title: Purification and characterization of an endoglucanase from Streptomyces lividans 66 and DNA sequence of the gene.
abstract: The endoglucanase isolated from culture filtrates of Streptomyces lividans IAF74 was shown to have an Mr of 46,000 and a pI of 3.3. The specific enzyme activity of 539 IU/mg, determined by the reducing assay method on carboxymethyl cellulose, is among the highest reported in the literature. The cellulase showed typical endo-type activity when reacting on oligocellodextrins. Optimal enzyme activity was obtained at 50 degrees C and pH 5.5. The kinetic constants for this endoglucanase, determined with carboxymethyl cellulose as the substrate, were a Vmax of 24.9 IU/mg of enzyme and a Km of 4.2 mg/ml. Activity was found against neither methylumbelliferyl- nor p-nitrophenyl-cellobiopyranoside nor with xylan. The DNA sequence contains one possible reading frame validated by the N terminus of the mature purified protein. However, neither ATG nor GTG starting codons were identified near the ribosome-binding site. A putative TTG codon was found as a good candidate for the start codon. Comparison of the primary amino acid sequence of the endoglucanase of S. lividans revealed that the N terminus contains a bacterial cellulose-binding domain. The catalytic domain at the C terminus showed similarity to endoglucanases from a Bacillus sp. Thus, the endoglucanase CelA belongs to family A of cellulases as described before (N. R. Gilkes, B. Henrissat, D. G. Kilburn, R. C. Miller, Jr., and R. A. J. Warren, Microbiol. Rev. 55:303-315, 1991.
authors.affiliation: Centre de recherche en microbiologie appliquée, Institut Armand-Frappier, Université du Québec, Laval-des-Rapides, Canada.
authors.forename: M
authors.initials: M
authors.lastname: Théberge
authors.forename: P
authors.initials: P
authors.lastname: Lacaze
authors.forename: F
authors.initials: F
authors.lastname: Shareck
authors.forename: R
authors.initials: R
authors.lastname: Morosoli
authors.forename: D
authors.initials: D
authors.lastname: Kluepfel
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D002480:Cellulase / Q000302:isolation & purification / Q000378:metabolism*; D004269:DNA, Bacterial; D004591:Electrophoresis, Polyacrylamide Gel; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D013302:Streptomyces / Q000201:enzymology*
chemical_list: D004269:DNA, Bacterial; D002480:Cellulase