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Purification and characterization of the major β‐1,4‐endoglucanase from Thermomonospora curvata
Author(s) -
Lin ShihBin,
Stutzenberger F.J.
Publication year - 1995
Publication title -
journal of applied bacteriology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.889
H-Index - 156
eISSN - 1365-2672
pISSN - 0021-8847
DOI - 10.1111/j.1365-2672.1995.tb03160.x
Subject(s) - chemistry , cellulase , chromatography , cellulose , carboxymethyl cellulose , ammonium , amino acid , dithiothreitol , thermophile , enzyme , biochemistry , organic chemistry , sodium
The major β‐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°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°C, pH 6.0 and had a half‐life of 30 min at 80°C; temperature and pH optima were 70–73°C and 6.0–6.5, respectively. The mol. wt was 1 and the pI was 4.0. The K m and V max values were 7.33 mg ml −1 and 833 μmol 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.