Rapid kinetic study of Cyclomaltodextrinase from alkalophilic Bacillus sp. I-5 by stopped-flow spectrophotometer

Abstract

The dissociation kinetics of the oligomeric states of Cyclomaltodextrinase I-5 (CDase I-5) and the quaternary structures of this enzyme have been investigated. CDase exists in oligomer, as an assembly of six unit of dimer and the equilibrium between the dimer and dodecamer is altered rapidly in the presence of salt like KCl. Changes in oligomeric state can be monitored by KCl change in intrinsic fluorescence of tryptophan residues in CDase I-5. The Circular dichroism demonstrated that this change measurement were distinguished from the denaturation at 1M KCl. The dissociation of this enzyme was also analysed by analytical ultracentrifugation and gel permeation chromatography. The rate constant (kd) of dissociation from dodecamer into dimer was determined at various KCl concentration by stopped-flow spectrophotometer. The dissociation constants in the presence of at 0.2 M KCl and at 1.0 M KCl were 5.96 s-1 and 7.99 s-1, respectively. The results indicated that the dodecameric CDase dissociated into dimer more favorably with increasing concentration of salts.