Bowman-Birk proteinase inhibitor from Clitoria fairchildiarta seeds: Isolation, biochemical properties and insecticidal potential

Autor(es)

Dantzger, Miriam ^{[1, 2]} ; Vasconcelos, Ilka Maria ^[3] ; Scorsato, Valeria ^{[4, 5]} ; Aparicio, Ricardo ^[4] ; Marangoni, Sergio ^[1] ; Rodrigues Macedo, Maria Ligia ^{[1, 2]}

Afiliação do(s) autor(es)

^[1] Univ Estadual Campinas, Inst Biol, Dept Biochem, BR-13083970 Campinas, SP - Brazil

^[2] Univ Mato Grosso Sul, Ctr Biol & Hlth Sci, Dept Food Technol & Publ Hlth, BR-79070900 Campo Grande, MS - Brazil

^[3] Univ Ceara, Dept Biochem & Mol Biol, BR-60451970 Fortaleza, CE - Brazil

^[4] Univ Estadual Campinas, Inst Chem, Lab Struct Biol & Crystallog, BR-13083970 Campinas, SP - Brazil

^[5] Univ Estadual Campinas, Inst Biol, BR-13083970 Campinas, SP - Brazil

• Tipo de documento: Artigo Científico
• Fonte: Phytochemistry; v. 118, p. 224-235, OCT 2015.
• Citações Web of Science: 9

Resumo

Herein described is the biochemical characterisation, including in vitro and in vivo assays, for a proteinase inhibitor purified from Clitoria fairchildiana seeds (CFPI). Purification was performed by hydrophobic interaction and gel filtration chromatography. Kinetic studies of the purified inhibitor showed a competitive-type inhibitory activity against bovine trypsin and chymotrypsin, with an inhibition stoichiometry of 1:1 for both enzymes. The inhibition constants against trypsin and chymotrypsin were 3.3 x 10(-10) and 1.5 x 10(-10) M, respectively, displaying a tight binding property. SDS-PAGE showed that CFPI has a single polypeptide chain with an apparent molecular mass of 15 kDa under non-reducing conditions. However, MALDI-TOF analysis demonstrated a molecular mass of 7.973 kDa, suggesting that CFPI is dimeric in solution. The N-terminal sequence of CFPI showed homology with members of the Bowman-Birk inhibitor family. CFPI remained stable to progressive heating for 30 min to each temperature range of 37 up to 100 degrees C and CD analysis exhibited no changes in spectra at 207 nm after heating at 90 degrees C and subsequent cooling. Moreover, CFPI was active over a wide pH range (2-10). In contrast, reduction with DTT resulted in a loss of inhibitory activity against trypsin and chymotrypsin. CFPI also exhibited significant inhibitory activity against larval midgut trypsin enzymes from Anagasta kuehniella (76%), Diatraea saccharalis (59%) and Heliothis virescens (49%). Its insecticidal properties were further analysed by bioassays and confirmed by negative impact on A. kuehniella development. (C) 2015 Elsevier Ltd. All rights reserved. (AU)

• Processo FAPESP: 11/09361-0 - Inibidor de tripsina de Entada acaciifolia (EATI): caracterização estrutural e avaliação do potencial inseticida contra a lagarta-do-cartucho (Spodoptera frugiperda)
• Beneficiário: Sergio Marangoni
• Linha de fomento: Auxílio à Pesquisa - Regular