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Journal of Bacteriology, February 2002, p. 777-784, Vol. 184, No. 3
0021-9193/01/$04.00+0     DOI: 10.1128/JB.184.3.777-784.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

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

Naeem Rashid, Joel Cornista, Satoshi Ezaki, Toshiaki Fukui, Haruyuki Atomi, and Tadayuki Imanaka*

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 (CGTTk) was identified and characterized. The gene (cgtTk) encoded a protein of 713 amino acid residues harboring the four conserved regions found in all members of the {alpha}-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. CGTTk could hydrolyze soluble starch and glycogen but failed to hydrolyze pullulan. Most importantly, although CGTTk 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 (CGTTk{Delta}C) with 23 C-terminal amino acid residues deleted. CGTTk{Delta}C displayed similar properties in terms of starch-binding activity, substrate specificity, and thermostability, but unexpectedly showed higher starch-degrading activity than the parental CGTTk. In contrast, the cyclization activity of CGTTk{Delta}C was abolished. The results indicate that the presence of the structurally novel C-terminal domain is essential for CGTTk to properly catalyze the cyclization reaction.

* Corresponding author. Mailing address: Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Yoshida-Honmachi, Sakyo-ku, Kyoto 606-8501, Japan. Phone: 81-75-753-5568. Fax: 81-75-753-4703. E-mail: imanaka{at}sbchem.kyoto-u.ac.jp.

Journal of Bacteriology, February 2002, p. 777-784, Vol. 184, No. 3
0021-9193/01/$04.00+0     DOI: 10.1128/JB.184.3.777-784.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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