Date of Award
Doctor of Philosophy
Food Science and Human Nutrition
Zivko L. Nikolov
Cyclodextrin glucanotransferase (CGT) folds into five domains A, B, C, D and E. Domain E has the ability to bind starch and cyclodextrins (CDs) and is considered the starch-binding domain of CGT. The role of domain E in the catalysis of Bacillus macerans cyclodextrin glucanotransferase was investigated by constructing five CGT mutants. The truncated CGT (CGT[delta]E) mutant was constructed by deleting domain E, and the chimeric CGT (CGT-SBD) by replacing domain E with the starch-binding domain (SBD) of Aspergillus glucoamylase I. The modified CGT (CGT+6) was constructed by inserting a six-amino-acid pseudo linker between domains D and E. The Tyr634 residue in the maltose binding site in Domain E (MBS2) was replaced with a glycine and phenylalanine residue, respectively, to produce the Y634G and Y634F mutants;CGT[delta]E exhibited no detectable activity, whereas CGT-SBD had very low cyclization, starch-degrading, and coupling activities. CGT+6 exhibited about 50-60% of the wild-type CGT (WT-CGT) activity, and its thermostability was lower than that of WT-CGT. The Km value of the cyclization activity of CGT+6 was about 2.6 fold greater than that of WT-CGT. The overall properties of the Y634F mutant were similar to that of WT-CGT. The thermostability of the Y634G mutant was significantly lower than that of WT-CGT, Y634F, and CGT+6. The starch substrate provided little stabilization to Y634G indicating that the aromatic side chains of Tyr634 or Phe634 participated in binding soluble starch. The Km value of the cyclization reaction with Y634G was about 1.4 times greater than that of WT-CGT. The results of this study suggest that domain E cooperates with the catalytic domain in binding long substrates. The groove, extending from the active site to domain E, can be considered as an extension of the active site cleft which assists in the binding and/or directing the binding of long substrates in the active site. The optimal orientation and distance of domain E from the catalytic domain appears important for maintaining the integrity of the groove structure.
Digital Repository @ Iowa State University, http://lib.dr.iastate.edu/
Chang, Hai-yin, "The role of domain E in the activity of Bacillus macerans cyclodextrin glucanotransferase " (1997). Retrospective Theses and Dissertations. 11781.