title: Purification and characterization of a highly thermostable chitinase from the stomach of the red scorpionfish Scorpaena scrofa with bioinsecticidal activity toward cowpea weevil Callosobruchus maculatus (Coleoptera: Bruchidae).
abstract: This present study is the first attempt to report on the purification and characterization of a chitinase from the stomach of the red scorpionfish Scorpaena scrofa. A 50-kDa chitinase (SsChi50) was purified to homogeneity, and matrix assisted laser desorption ionization-time of flight/mass spectrometry (MALDI-TOF/MS) analysis showed that SsChi50 was a monomer with a molecular mass of 50,103 Da. The 25 N-terminal residues of SsChi50 displayed high homology with family-18 chitinases. Optimal activity was obtained at pH 5.0 at 80 °C. SsChi50 was stable at pH and temperature ranges of 3.0 to 7.0 and 70 to 90 °C for 48 and 4 h respectively. Among the inhibitors and metals tested, p-chloromercuribenzoic acid, N-ethylmaleimide, Hg(2+), and Hg(+) completely inhibited enzyme activity. Chitinase activity was high on colloidal chitin, glycol chitin, glycol chitosane, chitotriose, and chitooligosaccharide. Chitinase activity towards synthetic substrates in the order of p-NP-(GlcNAc)(n) (n = 2-4) was p-NP-(GlcNAc)(2) > p-NP-(GlcNAc)(4) > p-NP-(GlcNAc)(3). Our results suggest that the SsChi50 enzyme preferentially hydrolyzed the second glycosidic link from the non-reducing end of (GlcNAc)(n). This enzyme obeyed Michaelis-Menten kinetics, the K(m) and k(cat) values being 0.412 mg, colloidal chitin mL(-1) and 5.33 s(-1) respectively. An in vivo bioinsecticidal assay was developed for SsChi50 against Callosobruchus maculatus adults. The enzyme showed bioinsecticidal activity toward Callosobruchus maculatus, indicating the possibility of using it in biotechnological strategies for insect management for stored cowpea seeds.
authors.affiliation: Laboratory of Environmental Biotechnology and Process Engineering (LEBPE), Department of Environmental Engineering, Ecole Nationale Polytechnique (ENP), Avenue Pasteur, Hacène Badi, PO Box 182, El Harrach, 16200 Algiers, Algeria. hassibalar@yahoo.fr
authors.forename: Hassiba
authors.initials: H
authors.lastname: Laribi-Habchi
authors.forename: Maya
authors.initials: M
authors.lastname: Dziril
authors.forename: Abdelmalek
authors.initials: A
authors.lastname: Badis
authors.forename: Samia
authors.initials: S
authors.lastname: Mouhoub
authors.forename: Nabil
authors.initials: N
authors.lastname: Mameri
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002686:Chitin / Q000378:metabolism; D002688:Chitinases / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D007887:Fabaceae / Q000469:parasitology; D029941:Fish Proteins / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007306:Insecticides / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D007700:Kinetics; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010473:Perciformes / Q000378:metabolism*; D010572:Pest Control, Biological; D013270:Stomach / Q000737:chemistry / Q000201:enzymology*; D013379:Substrate Specificity; D048889:Weevils / Q000187:drug effects* / Q000502:physiology
chemical_list: D029941:Fish Proteins; D007306:Insecticides; D002686:Chitin; D002688:Chitinases