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Heterologous expression and biochemical characterization of an endo‐β‐1,4‐glucanase from Thermobifida fusca
Author(s) -
Yan Peng'an,
Su Lingqia,
Chen Jian,
Wu Jing
Publication year - 2013
Publication title -
biotechnology and applied biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.468
H-Index - 70
eISSN - 1470-8744
pISSN - 0885-4513
DOI - 10.1002/bab.1097
Subject(s) - cellulase , carboxymethyl cellulose , chemistry , enzyme , fermentation , substrate (aquarium) , enzyme assay , heterologous expression , ether , chromatography , biochemistry , sodium dodecyl sulfate , sodium , recombinant dna , organic chemistry , biology , ecology , gene
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 K m and V max 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.