Identification of active site carboxylic residues in Trichoderma reesei endoglucanase Cel12A by site-directed mutagenesis

cDNA of Cel12A (formerly EG III), one of five endoglucanases of Trichoderma reesei, was expressed in Escherichia coli by using the tac promoter of E. coli. Transformants of E. coli harboring a plasmid pAGmegl3 containing mature form Cel12A cDNA produced Cel12A protein largely as insoluble inclusion bodies in the cytoplasm of the cells. The insoluble fraction was solubilized with urea from which Cel12A was purified by cation chromatography to electrophoretic homogeneity. The purified enzyme was immunologically and enzymologically identical to that of Cel12A purified from T. reesei. E116 and E200 of Cel12A of T. reesei are completely conserved in family-12 cellulases. In order to investigate the role of these two glutamate residues in the enzymic function of Cel12A, two mutant enzymes were produced at each position, namely E116D/Q and E200D/Q. The specific activity of these mutant enzymes is reduced by more than 98%, revealing the importance of these two residues to the catalytic function of Cel12A. The data demonstrated that E116 and E200 are the nucleophilic and acid-base residues, respectively.