EFFECTS OF MUTATIONS IN THE STARCH-BINDING DOMAIN OF BACILLUS-MACERANS CYCLODEXTRIN GLYCOSYLTRANSFERASE

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.