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Journal of Bacteriology, June 2000, p. 3045-3054, Vol. 182, No. 11
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Expression and Characterization of the Chitin-Binding Domain of Chitinase A1 from Bacillus circulans WL-12

Masayuki Hashimoto,1 Takahisa Ikegami,2 Shizuka Seino,1 Nobuhumi Ohuchi,3 Harumi Fukada,4 Junji Sugiyama,5 Masahiro Shirakawa,2 and Takeshi Watanabe1,3,*

Department of Biosystem Science, Graduate School of Science and Technology,1 and Department of Applied Biological Chemistry, Faculty of Agriculture,3 Niigata University, 8050 Ikarashi-2, Niigata 950-2181, Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0101,2 Laboratory of Biophysical Chemistry, College of Agriculture, Osaka Prefecture University, Sakai, Osaka 599-8531,4 and Wood Research Institute, Kyoto University, Uji, Kyoto, 611-0011,5 Japan

Received 12 July 1999/Accepted 5 March 2000

Chitinase A1 from Bacillus circulans WL-12 comprises an N-terminal catalytic domain, two fibronectin type III-like domains, and a C-terminal chitin-binding domain (ChBD). In order to study the biochemical properties and structure of the ChBD, ChBDChiA1 was produced in Escherichia coli using a pET expression system and purified by chitin affinity column chromatography. Purified ChBDChiA1 specifically bound to various forms of insoluble chitin but not to other polysaccharides, including chitosan, cellulose, and starch. Interaction of soluble chitinous substrates with ChBDChiA1 was not detected by means of nuclear magnetic resonance and isothermal titration calorimetry. In addition, the presence of soluble substrates did not interfere with the binding of ChBDChiA1 to regenerated chitin. These observations suggest that ChBDChiA1 recognizes a structure which is present in insoluble or crystalline chitin but not in chito-oligosaccharides or in soluble derivatives of chitin. ChBDChiA1 exhibited binding activity over a wide range of pHs, and the binding activity was enhanced at pHs near its pI and by the presence of NaCl, suggesting that the binding of ChBDChiA1 is mediated mainly by hydrophobic interactions. Hydrolysis of beta -chitin microcrystals by intact chitinase A1 and by a deletion derivative lacking the ChBD suggested that the ChBD is not absolutely required for hydrolysis of beta -chitin microcrystals but greatly enhances the efficiency of degradation.

* Corresponding author. Mailing address: Department of Applied Biological Chemistry, Faculty of Agriculture, Niigata University, 8050 Ikarashi-2, Niigata 950-2181, Japan. Phone: (81)252626647. Fax: (81)252626854. E-mail: wata{at}agr.niigata-u.ac.jp.

Journal of Bacteriology, June 2000, p. 3045-3054, Vol. 182, No. 11
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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