Abstract
Amylomaltase can be used to synthesize large ring cyclodextrins (LR-CDs), applied as drug solubilizer, gene delivery vehicle and protein aggregation suppressor. This study aims to determine the functional amino acid positions of Corynebacterium glutamicum amylomaltase (CgAM) involved in LR-CD synthesis by site-directed mutagenesis approach and molecular dynamic simulation. Mutants named Δ167, Y23A, P228Y, E231Y, A413F and G417F were constructed, purified, and characterized. The truncated CgAM, Δ167 exhibited no starch transglycosylation activity, indicating that the N-terminal domain of CgAM is necessary for enzyme activity. The P228Y, A413F and G417F produced larger LR-CDs from CD36-CD40 as compared to CD29 by WT. A413F and G417F mutants produced significantly low LR-CD yield compared to the WT. The A413F mutation affected all tested enzyme activities (starch tranglycosylation, disproportionation and cyclization), while the G417F mutation hindered the cyclization activity. P228Y mutation significantly lowered the k cat of disproportionation activity, while E231Y mutant exhibited much higher k cat and K m values for starch transglycosylation, compared to that of the WT. In addition, Y23A mutation affected the kinetic parameters of starch transglycosylation and cyclization. Molecular dynamic simulation further confirmed these mutations' impacts on the CgAM and LR-CD interactions. Identified functional amino acids for LR-CD synthesis may serve as a model for future modification to improve the properties and yield of LR-CDs.