The role of domain E in the activity of Bacillus macerans cyclodextrin glucanotransferase

Abstract

Cyclodextrin glucanotransferase (CGT) is an industrial enzyme used for the production of cyclodextrins (CDs). 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. Two maltose binding sites (MBS1 and MBS2) have been identified in domain E. Domain E participates in the formation of a deep groove which is located on the protein surface of CGT and extends from the active site to MBS2. To investigate the function of domain E in the catalysis of CGT, CGT mutants were constructed using a CGT from Bacillus macerans. The truncated CGT (CGTAE), was constructed by deleting domain E. In the chimeric CGT (CGT-SBD), domain E was replaced with the starch binding domain (SBD) of glucoamylase I (GAI), which has a similar structure and function to domain E. The modified CGT (CGT+6) was constructed by inserting six amino acids between domains D and E. The six amino acids were inserted as a pseudo linker in order to perturb the alignment of the groove by increasing the distance between domains D and E. CGTAE exhibited no detectable activity, whereas CGT-SBD had very low cyclization, starch-degrading, and coupling activities. CGT+6 exhibited about 5060% of the wild type CGT (WT-CGT) activity, and its thennostability was lower than that of WT-CGT. The K^, value for the cyclization activity of CGT+6 was about 2.6 fold greater than that of WT-CGT. The efficiency of the cyclization reaction (kgat /K„) was also significantly reduced.