title: A Novel Three Domains Glycoside Hydrolase Family 3 from Sclerotinia sclerotiorum Exhibits β-Glucosidase and Exoglucanase Activities: Molecular, Biochemical, and Transglycosylation Potential Analysis.
abstract: The filamentous fungus Sclerotinia sclerotiorum produces a complete set of cellulolytic enzymes. We report here the purification and the biochemical characterization of a new β-glucosidase from S. sclerotiorum which belongs to the family 3 of glycoside hydrolases and that was named as SsBgl3. After two size-exclusion chromatography steps, purified protein bands of 80 and 90 kDa from SDS-PAGE were subjected to a mass spectrometry analysis. The results displayed four peptides from the upper band belonging to a polypeptide of 777 amino acids having a calculated molecular weight of 83.7 kDa. Biochemical analysis has been carried out to determine some properties. We showed that this SsBgl3 protein displayed both β-glucosidase and exoglucanase activities with optimal activity at 55 °C and at pH 5. The transglycosylation activity was investigated using gluco-oligosaccharides TLC analysis. The molecular modeling and comparison with different crystal structures of β-glucosidases showed that SsBgl3 putative protein present three domains. They correspond to an (α/β)8 domain TIM barrel, a five-stranded α/β sandwich domain (both of which are important for active-site organization), and a C-terminal fibronectin type III domain. Enzyme engineering will be soon investigated to identify the key residues for the catalytic reactions.
authors.affiliation: Laboratoire d'Ingénierie des Protéines et des Molécules Bioactives (LIP-MB), LR11ES24, National Institute of Applied Sciences and Technology, University of Carthage, 1080, Tunis Cedex, Tunisia. chahed_haifa@yahoo.fr.
authors.forename: Haifa
authors.initials: H
authors.lastname: Chahed
authors.affiliation: Laboratoire d'Ingénierie des Protéines et des Molécules Bioactives (LIP-MB), LR11ES24, National Institute of Applied Sciences and Technology, University of Carthage, 1080, Tunis Cedex, Tunisia.
authors.forename: Aymen
authors.initials: A
authors.lastname: Ezzine
authors.affiliation: Laboratoire Polymères Biopolymères Surfaces (PBS), UMR 6270 CNRS, Université de Rouen, 76821, Mont-Saint-Aignan Cedex, France.
authors.forename: Mohamed Amine Ben
authors.initials: MA
authors.lastname: Mlouka
authors.affiliation: Laboratoire Polymères Biopolymères Surfaces (PBS), UMR 6270 CNRS, Université de Rouen, 76821, Mont-Saint-Aignan Cedex, France.
authors.forename: Christophe
authors.initials: C
authors.lastname: Rihouey
authors.affiliation: Laboratoire Polymères Biopolymères Surfaces (PBS), UMR 6270 CNRS, Université de Rouen, 76821, Mont-Saint-Aignan Cedex, France.
authors.forename: Julie
authors.initials: J
authors.lastname: Hardouin
authors.affiliation: Laboratoire Polymères Biopolymères Surfaces (PBS), UMR 6270 CNRS, Université de Rouen, 76821, Mont-Saint-Aignan Cedex, France.
authors.forename: Thierry
authors.initials: T
authors.lastname: Jouenne
authors.affiliation: Laboratoire d'Ingénierie des Protéines et des Molécules Bioactives (LIP-MB), LR11ES24, National Institute of Applied Sciences and Technology, University of Carthage, 1080, Tunis Cedex, Tunisia.
authors.forename: M Nejib
authors.initials: MN
authors.lastname: Marzouki
mesh_terms: D001203:Ascomycota / Q000201:enzymology*; D002850:Chromatography, Gel; D003198:Computer Simulation; D004591:Electrophoresis, Polyacrylamide Gel; D005656:Fungal Proteins / Q000737:chemistry* / Q000302:isolation & purification; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000302:isolation & purification; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D011487:Protein Conformation; D040901:Proteomics; D015203:Reproducibility of Results; D013379:Substrate Specificity; D001617:beta-Glucosidase / Q000737:chemistry* / Q000302:isolation & purification
chemical_list: D005656:Fungal Proteins; D006026:Glycoside Hydrolases; D001617:beta-Glucosidase
keywords: Glycoside hydrolase; Modeling; Sclerotinia sclerotiorum; Transglycosylation; β-glucosidase