A highly active α-cyclodextrin preferring cyclomaltodextrinase from Geobacillus thermopakistaniensis

Cyclomaltodextrinases show diverse hydrolyzing and/or transglycosylation activities against cyclodextrins, starch and pullulan. A gene annotated as cyclomaltodextrinase from Geobacillus thermopakistaniensis was cloned and overexpressed in Escherichia coli. The gene product, CDase_Gt, was purified and biochemically characterized. The recombinant enzyme exhibited highest activity with α-cyclodextrin at 55 °C and pH 6.0. Specific hydrolytic activities towards α-, β- and γ-cyclodextrin were 1200, 735 and 360 μmol min^-1 mg^-1, respectively. To the best of our knowledge, the activity against α-cyclodextrin is the highest among the reported enzymes. Next to cyclodextrins, pullulan was the most preferred substrate with a specific activity of 105 μmol min^-1 mg^-1. CDase_Gt was capable of hydrolysis of maltotriose and acarbose as well as transglycosylation of their hydrolytic products. At 65 °C, there was no significant loss in enzyme activity even after overnight incubation. Activity of CDase_Gt was not metal ions dependent, however, the presence of Mn²⁺ significantly enhanced the α-CDase activity. EDTA had no significant effect on the CDase_Gt activity, however, it enhanced the thermostability of the enzyme. CDase_Gt existed in monomeric as well as dimeric form in solution. Dimeric form is more active compared to the monomeric one. Equilibrium between the two forms seems to be concentration dependent.