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Journal of Bacteriology, September 2003, p. 5175-5181, Vol. 185, No. 17
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.17.5175-5181.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Characterization of an Exo-ß-D-Glucosaminidase Involved in a Novel Chitinolytic Pathway from the Hyperthermophilic Archaeon Thermococcus kodakaraensis KOD1

Takeshi Tanaka, 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 17 March 2003/ Accepted 16 June 2003

We previously clarified that the chitinase from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 produces diacetylchitobiose (GlcNAc2) as an end product from chitin. Here we sought to identify enzymes in T. kodakaraensis that were involved in the further degradation of GlcNAc2. Through a search of the T. kodakaraensis genome, one candidate gene identified as a putative ß-glycosyl hydrolase was found in the near vicinity of the chitinase gene. The primary structure of the candidate protein was homologous to the ß-galactosidases in family 35 of glycosyl hydrolases at the N-terminal region, whereas the central region was homologous to ß-galactosidases in family 42. The purified protein from recombinant Escherichia coli clearly showed an exo-ß-D-glucosaminidase (GlcNase) activity but not ß-galactosidase activity. This GlcNase (GlmATk), a homodimer of 90-kDa subunits, exhibited highest activity toward reduced chitobiose at pH 6.0 and 80°C and specifically cleaved the nonreducing terminal glycosidic bond of chitooligosaccharides. The GlcNase activity was also detected in T. kodakaraensis cells, and the expression of GlmATk was induced by GlcNAc2 and chitin, strongly suggesting that GlmATk is involved in chitin catabolism in T. kodakaraensis. These results suggest that T. kodakaraensis, unlike other organisms, possesses a novel chitinolytic pathway where GlcNAc2 from chitin is first deacetylated and successively hydrolyzed to glucosamine. This is the first report that reveals the primary structure of GlcNase not only from an archaeon but also from any organism.

* 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, September 2003, p. 5175-5181, Vol. 185, No. 17
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.17.5175-5181.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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