title: Recombinant β-1,3-1,4-glucanase from Theobroma cacao impairs Moniliophthora perniciosa mycelial growth.
abstract: In this work, we identified a gene from Theobroma cacao L. genome and cDNA libraries, named TcGlu2, that encodes a β-1,3-1,4-glucanase. The TcGlu2 ORF was 720 bp in length and encoded a polypeptide of 239 amino acids with a molecular mass of 25.58 kDa. TcGlu2 contains a conserved domain characteristic of β-1,3-1,4-glucanases and presented high protein identity with β-1,3-1,4-glucanases from other plant species. Molecular modeling of TcGlu2 showed an active site of 13 amino acids typical of glucanase with β-1,3 and 1,4 action mode. The recombinant cDNA TcGlu2 obtained by heterologous expression in Escherichia coli and whose sequence was confirmed by mass spectrometry, has a molecular mass of about 22 kDa (with His-Tag) and showed antifungal activity against the fungus Moniliophthora perniciosa, causal agent of the witches' broom disease in cacao. The integrity of the hyphae membranes of M. perniciosa, incubated with protein TcGlu2, was analyzed with propidium iodide. After 1 h of incubation, a strong fluorescence emitted by the hyphae indicating the hydrolysis of the membrane by TcGlu2, was observed. To our knowledge, this is the first study of a cacao β-1,3-1,4-glucanase expression in heterologous system and the first analysis showing the antifungal activity of a β-1,3-1,4-glucanase, in particular against M. perniciosa.
authors.affiliation: Centro de Biotecnologia e Genética, Universidade Estadual de Santa Cruz (UESC), Rodovia Ilhéus-Itabuna, km 16, Ilhéus, BA, 45662-900, Brazil.
authors.forename: Dahyana Santos
authors.initials: DS
authors.lastname: Britto
authors.forename: Carlos Priminho
authors.initials: CP
authors.lastname: Pirovani
authors.forename: Bruno Silva
authors.initials: BS
authors.lastname: Andrade
authors.forename: Tassiara Pereira
authors.initials: TP
authors.lastname: Dos Santos
authors.forename: Cristina
authors.initials: C
authors.lastname: Pungartnik
authors.forename: Júlio Cezar M
authors.initials: JC
authors.lastname: Cascardo
authors.forename: Fabienne
authors.initials: F
authors.lastname: Micheli
authors.forename: Abelmon S
authors.initials: AS
authors.lastname: Gesteira
mesh_terms: D000363:Agaricales / Q000187:drug effects* / Q000254:growth & development; D002099:Cacao / Q000201:enzymology* / Q000382:microbiology; D004926:Escherichia coli; D005453:Fluorescence; D043326:Glucan 1,3-beta-Glucosidase / Q000235:genetics / Q000494:pharmacology*; D013058:Mass Spectrometry; D008958:Models, Molecular; D025282:Mycelium / Q000187:drug effects* / Q000254:growth & development; D011419:Propidium; D011994:Recombinant Proteins / Q000235:genetics / Q000494:pharmacology*
chemical_list: D011994:Recombinant Proteins; D011419:Propidium; D043326:Glucan 1,3-beta-Glucosidase