(The)Role of oligomerization in the high activity and thermal stability of cyclomaltodextrinase from alkalophilic bacillus sp. I-5

Abstract

A Cyclomaltodextrinase from alkalophilic Bacillus sp. I-5 (CDase I-5) which catalyzes the hydrolysis of cyclomaltodextrin (CD) to maltose was found to be a dodecamer as a supramolecular assembly consisted of six units of dimer. The previous study has shown that the dodecamer dissociates into dimer at pH 6.0 and associates to dodecameric form at pH 7.0. In order to investigate the dissociation/association process in details, CDase I-5 mutant was generated by site-directed mutagenesis in which H49, H89, H539 in N- and C-terminal region were substituted with valine and H547 in C-terminal region was replaced with lysine. The mutant enzymes reached equilibrium state of dodecamer-dimer depending on enzyme concentration at pH 7.0. The dissociation constant (Kd) was estimated to be 1.84¡¿10?28M5. The specific activity of the enzyme was compared between dodecameric and dimeric form after the dimeric form was prepared at different pH condition. The results revealed that dodecamer of wild-type enzyme had three times higher specific activity of hydrolyzing b-CD than dimer. The denaturation enthalpy (¥ÄH) of the wild-type dodecameric enzyme increased two fold and denaturation temperature (Tm) was shifted to high temperature by 16¡É compared with dimer. In case of CDase I-5 mutant enzyme, dodecamer had three times higher specific activity of hydrolyzing b-CD and two times larger ¥ÄH as shown in the wild-type enzyme. Whereas Tm for the mutant enzyme was 5¡É higher compared with that of dimer. Unlike other CD degrading enzymes which have dimeric structure, CDase I-5 have been evolved into supramolecular structure for adaptation to alkaline evironment, thereby enhancing activity and thermostability through oligomerization.