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Abstract
Cyclodextrin glycosyltransferase (CGTase) is an industrially important enzyme that
produces cyclodextrins (CD) from starch by intramolecular transglycosylation. CGTase
consists of five globular domains labeled A through E. To better understand the role
of domain E in CGTase catalysis, we have constructed several mutants of Bacillus macerans
CGTase. Removing the entire E domain resulted in an inactive enzyme. Adding six amino
acids between domains D and E caused a decrease in activity and thermostability. Replacing
domain E with the similar starch-binding domain from Aspergillus awamori glucoamylase
I caused a drastic decrease in activity, indicating the necessity of correct alignment
of bound substrate. Substituting tyrosine residue 634 (Tyr634) with phenylalanine
had very little effect on activity or thermostability. Substituting Tyr634 with glycine
resulted in a 25% increase of specific cyclization and starch-hydrolyzing activities
compared with that of the wild-type enzyme. The latter mutant was less thermostable.
The results of this study indicate that domain E is important for the stability and
integrity of B. macerans CGTase.