H. Chang et al., EFFECTS OF MUTATIONS IN THE STARCH-BINDING DOMAIN OF BACILLUS-MACERANS CYCLODEXTRIN GLYCOSYLTRANSFERASE, Journal of biotechnology, 65(2-3), 1998, pp. 191-202
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 catal
ysis, we have constructed several mutants of Bacillus macerans CGTase.
Removing the entire E domain resulted in an inactive enzyme. Adding s
ix amino acids between domains D and E caused a decrease in activity a
nd thermostability. Replacing domain E with the similar starch-binding
domain from Aspergillus awamori glucoamylase I caused a drastic decre
ase in activity, indicating the necessity of correct alignment of boun
d substrate. Substituting tyrosine residue 634 (Tyr634) with phenylala
nine had very little effect on activity or thermostability. Substituti
ng Tyr634 with glycine resulted in a 25% increase of specific cyclizat
ion and starch-hydrolyzing activities compared with that of the wild-t
ype enzyme. The latter mutant was less thermostable. The results of th
is study indicate that domain E is important for the stability and int
egrity of B. macerans CGTase, (C) 1998 Elsevier Science B.V. All right
s reserved.