The maltose-binding site 1 (MBS1) located in the E-domain of beta-cyclodextrin glucanotransferase (beta-CGTase) from Bacillus firmus var. alkalophilus was modified through site-directed mutagenesis, and five mutants, deleting whole MBS1 and four replacing tryptophan residue (W) 652 with the non-hydrophobic glycine (G) and the hydrophobic phenylalanine (F), tyrosine (Y), and leucine (L), respectively, were constructed. The catalytic function of mutants deleting the MBS1 and replacing with the non-hydrophobic glycine changed significantly, however, other mutants remained unchanged. The MBS1 in E-domain is closely connected with the cyclization reaction of beta-CGTase rather than the coupling or starch-hydrolysis reactions.