title: Purification and characterization of the major beta-1,4-endoglucanase from Thermomonospora curvata.
abstract: The major beta-1,4-endoglucanase (EG) of the thermophilic actinomycete, Thermomonospora curvata, contributed over 80% of the total EG activity recovered from cell-free culture fluid after growth on cellulose. The enzyme was purified to electrophoretic homogeneity by ammonium sulphate precipitation, ion-exchange chromatography and size exclusion HPLC. This monomeric enzyme had a specific activity of 750 IU mg(-1) when assayed with 2.5% (w/v) carboxymethyl cellulose (CMC) at 70 degrees C, pH 6.0. Highest activity was observed on CMC with a degree of polymerization of 3200. The EG was stable for 48 h at 60 degrees C, pH 6.0 and had a half-life of 30 min at 80 degrees C; temperature and pH optima were 70-73 degrees C and 6.0-6.5, respectively. The mol. wt was 100,000 and the pI was 4.0. The Km and Vmax values were 7.33 mg/ml(-1) and 833 microns min(-1), respectively. EG activity was inhibited by Fe(2+), Hg(2+), Ag(+) and Pb(2+), and enhanced by dithiothreitol and Zn(2+). The first 12 amino acid residues at the N-terminus were: Asp-Glu-Val-Asp-Glu-Ile-Arg-Asn-Gly-Asp-Phe-Ser. Glutamic and aspartic acid constituted 24% of the total amino acid composition; no amino sugar was found.
authors.affiliation: Department of Microbiology, Clemson University, SC, USA.
authors.forename: S B
authors.initials: SB
authors.lastname: Lin
authors.forename: F J
authors.initials: FJ
authors.lastname: Stutzenberger
mesh_terms: D000192:Actinomycetales / Q000201:enzymology*; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D002266:Carboxymethylcellulose Sodium / Q000378:metabolism; D002412:Cations / Q000494:pharmacology; D002480:Cellulase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D013696:Temperature
chemical_list: D000596:Amino Acids; D002412:Cations; D002480:Cellulase; D002266:Carboxymethylcellulose Sodium