Identification of active site carboxylic residues in Trichoderma reesei endoglucanase Cell2A by site-directed mutagenesis

摘要

cDNA of Cell2A (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 pAGmeg13 containing mature form Cell2A cDNA produced Cell2A protein largley as insoluble inclusion bodies in the cytoplasm of the cells. The insoluble fraction was solubilized with urea from which Cell2A was purified by cation chromatography to electrophoretic homogeneity. The purified enzyme was immunologically and enzymologically identical to that of Cell2A purified from T. reesei. E116 and E200 of Cell2A of T. seesei are completely conserved in family-12 cellulases. In order to investigate the role of these two glutamate residues in the enzymic funciton of Cell2A, 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 funciton of Cell2A. The data demonstrated that E116 and E200 are the nucleophilic and acid-base residues, respectively.