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 Ni2+-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.