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Characterization of an Archaeal Cyclodextrin Glucanotransferase with a Novel C-Terminal Domain

Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Yoshida-Honmachi, Sakyo-ku, Kyoto 606-8501, Japan

Received 11 June 2001/ Accepted 31 October 2001

A gene encoding a cyclodextrin glucanotransferase (CGTase) from Thermococcus kodakaraensis KOD1 (CGT_Tk) was identified and characterized. The gene (cgt_Tk) encoded a protein of 713 amino acid residues harboring the four conserved regions found in all members of the -amylase family. However, the C-terminal domain corresponding to domain E of previously known CGTases displayed a completely distinct primary structure. In order to elucidate the catalytic function of the gene product, the recombinant enzyme was purified by anion-exchange chromatography, and its enzymatic properties were investigated. The enzyme displayed significant starch-degrading activity (750 U/mg of protein) with an optimal temperature and pH of 80°C and 5.5 to 6.0, respectively. The presence of Ca²⁺ enhanced the enzyme activity and elevated the optimum temperature to 85 to 90°C. With the addition of Ca²⁺, the enzyme showed extreme thermostability, with almost no loss of enzymatic activity after 80 min at 85°C, and a half-life of 20 min at 100°C. CGT_Tk could hydrolyze soluble starch and glycogen but failed to hydrolyze pullulan. Most importantly, although CGT_Tk harbored a unique C-terminal domain, we found that the protein also exhibited significant CGTase activity, with ß-cyclodextrin as the main product. In order to identify the involvement, if any, of the C-terminal region in the CGTase activity, we analyzed a truncated protein (CGT_TkC) with 23 C-terminal amino acid residues deleted. CGT_TkC displayed similar properties in terms of starch-binding activity, substrate specificity, and thermostability, but unexpectedly showed higher starch-degrading activity than the parental CGT_Tk. In contrast, the cyclization activity of CGT_TkC was abolished. The results indicate that the presence of the structurally novel C-terminal domain is essential for CGT_Tk to properly catalyze the cyclization reaction.

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