Please use this identifier to cite or link to this item: http://repositorio.unicamp.br/jspui/handle/REPOSIP/242687
Type: Artigo
Title: Bowman-birk proteinase inhibitor from Clitoria fairchildiarta seeds: isolation, biochemical properties and insecticidal potential
Author: Dartzger, Mirian
Vasconcelos, Ilka Maria
Scorsato, valéria
Aparicio, Ricardo
Marangoni, Sergio
Macedo, Maria Lígia Rodrigues
Abstract: 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.
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
Subject: Clitoria fairchildiana
Fabaceae
Proteínas - Purificação
Inibidores de proteinase
Lepidópteros
Pragas de insetos - Controle biológico
Country: Reino Unido
Editor: Elsevier
Citation: Bowman-birk Proteinase Inhibitor From Clitoria Fairchildiarta Seeds: Isolation, Biochemical Properties And Insecticidal Potential. Pergamon-elsevier Science Ltd, v. 118, p. 224-235 OCT-2015.
Rights: fechado
Identifier DOI: 10.1016/j.phytochem.2015.08.013
Address: http://www.sciencedirect.com/science/article/pii/S003194221530073X
Date Issue: 2015
Appears in Collections:IQ - Artigos e Outros Documentos
IB - Artigos e Outros Documentos

Files in This Item:
There are no files associated with this item.


Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.