Analysis of the carbohydrate-binding-module from Fragaria x ananassa α-L-arabinofuranosidase 1.
α-L-arabinofuranosidases (EC 3.2.1.55) are enzymes involved in the catabolism of several cell-wall polysaccharides such as pectins and hemicelluloses, catalyzing the hydrolysis of terminal non-reducing α-L-arabinofuranosil residues. Bioinformatic analysis of the aminoacidic sequences of Fragaria x ananassa α-L-arabinofuranosidases predict a putative carbohydrate-binding-module of the family CBM_4_9, associated to a wide range of carbohydrate affinities. In this study, we report the characterization of the binding affinity profile to different cell wall polysaccharides of the putative CBM of α-L-arabinofuranosidase 1 from Fragaria x ananassa (CBM-FaARA1). The sequence encoding for the putative CBM was cloned and expressed in Escherichia coli, and the resultant recombinant protein was purified from inclusion bodies by a Nickel affinity chromatography under denaturing conditions. The refolded recombinant protein was then subjected to binding assays and affinity gel electrophoresis, which indicated its ability to bind cellulose and also high affinity for homogalacturonans.
INFIVE (CONICET-UNLP), Instituto de Fisiología Vegetal, Diag. 113 n°495 - C.c 327, 1900, La Plata, Argentina.
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INFIVE (CONICET-UNLP), Instituto de Fisiología Vegetal, Diag. 113 n°495 - C.c 327, 1900, La Plata, Argentina.
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Perini
IIB-INTECH (CONICET-UNSAM), Instituto de Investigaciones Biotecnológicas-Instituto Tecnológico de Chascomús, Avenida Intendente Marino km 8,2, B7130IWA, Chascomús, Pcia. Buenos Aires, Argentina; Facultad de Ciencias Exactas, Universidad Nacional de La Plata (UNLP), 47 y 115, 1900, La Plata, Argentina.
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Martínez
INFIVE (CONICET-UNLP), Instituto de Fisiología Vegetal, Diag. 113 n°495 - C.c 327, 1900, La Plata, Argentina; Facultad de Ciencias Exactas, Universidad Nacional de La Plata (UNLP), 47 y 115, 1900, La Plata, Argentina. Electronic address: pmcivello@agro.unlp.edu.ar.
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Civello
D000327:Adsorption; D000595:Amino Acid Sequence; D001681:Biological Assay; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D003198:Computer Simulation; D004591:Electrophoresis, Polyacrylamide Gel; D031985:Fragaria / Q000201:enzymology*; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D058849:Protein Refolding; D055550:Protein Stability; D011956:Receptors, Cell Surface / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012995:Solubility; D013696:Temperature
D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; C055213:saccharide-binding proteins; D006026:Glycoside Hydrolases; C019713:alpha-N-arabinofuranosidase
Carbohydrate binding module (CBM); Cell wall; Heterologous expression; Homogalacturonans; Strawberry; α-L-arabinofuranosidas