title: Characterization of two Acanthoscelides obtectus alpha-amylases and their inactivation by wheat inhibitors.
abstract: Wheat alpha-amylase inhibitors represent an important tool in engineering crop plants against bean bruchids. Because Acanthoscelides obtectus is a devastating storage bean insect-pest, we attempted to purify and characterize its gut alpha-amylases, to study their interaction with active proteinaceous inhibitors. Two digestives alpha-amylases (AoA1 and AoA2) were purified from gut larvae, showing molecular masses of 30 and 45 kDa for each one, respectively. The stoichiometry interaction between these alpha-amylases with two wheat inhibitors (0.19 and 0.53) showed a binding complex of 1:1 enzyme:inhibitor. In vivo activities of these inhibitors against A. obtectus were also evaluated using a rich ammonium sulfate inhibitor fraction (F(20)(-)(40)) and purified inhibitors after reversed phase high-performance liquid chromatography columns. Incorporation of three different inhibitor concentrations (0.25, 0.5, and 1.0% w/w) into artificial seeds showed that addition of the purified 0.19 inhibitor at the highest concentration (1.0%) reduced the larval weight by 80%. Similar data were observed when 0.53 inhibitor was incorporated at 0.5%. When the concentration of purified 0.53 was enhanced to 1.0%, no larvae or adult emergence were observed. Our data suggest that these alpha-amylase inhibitors present great potential for use in Phaseolus genetic improvement programs.
authors.affiliation: EMBRAPA/Recursos Genéticos e Biotecnologia, Brasília-DF, Brazil, Universidade Católica de Brasília, Brasília-DF, Brazil. ocfranco@pos.ucb.br
authors.forename: Octávio L
authors.initials: OL
authors.lastname: Franco
authors.forename: Francislete R
authors.initials: FR
authors.lastname: Melo
authors.forename: Paulo A
authors.initials: PA
authors.lastname: Mendes
authors.forename: Norma S
authors.initials: NS
authors.lastname: Paes
authors.forename: Massaru
authors.initials: M
authors.lastname: Yokoyama
authors.forename: Marise V
authors.initials: MV
authors.lastname: Coutinho
authors.forename: Carlos
authors.initials: C
authors.lastname: Bloch
authors.forename: Maria F
authors.initials: MF
authors.lastname: Grossi-de-Sá
mesh_terms: D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D007814:Larva / Q000201:enzymology; D027805:Phaseolus; D014908:Triticum / Q000737:chemistry*; D048889:Weevils / Q000201:enzymology*; D000516:alpha-Amylases / Q000037:antagonists & inhibitors* / Q000302:isolation & purification
chemical_list: D004791:Enzyme Inhibitors; D000516:alpha-Amylases