title: Purification, characterization, and molecular cloning of an extracellular chitinase from Bacillus licheniformis stain LHH100 isolated from wastewater samples in Algeria.
abstract: An extracellular chitinase (ChiA-65) was produced and purified from a newly isolated Bacillus licheniformis LHH100. Pure protein was obtained after heat treatment and ammonium sulphate precipitation followed by Sephacryl S-200 chromatography. Based on matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/MS) analysis, the purified enzyme is a monomer with a molecular mass of 65,195.13 Da. The sequence of the 27 N-terminal residues of the mature ChiA-65 showed high homology with family-18 chitinases. Optimal activity was achieved at pH 4 and 75 °C. 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 ChiA-65 preferentially hydrolyzed the second glycosidic link from the non-reducing end of (GlcNAc)n. ChiA-65 obeyed Michaelis-Menten kinetics, the Km and kcat values being 0.385 mg, colloidal chitin/ml and 5000 s(-1), respectively. The chiA-65 gene encoding ChiA-65 was cloned in Escherichia coli and its sequence was determined. Above all, ChiA-65 exhibited remarkable biochemical properties suggesting that this enzyme is suitable for bioconversion of chitin waste.
authors.affiliation: Laboratory of Environmental Biotechnology and Process Engineering, Department of Environmental Engineering, Ecole Nationale Polytechnique, Avenue Pasteur, Hacène Badi, PO Box 182, El Harrach Algiers, 16200, Algeria; Department of Industrial Chemistry, Faculty of Engineering Science, University of Saàd Dahlab of Blida, PO Box 270 Blida, 09000, Algeria. Electronic address: hassibalar@yahoo.fr.
authors.forename: Hassiba
authors.initials: H
authors.lastname: Laribi-Habchi
authors.affiliation: Laboratory of Cellular and Molecular Biology, Microbiology Team, Faculty of Biological Sciences, University of Science and Technology of Houari Boumediene, PO Box 32, El Alia, Bab Ezzouar Algiers, 16000, Algeria.
authors.forename: Amel
authors.initials: A
authors.lastname: Bouanane-Darenfed
authors.affiliation: Laboratory of Environmental Biotechnology and Process Engineering, Department of Environmental Engineering, Ecole Nationale Polytechnique, Avenue Pasteur, Hacène Badi, PO Box 182, El Harrach Algiers, 16200, Algeria; Centre de Recherche en technologie des Semi-conducteurs pour l'Energétique (CRTSE). 2, Bd Frantz Fanon BP140, Alger - 7 merveilles, 16038, Algeria. Electronic address: nadjibdrouiche@yahoo.fr.
authors.forename: Nadjib
authors.initials: N
authors.lastname: Drouiche
authors.affiliation: Chemical Engineering Department, University of Technology of Compiegne PO Box 20.529, 60205 Compiegne Cedex, France.
authors.forename: André
authors.initials: A
authors.lastname: Pauss
authors.affiliation: Laboratory of Environmental Biotechnology and Process Engineering, Department of Environmental Engineering, Ecole Nationale Polytechnique, Avenue Pasteur, Hacène Badi, PO Box 182, El Harrach Algiers, 16200, Algeria; Centre de Recherche en technologie des Semi-conducteurs pour l'Energétique (CRTSE). 2, Bd Frantz Fanon BP140, Alger - 7 merveilles, 16038, Algeria.
authors.forename: Nabil
authors.initials: N
authors.lastname: Mameri
mesh_terms: D000462:Algeria; D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology* / Q000302:isolation & purification*; D002688:Chitinases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004795:Enzyme Stability / Q000187:drug effects; D005110:Extracellular Space / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D007477:Ions; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010802:Phylogeny; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity / Q000187:drug effects; D013696:Temperature; D062065:Wastewater / Q000382:microbiology*
chemical_list: D002688:Chitinases; D007477:Ions; D008670:Metals; D012336:RNA, Ribosomal, 16S; D062065:Wastewater
keywords: Bacillus licheniformis; Chitinase; Purification