Heterologous expression and site-directed mutagenesis of endoglucanase CelA from Clostridium thermocellum.
The endoglucanase CelA from Clostridium thermocellum was strongly expressed in Bacillus subtilis. The enzyme was purified by Ni(2+)-affinity chromatography. Site-directed substitution of D278 with an asparagine or an alanine residue surprisingly did not decrease the apparent k(cat) value. Further substitutions of two other potentially critical residues, Y215 and D152, resulted in a 2-fold decrease in apparent k(cat) value for Y215P and complete loss of activity for D152N.
Yao
Qiang
Q
State Key Laboratory of Microbial Technology, School of Life Science, Shandong University, Jinan, Shandong, 250100, P.R. China.
Sun
Tingting
T
Chen
Guanjun
G
Liu
Weifeng
W
D001709:Biotechnology / Q000379:methods*; D020134:Catalytic Domain; D002480:Cellulase / Q000737:chemistry* / Q000235:genetics*; D002482:Cellulose / Q000737:chemistry; D048013:Acetivibrio thermocellus / Q000201:enzymology* / Q000235:genetics*; D004926:Escherichia coli / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009532:Nickel / Q000737:chemistry
D009532:Nickel; D002482:Cellulose; C074775:CelA endoglucanase; D002480:Cellulase