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Applied and Environmental Microbiology, October 2005, p. 5787-5793, Vol. 71, No. 10
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.10.5787-5793.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Cloning, Sequencing, and Expression of a Eubacterium cellulosolvens 5 Gene Encoding an Endoglucanase (Cel5A) with Novel Carbohydrate-Binding Modules, and Properties of Cel5A

Kazutoyo Yoda,¹ Atsushi Toyoda,^2* Yoshihiro Mukoyama,² Yutaka Nakamura,² and Hajime Minato²

United Graduate School of Agricultural Science, Tokyo University of Agriculture and Technology, Fuchuu-city, Tokyo 183-8509, Japan,¹ College of Agriculture, Ibaraki University, Ami, Ibaraki 300-0393, Japan²

Received 17 February 2005/ Accepted 12 May 2005

A novel Eubacterium cellulosolvens 5 gene encoding an endoglucanase (Cel5A) was cloned and expressed in Escherichia coli, and its enzymatic properties were characterized. The cel5A gene consists of a 3,444-bp open reading frame and encodes a 1,148-amino-acid protein with a molecular mass of 127,047 Da. Cel5A is a modular enzyme consisting of an N-terminal signal peptide, two glycosyl hydrolase family 5 catalytic modules, two novel carbohydrate-binding modules (CBMs), two linker sequences, and a C-terminal sequence with an unknown function. The amino acid sequences of the two catalytic modules and the two CBMs are 94% and 73% identical to each other, respectively. Two regions that consisted of one CBM and one catalytic module were tandemly connected via a linker sequence. The CBMs did not exhibit significant sequence similarity with any other CBMs. Analyses of the hydrolytic activity of the recombinant Cel5A (rCel5A) comprising the CBMs and the catalytic modules showed that the enzyme is an endoglucanase with activities with carboxymethyl cellulose, lichenan, acid-swollen cellulose, and oat spelt xylan. To investigate the functions of the CBMs and the catalytic modules, truncated derivatives of rCel5A were constructed and characterized. There were no differences in the hydrolytic activities with various polysaccharides or in the hydrolytic products obtained from cellooligosaccharides between the two catalytic modules. Both CBMs had the same substrate affinity with intact rCel5A. Removal of the CBMs from rCel5A reduced the catalytic activities with various polysaccharides remarkably. These observations show that CBMs play an important role in the catalytic function of the enzyme.

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* Corresponding author. Mailing address: College of Agriculture, Ibaraki University, Ami, Ibaraki 300-0393, Japan. Phone and fax: 81-29-888-8584. E-mail: atoyoda{at}mx.ibaraki.ac.jp.

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Applied and Environmental Microbiology, October 2005, p. 5787-5793, Vol. 71, No. 10
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.10.5787-5793.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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