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Applied and Environmental Microbiology, May 1999, p. 1991-1997, Vol. 65, No. 5
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Purification and Characterization of an Extremely Thermostable Cyclomaltodextrin Glucanotransferase from a Newly Isolated Hyperthermophilic Archaeon, a Thermococcus sp.

Yoshihisa Tachibana,1,* Akiko Kuramura,1 Naoki Shirasaka,1 Yuji Suzuki,2 Tomoko Yamamoto,3 Shinsuke Fujiwara,3 Masahiro Takagi,3 and Tadayuki Imanaka4

Research and Development Center, Nagase Co., Ltd., 2-2-3 Murotani, Nishi-ku, Kobe 651-2241,1 Nagase Biochemicals, Ltd., 1-52 Osadano-cho, Fukuchiyama, Kyoto 620-0853,2 Department of Biotechnology, Faculty of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871,3 and Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto 606-8501,4 Japan

Received 4 December 1998/Accepted 25 February 1999

The extremely thermophilic anaerobic archaeon strain B1001 was isolated from a hot-spring environment in Japan. The cells were irregular cocci, 0.5 to 1.0 µm in diameter. The new isolate grew at temperatures between 60 and 95°C (optimum, 85°C), from pH 5.0 to 9.0 (optimum, pH 7.0), and from 1.0 to 6.0% NaCl (optimum, 2.0%). The G+C content of the genomic DNA was 43.0 mol%. The 16S rRNA gene sequencing of strain B1001 indicated that it belongs to the genus Thermococcus. During growth on starch, the strain produced a thermostable cyclomaltodextrin glucanotransferase (CGTase). The enzyme was purified 1,750-fold, and the molecular mass was determined to be 83 kDa by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Incubation at 120°C with SDS and 2-mercaptoethanol was required for complete unfolding. The optimum temperatures for starch-degrading activity and cyclodextrin synthesis activity were 110 and 90 to 100°C, respectively. The optimum pH for enzyme activity was pH 5.0 to 5.5. At pH 5.0, the half-life of the enzyme was 40 min at 110°C. The enzyme formed mainly alpha -cyclodextrin with small amounts of beta - and gamma -cyclodextrins from starch. This is the first report on the presence of the extremely thermostable CGTase from hyperthermophilic archaea.

* Corresponding author. Mailing address: Research and Development Center, Nagase Co., Ltd., 2-2-3 Murotani, Nishi-ku, Kobe 651-2241, Japan. Phone: (81)-78-992-3164. Fax: (81)-78-992-1050. E-mail: yoshihisa.tachibana{at}nagase.co.jp.

Applied and Environmental Microbiology, May 1999, p. 1991-1997, Vol. 65, No. 5
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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