Enhancement of the Structure and Biochemical Function of Cyclomaltodextrinase from the Anoxybacillus Flavithermus ZNU-NGA with Site-Directed Mutagenesis.

This study was conducted to examine the role of the central domain of cyclomaltodextrinase in terms of stability, substrate specificity, becoming dodecameric form, and enzyme activity. To this end, H403R/L309V double-point mutation and T280Q single-point mutation were performed at the central domain and (β/α)8-barrel. The results indicated that the activity of the H403R/L309V mutant at the optimal pH and temperature increased by about 25% and 40%, respectively. Plus, the irreversible thermal inactivation of the H403R/L309V mutant at 60 °C and 160 min was approximately twice of the enzyme without mutation. Both mutants underwent significant structural change relative to the wild enzyme and subsequently a significant catalytic activity. However, the catalytic efficiency (kcat/Km) of the H403R/L309V mutant increased in the presence of beta- and gamma-cyclomaltodextrin substrates compared to the wild enzyme and T280Q mutant. As a result, by applying the L309V mutant and given the smaller size of the valine, leucine spatial inhibition in the wild protein seems to decline, and also it facilitates the substrate access to active site amino acids. Moreover, as gamma substrate is larger, eliminating the effect of spatial inhibition on this substrate has a greater effect on improving the catalytic activity of this enzyme.