Functional relationships between cyclodextrin glucanotransferase from an alkalophilic Bacillus and α‐amylases Site‐directed mutagenesis of the conserved two Asp and one Glu residues

Comparison of the amino acid sequences of cyclodextrin glucanotransferases (CGTases) with those of α‐amylases revealed that two Asp and one Glu residues, which are considered to be the catalytic residues in α‐amylases, were also conserved in CGTases. To analyze the function of the three conserved amino acid residues in CGTases, site‐directed mutagenesis was carried out. The three mutant CGTases, in which Asp 229 , Glu 257 and Asp 328 were individually replaced by Asn or Gln, completely lost both their starch‐degrading and β‐cyclodextrin‐forming activities, whereas another mutant CGTase, in which Glu 264 replaced by Gln, retained these activities. The three inactive enzymes retained the ability to be bound to starch. These results suggest that Asp 229 , Glu 257 and Asp 328 play an important role in the enzymatic reaction catalyzed by CGTase and that a similar catalytic mechanism is present in both CGTases and α‐amylases.