different processes of trypsin-like proteases purification from midgut A. gemmatalis and characterization of these proteases were carried out in this work. Two fractions were isolated during one of the gel-filtration processes, fraction FI (anionic), with amidasic activity (substrate: L-BApNA) partially purified by ion-exchange (Mono-Q HR 5/5 column) and affinity (p-aminebenzamidine column) showed in SDS-PAGE bands with molecular weight of 17.1 kDa and 65.4 kDa; the molecular weight of 17.1 kDa is probably hydrolysis product and the molecular weight of 65.4 kDa aggregation product. The fraction FII (anionic) also partially purified by anionic exchange and affinity presented two bands of 33.5 kDa and 31.3 kDa. Antimicrobial assay carried out against S. aureus and E. coli showed negative result for the enzymatic extract and for a partially purified sample of FII. In assay with L. monocystogenes and E. faecalis, the result was positive for the enzymatic extract and negative for a partially purified sample of FII. In a second purification process, a cationic trypsin-like serine protease with molecular weight of 30 kDa (SDS-PAGE) was purified from midgut A. gemmatalis by combining affinity chromatography (p-aminebenzamidine column), cationexchange chromatography (Mono-S HR 5/5 column), in FPLC system and reverse-phase chromatography (C-18 column), in HPLC system. The cationic sample partially purified by affinity showed in SDS-PAGE, bands with molecular weight of 24.8 kDa, 26.3 kDa, 30 kDa, and 33.4 kDa. The analysis with PMSF, TLCK, and TPCK, in SDS-PAGE co-polymerized with gelatin, indicated that the enzymes are trypsin-like serine protease. Glycosylation assay, in SDS-PAGE, showed positive results for bands of 24.8 kDa and 26.3 kDa. The specific activity found was 13.2 μM.s-1.μg-1 and the purification factor, 123.4 times. In the kinetic determination, the value of KM app found for the cationic trypsin-like after ion-exchange chromatography was 0.46 mM. Mass spectrometry analysis of the material purified by HPLC reverse phase chromatography, afforded the mass of 1750.8 Da (ESI-TOF), 7997.23 Da (MALDI-TOF) and 23324.74 Da (ESI-TOF), suggesting degradation of this material during processing