Purificação parcial e caracterização cinética da inibição de proteases intestinais tripsina-like de Anticarsia gemmatalis

• Ano de defesa: 2011
• Autor(a) principal: Vargas, Adriana Maria Patarroyo
• Orientador(a): Oliveira, Maria Goreti de Almeida
• Banca de defesa: Santana, Luiz Alberto , Siqueira-batista, Rodrigo , Guia, Thiago Rennó dos Mares
• Tipo de documento: Dissertação
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Federal de Viçosa
• Programa de Pós-Graduação: Mestrado em Bioquímica Agrícola
• Departamento: Bioquímica e Biologia molecular de plantas; Bioquímica e Biologia molecular animal
• País: BR
• Palavras-chave em Português: Protease; Tripsina-like; Anticarsia gemmatalis
• Palavras-chave em Inglês: Protease; Trypsin-like; Anticarsia gemmatalis
• Área do conhecimento CNPq: CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA::ENZIMOLOGIA
• Link de acesso: http://locus.ufv.br/handle/123456789/2432

Anticarsia gemmatalis (Lepidoptera), the velvetbean caterpillar, is considered the main pest of soybean culture, causing important yield losses due to herbivorous attack. 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 impairs the digestion with consequences not only to the larvae growth and development but also to the adult fertility and fecundity. To use the control strategy via digestive enzymes it’s necessary to study the structure/function of these macromolecules. Enzyme kinetics, as well as the characterization of the kinetics of inhibition of these proteases would offer a better comprehension of active centers and the mechanisms of action of these enzymes. In this context, this work aimed to study the kinetics of inhibition of A. gemmatalis gut serine proteases using different synthetic and protein inhibitors and to identify a potent inhibitor to be used in the defense process of the soybean plants against A. gemmatalis. For this purpose, soluble extract from the gut of A. gemmatalis larvae was submitted to affinity chromatography in p-aminobenzamidine agarose and the eluted fraction was submitted to electrophoresis. The presence of two bands with approximate molecular mass of 35kDa and 66kDa was observed. In the kinetics characterization the KM value for the substrate L-BApNA was of 0,503mM, Vmax was 46,650nM.s-1, Vmax/[E] was 9,256nM.s-1.mg-1.L and Vmax/[E]/KM was 18,402nM. s-1.mg-1.L. mM-1. In the inhibition study the inhibitors benzamidine, berenil, SKTI and SBBI obtained Ki of 11,2μM, 32,4μM, 0,25nM and 1,4nM, respectively, all calculated using Dixon’s method. It was used to characterize the inhibition type the double reciprocal plot of Lineweaver-Burk and the graph of the slopes of Lineweaver-Burk versus the inhibitor concentration. For the four tested inhibitors the inhibition was characterized as linear competitive in the concentration range used. The determined values of Ki showed that SKTI was the best inhibitor of the activity of trypsin-like of purified serine-proteases from medium gut of A. gemmatalis becoming a promising target of study for the production of mimetic peptide inhibitors.