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Applied and Environmental Microbiology, May 2006, p. 3515-3523, Vol. 72, No. 5
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.5.3515-3523.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Characterization of an Exo-ß-1,3-Galactanase from Clostridium thermocellum

Hitomi Ichinose,¹ Atsushi Kuno,² Toshihisa Kotake,³ Makoto Yoshida,¹ Kazuo Sakka,⁴ Jun Hirabayashi,² Yoichi Tsumuraya,³ and Satoshi Kaneko^1*

Food Biotechnology Division, National Food Research Institute, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan,¹ Research Center for Glycoscience, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan,² Faculty of Science, Saitama University, 255 Shimo-okubo, Saitama 338-8570, Japan,³ Faculty of Bioresources, Mie University, 1515 Kamihama, Tsu 514-8507, Japan⁴

Received 16 August 2005/ Accepted 11 March 2006

A gene encoding an exo-ß-1,3-galactanase from Clostridium thermocellum, Ct1,3Gal43A, was isolated. The sequence has similarity with an exo-ß-1,3-galactanase of Phanerochaete chrysosporium (Pc1,3Gal43A). The gene encodes a modular protein consisting of an N-terminal glycoside hydrolase family 43 (GH43) module, a family 13 carbohydrate-binding module (CBM13), and a C-terminal dockerin domain. The gene corresponding to the GH43 module was expressed in Escherichia coli, and the gene product was characterized. The recombinant enzyme shows optimal activity at pH 6.0 and 50°C and catalyzes hydrolysis only of ß-1,3-linked galactosyl oligosaccharides and polysaccharides. High-performance liquid chromatography analysis of the hydrolysis products demonstrated that the enzyme produces galactose from ß-1,3-galactan in an exo-acting manner. When the enzyme acted on arabinogalactan proteins (AGPs), the enzyme produced oligosaccharides together with galactose, suggesting that the enzyme is able to accommodate a ß-1,6-linked galactosyl side chain. The substrate specificity of the enzyme is very similar to that of Pc1,3Gal43A, suggesting that the enzyme is an exo-ß-1,3-galactanase. Affinity gel electrophoresis of the C-terminal CBM13 did not show any affinity for polysaccharides, including ß-1,3-galactan. However, frontal affinity chromatography for the CBM13 indicated that the CBM13 specifically interacts with oligosaccharides containing a ß-1,3-galactobiose, ß-1,4-galactosyl glucose, or ß-1,4-galactosyl N-acetylglucosaminide moiety at the nonreducing end. Interestingly, CBM13 in the C terminus of Ct1,3Gal43A appeared to interfere with the enzyme activity toward ß-1,3-galactan and -L-arabinofuranosidase-treated AGP.

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* Corresponding author. Mailing address: National Food Research Institute, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan. Phone: 81-298-38-8022. Fax: 81-298-38-7996. E-mail: sakaneko{at}affrc.go.jp.

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Applied and Environmental Microbiology, May 2006, p. 3515-3523, Vol. 72, No. 5
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.5.3515-3523.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

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