title: Heterologous expression and biochemical characterization of an endo-β-1,4-glucanase from Thermobifida fusca.
abstract: The endoglucanase Cel5A from Thermobifida fusca was cloned and expressed in Escherichia coli BL21(DE3). The carboxymethyl cellulase (CMCase) activity in shake flasks and 3-L fermentation scale reached 46.8 and 656.6 IU/mL, respectively. The CMCase activity in 3-L fermentation scale represented the highest yield of T. fusca Cel5A reported so far. Recombinant Cel5A was purified and characterized in detail. The optimum temperature of recombinant enzyme was 80 °C, and the half-life of the enzyme was 132 H at 50 °C and 65 H at 60 °C. The activity of recombinant Cel5A was retained more than 90% over the range of pH 5.0-10.0 with maximal activity at pH 5.5. Using carboxymethyl cellulose as the substrate, the Km and Vmax values were 5.1 mg/mL and 48.7 IU/mg, respectively. The enzyme showed superstability in surfactants and was retained above 90% activity after treatment with sodium dodecyl sulfate, linear alkyl benzene sulfonate, fatty alcohol polyoxyethylene (9) ether, and polyoxyethylene (10) nonyl phenyl ether at 25 °C for 1 H, indicating that the enzyme could be a valuable component in detergents. The potential mechanism of this stability was investigated by analysis of the electrostatic potential of the surface of the enzyme.
authors: State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, PR China.
authors: Peng'an
authors: P
authors: Yan
authors: Lingqia
authors: L
authors: Su
authors: Jian
authors: J
authors: Chen
authors: Jing
authors: J
authors: Wu
mesh_terms: D000192:Actinomycetales / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism*; D002480:Cellulase / Q000235:genetics* / Q000378:metabolism*; D003001:Cloning, Molecular / Q000379:methods; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005285:Fermentation / Q000235:genetics; D006207:Half-Life; D006863:Hydrogen-Ion Concentration; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013696:Temperature
chemical_list: D011994:Recombinant Proteins; C407024:endoglucanase Cel5A; D002480:Cellulase