title: Molecular study and overexpression of the Clostridium cellulolyticum celF cellulase gene in Escherichia coli.
abstract: The CelF-encoding sequence was isolated from Clostridium cellulolyticum genomic DNA using the inverse PCR technique. The gene lies between cipC (the gene encoding the cellulosome scaffolding protein) and celC (coding for the endoglucanase C) in the large cel cluster of this mesophilic cellulolytic Clostridium species. Comparisons between the deduced amino acid sequence of the mature CelF (693 amino acids, molecular mass 77626) and those of other beta-glycanases showed that this enzyme belongs to the recently proposed family L of cellulases (family 48 of glycosyl hydrolases). The protein was overproduced in Escherichia coli using the T7 expression system. It formed both cytoplasmic and periplasmic inclusion bodies when induction was performed at 37 degrees C. Surprisingly, the protein synthesized from the cytoplasmic production vector was degraded in the Ion protease-deficient strain BL21(DE3). The induction conditions were optimized with regard to the concentration of inductor, cell density, and temperature and time of induction in order to overproduce an active periplasmic protein (CelFp) which was both soluble and stable. It was collected using the osmotic shock method. The enzymic degradation of various cellulosic substrates by CelFp was studied. CelFp degraded swollen Avicel more efficiently than substituted soluble CM-cellulose or crystalline Avicel and was not active on xylan. Its activity is therefore quite different from that of endoglucanases, which are most active on CM-cellulose.
authors.affiliation: Bioénergétique et Ingéniérie des protéines, Centre National de la Recherche Scientifique, 31 chemin Joseph Aiguier, BP71, 13402 Marseille Cedex 20, France.
authors.forename: Corinne
authors.initials: C
authors.lastname: Reverbel-Leroy
authors.affiliation: Bioénergétique et Ingéniérie des protéines, Centre National de la Recherche Scientifique, 31 chemin Joseph Aiguier, BP71, 13402 Marseille Cedex 20, France.
authors.forename: Anne
authors.initials: A
authors.lastname: Belaich
authors.affiliation: Laboratoire d'lngéniérie et Dynamique des Systèmes Membranaires, Centre National de la Recherche Scientifique, 31 chemin Joseph Aiguier, BP71, 13402 Marseille Cedex 20, France.
authors.forename: Alain
authors.initials: A
authors.lastname: Bernadac
authors.affiliation: Bioénergétique et Ingéniérie des protéines, Centre National de la Recherche Scientifique, 31 chemin Joseph Aiguier, BP71, 13402 Marseille Cedex 20, France.
authors.forename: Christian
authors.initials: C
authors.lastname: Gaudin
authors.affiliation: Université de Provence, Marseille, France 3.|Bioénergétique et Ingéniérie des protéines, Centre National de la Recherche Scientifique, 31 chemin Joseph Aiguier, BP71, 13402 Marseille Cedex 20, France.
authors.forename: Jean-Pierre
authors.initials: JP
authors.lastname: Belaich
authors.affiliation: Université de Provence, Marseille, France 3.|Bioénergétique et Ingéniérie des protéines, Centre National de la Recherche Scientifique, 31 chemin Joseph Aiguier, BP71, 13402 Marseille Cedex 20, France.
authors.forename: Chantal
authors.initials: C
authors.lastname: Tardif
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002480:Cellulase / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D003013:Clostridium / Q000201:enzymology* / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics* / Q000648:ultrastructure; D015870:Gene Expression; D005798:Genes, Bacterial; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D005810:Multigene Family; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D012995:Solubility
chemical_list: D017931:DNA Primers; D004269:DNA, Bacterial; D002480:Cellulase