title: Functional and Structural Characterization of a Thermostable Phospholipase A2from a Sparidae Fish (Diplodus annularis).
abstract: Novel phospholipase (PLA2) genes from the Sparidae family were cloned. The sequenced PLA2revealed an identity with pancreatic PLA2group IB. To better understand the structure/function relationships of these enzymes and their evolution, the Diplodus annularis PLA2(DaPLA2) was overexpressed in E. coli. The refolded enzyme was purified by Ni-affinity chromatography and has a molecular mass of 15 kDa as determined by MALDI-TOF spectrometry. Interestingly, unlike the pancreatic type, the DaPLA2was active and stable at higher temperatures, which suggests its great potential in biotechnological applications. The 3D structure of DaPLA2was constructed to gain insights into the functional properties of sparidae PLA2. Molecular docking and dynamic simulations were performed to explain the higher thermal stability and the substrate specificity of DaPLA2. Using the monolayer technique, the purified DaPLA2was found to be active on various phospholipids ranging from 10 to 20 mN·m-1, which explained the absence of the hemolytic activity for DaPLA2.
authors.affiliation: Laboratory of Biochemistry and Enzymatic Engineering of Lipases, ENIS , 3038 Sfax, Tunisia.|Enzymologie Interfaciale et Physiologie de la Lipolyse, UMR7282, CNRS, Aix-Marseille Université , 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 20, France.
authors.forename: Nabil
authors.identifier: http://orcid.org/0000-0003-1416-5378
authors.initials: N
authors.lastname: Smichi
authors.affiliation: Laboratoire des Venins et Biomolécules Thérapeutiques LR11IPT08, Université Tunis-El Manar, Institut Pasteur de Tunis , Tunis 1002, Tunisia.
authors.forename: Houcemeddine
authors.initials: H
authors.lastname: Othman
authors.affiliation: Laboratory of Biochemistry and Enzymatic Engineering of Lipases, ENIS , 3038 Sfax, Tunisia.
authors.forename: Neila
authors.initials: N
authors.lastname: Achouri
authors.affiliation: Univ Lyon, Université Lyon 1, Institut de Chimie et de Biochimie Moléculaires et Supramoléculaires (ICBMS), UMR 5246 CNRS, Métabolisme, Enzymes et Mécanismes Moléculaires (MEM2 , F-69622 Villeurbanne cedex, France.
authors.forename: Alexandre
authors.initials: A
authors.lastname: Noiriel
authors.affiliation: Univ Bordeaux, UMR 5200, Laboratoire de Biogenèse Membranaire , Bat. A3 Campus INRA de Bordeaux 71 avenue E., Bourlaux CS 2003233140 Villenave d'Ornon, France.
authors.forename: Vincent
authors.initials: V
authors.lastname: Arondel
authors.affiliation: Laboratoire des Venins et Biomolécules Thérapeutiques LR11IPT08, Université Tunis-El Manar, Institut Pasteur de Tunis , Tunis 1002, Tunisia.
authors.forename: Najet
authors.initials: N
authors.lastname: Srairi-Abid
authors.affiliation: Univ Lyon, Université Lyon 1, Institut de Chimie et de Biochimie Moléculaires et Supramoléculaires (ICBMS), UMR 5246 CNRS, Métabolisme, Enzymes et Mécanismes Moléculaires (MEM2 , F-69622 Villeurbanne cedex, France.
authors.forename: Abdelkarim
authors.initials: A
authors.lastname: Abousalham
authors.affiliation: Laboratory of Biochemistry and Enzymatic Engineering of Lipases, ENIS , 3038 Sfax, Tunisia.
authors.forename: Youssef
authors.initials: Y
authors.lastname: Gargouri
authors.affiliation: Laboratory of Biochemistry and Enzymatic Engineering of Lipases, ENIS , 3038 Sfax, Tunisia.
authors.forename: Nabil
authors.initials: N
authors.lastname: Miled
authors.affiliation: Laboratory of Biochemistry and Enzymatic Engineering of Lipases, ENIS , 3038 Sfax, Tunisia.
authors.forename: Ahmed
authors.initials: A
authors.lastname: Fendri
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D004795:Enzyme Stability; D029941:Fish Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D005399:Fishes; D006358:Hot Temperature; D007700:Kinetics; D062105:Molecular Docking Simulation; D008969:Molecular Sequence Data; D008970:Molecular Weight; D054467:Phospholipases A2 / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D016415:Sequence Alignment; D013379:Substrate Specificity
chemical_list: D029941:Fish Proteins; D054467:Phospholipases A2
keywords: E. coli expression; Sparidae; monolayer properties; phospholipase A2; refolding and purification; structural characterization; thermostability