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Previous issue date: 2008 === Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) === Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) === Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) === Fundo de Auxílio aos Docentes e Alunos (FADA) === Sementes de Caesalpinia echinata contem grande quantidade de proteinas incluindo enzimas e inibidores; mas, ate o momento pouco se sabia da composicao e atividade de peptidases nessas sementes. Nosso grupo ja purificou e caracterizou inibidores de calicreina plasmatica humana (CeKI), elastase (CeEI) e catepsina B (CeCBI) presentes nas sementes de Caesalpinia echinata. Depois dos inibidores, nos interessamos em estudar as peptidases. As proteinas das sementes foram extraidas em tampao Tris 0,02 M pH 7,5. A peptidase foi purificada do extrato por cromatografias de troca ionica, filtracao em gel e interacao hidrofobica. Alguns substratos cromogenicos foram testados e essa enzima apresentou alta atividade na hidrolise de H-D-Pro-Phe-Arg-pNan (Km = 55,7 μM, Vmax = 0,319 μmol min-1 mg proteina-1) e Bz- Ile-Glu-Gly-Arg-pNan (Km = 297 μM, Vmax = 0,544 μmol min-1 mg proteina-1). A enzima foi inativada por inibidores de serino-endopeptidases do tipo da tripsina (TLCK e benzamidina), mas nao por inibidores de cisteino (E64), metalo (EDTA) e aspartilendopeptidases (pepstatina A). A peptidase apresentou pH otimo entre 7,1 e 9,2, e se manteve estavel entre 30 e 45oC, por 30 min, perdendo totalmente a atividade acima de 55oC. As caracteristicas da enzima purificada permitem a classifica-la como uma serino-endopeptidase === Some proteases found in different longevity stages of the leguminous seeds play an important role on their viability. Caesalpinia echinata seeds contain high amounts of proteins, including enzymes and their inhibitors. When stored in laboratory environmental for one month or at 4ºC for 18 months, these seeds lose their viability. We had already identified kallikrein (CeKI) and elastase (CeEI) inhibitors on stored and freshly harvested C. echinata seeds. Now, we are interested in studying different proteolytic activities in these seeds, in several longevity stages, and purifying and characterizing one of them. Proteins from green seeds, freshly harvested or 18-months-stored were extracted on 20 mM Tris buffer pH 7.5. The best amydolytic activity was found in the older seeds.The peptidase was purified from the extracts by ion exchange, gel filtration and hydrophobic interaction chromatographies. Several chromogenic substrates were tested and this protein, presented high enzyme activity on the hydrolysis of Bz-Ile-Glu-Gly-Arg-pNan (Km=0.268 mM, Vmax=0.334 nmol/min). The enzyme was inactivated by serine peptidase inhibitors (AEBSF, SBTI and CeKI), but not by cystein (E-64) and metallo peptidase (EDTA) nor by elastase (CeEI) inhibitors. The characteristics of the enzyme allowed us to classify it is as serine protease. === CAPES: 11/06 === FAPESP: 04/11015-0 === FAPESP: 07/55496-0 === CNPq: 304923/2006-0 === BV UNIFESP: Teses e dissertações
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