title: Employing in vitro directed molecular evolution for the selection of α-amylase variant inhibitors with activity toward cotton boll weevil enzyme.
abstract: Numerous species of insect pests attack cotton plants, out of which the cotton boll weevil (Anthonomus grandis) is the main insect in Brazil and must be controlled to avert large economic losses. Like other insect pests, A. grandis secretes a high level of α-amylases in the midgut lumen, which are required for digestion of carbohydrates. Thus, α-amylase inhibitors (α-AIs) represent a powerful tool to apply in the control of insect pests. Here, we applied DNA shuffling and phage display techniques and obtained a combinatorial library containing 10⁸ α-AI variant forms. From this library, variants were selected exhibiting in vitro affinity for cotton boll weevil α-amylases. Twenty-six variant sequences were cloned into plant expression vectors and expressed in Arabidopsis thaliana. Transformed plant extracts were assayed in vitro to select specific and potent α-amylase inhibitors against boll weevil amylases. While the wild type inhibitors, used to create the shuffled library, did not inhibit the A. grandis α-amylases, three α-AI mutants, named α-AIC3, α-AIA11 and α-AIG4 revealed high inhibitory activities against A. grandis α-amylases in an in vitro assay. In summary, data reported here shown the potential biotechnology of new α-AI variant genes for cotton boll weevil control.
authors.affiliation: Embrapa Recursos Genéticos e Biotecnologia, Laboratório Interação Molecular Planta Praga, Asa Norte, Brasília, DF 70770-917, Brazil. Electronic address: cristina.mattar@embrapa.br.
authors.forename: Maria Cristina Mattar
authors.initials: MC
authors.lastname: da Silva
authors.forename: Rafael Perseghini
authors.initials: RP
authors.lastname: Del Sarto
authors.forename: Wagner Alexandre
authors.initials: WA
authors.lastname: Lucena
authors.forename: Daniel John
authors.initials: DJ
authors.lastname: Rigden
authors.forename: Fabíola Rodrigues
authors.initials: FR
authors.lastname: Teixeira
authors.forename: Caroline de Andrade
authors.initials: Cde A
authors.lastname: Bezerra
authors.forename: Erika Valéria Saliba
authors.initials: EV
authors.lastname: Albuquerque
authors.forename: Maria Fatima
authors.initials: MF
authors.lastname: Grossi-de-Sa
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D017360:Arabidopsis / Q000201:enzymology / Q000235:genetics; D061505:Cell Surface Display Techniques; D032621:DNA Shuffling; D019020:Directed Molecular Evolution; D004791:Enzyme Inhibitors / Q000378:metabolism* / Q000494:pharmacology; D014644:Genetic Variation; D003368:Gossypium; D007300:Insect Control; D008969:Molecular Sequence Data; D010936:Plant Extracts / Q000235:genetics / Q000378:metabolism; D030821:Plants, Genetically Modified; D020539:Sequence Analysis, Protein; D048889:Weevils / Q000201:enzymology*; D000516:alpha-Amylases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism
chemical_list: D004791:Enzyme Inhibitors; D010936:Plant Extracts; D000516:alpha-Amylases
keywords: AgA; Anthonomus grandis; Anthonomus grandis α-amylases; DNA shuffling; In vitro molecular evolution; Insect control; MS; Murashige Skoog; α-AI; α-Amylase Inhibitor; α-Amylase inhibitors