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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
-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
-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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