Purification and characterization of an endoglucanase from Streptomyces lividans 66 and DNA sequence of the gene.
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.
Théberge
M
M
Centre de recherche en microbiologie appliquée, Institut Armand-Frappier, Université du Québec, Laval-des-Rapides, Canada.
Lacaze
P
P
Shareck
F
F
Morosoli
R
R
Kluepfel
D
D
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*
D004269:DNA, Bacterial; D002480:Cellulase