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Journal of Bacteriology, September 2001, p. 5050-5057, Vol. 183, No. 17
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.17.5050-5057.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Extracellular Synthesis, Specific Recognition, and Intracellular Degradation of Cyclomaltodextrins by the Hyperthermophilic Archaeon Thermococcus sp. Strain B1001

Yoshiteru Hashimoto,1,dagger Tomoko Yamamoto,2 Shinsuke Fujiwara,2 Masahiro Takagi,2,Dagger and Tadayuki Imanaka3,*

CREST1 and Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Osaka 565-0871,2 and Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Sakyo-ku, Kyoto 606-8501,3 Japan

Received 11 December 2000/Accepted 4 June 2001

A unique extracellular and thermostable cyclomaltodextrin glucanotransferase (CGTase) from the hyperthermophilic archaeon Thermococcus sp. strain B1001 produces predominantly (>85%) alpha -cyclomaltodextrin (alpha -CD) from starch (Y. Tachibana, et al., Appl. Environ. Microbiol. 65:1991-1997, 1999). Nucleotide sequencing of the CGTase gene (cgtA) and its flanking region was performed, and a cluster of five genes was found, including a gene homolog encoding a cyclomaltodextrinase (CDase) involved in the degradation of CDs (cgtB), the gene encoding CGTase (cgtA), a gene homolog for a CD-binding protein (CBP) (cgtC), and a putative CBP-dependent ABC transporter involved in uptake of CDs (cgtDE). The CDase was expressed in Escherichia coli and purified. The optimum pH and temperature for CD hydrolysis were 5.5 and 95°C, respectively. The molecular weight of the recombinant enzyme was estimated to be 79,000. The CDase hydrolyzed beta -CD most efficiently among other CDs. Maltose and pullulan were not utilized as substrates. Linear maltodextrins with a small glucose unit were very slowly hydrolyzed, and starch was hydrolyzed more slowly. Analysis by thin-layer chromatography revealed that glucose and maltose were produced as end products. The purified recombinant CBP bound to maltose as well as to alpha -CD. However, the CBP exhibited higher thermostability in the presence of alpha -CD. These results suggested that strain B1001 possesses a unique metabolic pathway that includes extracellular synthesis, transmembrane uptake, and intracellular degradation of CDs in starch utilization. Potential advantages of this starch metabolic pathway via CDs are discussed.

* Corresponding author. Mailing address: Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto, University, 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.

dagger Present address: Institute of Applied Biochemistry, The University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8572, Japan.

Dagger Present address: School of Materials Science, Japan Advanced Institute of Science and Technology, Hokuriku, 1-1 Asahidai Tatsunokuchi, Ishikawa 923-1292, Japan.

Journal of Bacteriology, September 2001, p. 5050-5057, Vol. 183, No. 17
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.17.5050-5057.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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