Made available in DSpace on 2014-12-17T14:03:30Z (GMT). No. of bitstreams: 1 AnaCBC.pdf: 1320381 bytes, checksum: 881b82ecb0df2b87414957a1e02761bb (MD5) Previous issue date: 2008-10-20 / One Kunitz-type trypsin inhibitors (PmTI) was purified from Piptadenia moniliformis seeds, a tree of the sub-family Mimosoideae, by TCA precipitation, affinity chromatography on immobilized trypsin-Sepharose, DEAE cellulose (ion exchange) and Superose 12 (molecular exclusion) column FPLC/AKTA. The inhibitor has Mr of 25 kDa by SDS-PAGE and chromatography molecular exclusion. The N-terminal sequence of this inhibitor showed high homology with other family Kunitz inhibitors. This also stable variations in temperature and pH and showed a small decrease in its activity when incubated with DDT in the concentration of 100mM for 120 minutes. The inhibition of trypsin by PmTI was competitive, with Ki of 1.57 x10-11 M. The activity of trypsin was effectively inhibited by percentage of inhibition of 100%, among enzymes tested, was not detected inhibition for the bromelain, was weak inhibitor of pancreatic elastase (3.17% of inhibition) and inhibited by 76.42% elastase of neutrophils, and inhibited in a moderate, chymotrypsin and papain with percentage of inhibition of 42.96% and 23.10% respectively. In vitro assays against digestive proteinases from Lepidoptera, Diptera and Coleoptera pests were carried out. Several degrees of inhibition were found. For Anthonomus grandis and Ceratitis capitata the inhibition was 89.93% and 70.52%, respectively, and the enzymes of Zabrotes subfasciatus and Callosobruchus maculatus were inhibited by 5.96% and 9.41%, respectively, and the enzymes of Plodia. interpunctella and Castnia licus were inhibited by 59.94% and 23.67, respectively. In vivo assays, was observed reduction in the development of larvae in 4rd instar of C. capitata, when PmTI was added to the artificial diet, getting WD50 and LD50 of 0.30% and 0.33%, respectively. These results suggest that this inhibitor could be a strong candidate to plant management programs cross transgenic / Um inibidor de tripsina da fam?lia Kunitz (PmTI) foi purificado de sementes de Piptadenia moniliformis, uma ?rvore da sub-fam?lia Mimosoideae, atrav?s da precipita??o com ?cido tricloroac?tico (TCA), cromatografia de afinidade com tripsina acoplada em sepharose, coluna DEAE- celulose(troca i?nica) e Superose 12 (exclus?o molecular) em sistema FPLC/AKTA. O inibidor possui massa molecular de 25 kDa como confirmado atrav?s de SDS-PAGE e cromatografia de exclus?o molecular. A seq??ncia do N-terminal deste inibidor mostrou alta homologia com outros inibidores da fam?lia Kunitz. Este tamb?m ? est?vel as varia??es de temperatura e pH, e apresentou um pequeno decr?scimo na sua atividade quando incubado com DTT na concentra??o de 100 mM por 120 minutos. A inibi??o da tripsina foi do tipo competitiva com Ki de 1,57x10-11 mM. A atividade da tripsina foi inibida efetivamente com percentual de inibi??o de 100%, entre as outras enzimas testadas n?o foi detectada inibi??o para a bromela?na, foi fracamente inibidor da elastase pancre?tica (3,17% de inibi??o), inibiu em 76,42% elastase de neutr?filos, inibiu de forma moderada quimotripsina e papa?na com percentual de inibi??o de 42,96% e 23,10%, respectivamente. Ensaios in vitro foram realizados com as proteinases digestivas de Lepid?ptera, Cole?ptera e D?ptera. V?rios graus de inibi??o foram encontrados. Para Anthonomus grandis e Ceratitis capitata a inibi??o foi de 89,93% e 70,52%, respectivamente, e as enzimas de Zabrotes subfasciatus e Callosobruchus maculatus foram inibidas com percentuais de 5,96% e 9,41% respectivamente, e as enzimas de Plodia interpunctella e Castnia licus foram inibidas com percentuais de 59,94% e 23,67%, respectivamente. No ensaio in vivo, foi observada redu??o no desenvolvimento de larvas em 4? ?nstar de C. capitata, quando PmTI foi adicionado ? dieta artificial, obtendo WD50 de 0,30% e LD50 0,33% . Estes resultados sugerem que este inibidor possa ser um forte candidato para programas de melhoramentos de plantas via transgenia