Expression and characterization of the chitin-binding domain of chitinase A1 from Bacillus circulans WL-12.
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, ChBD(ChiA1) was produced in Escherichia coli using a pET expression system and purified by chitin affinity column chromatography. Purified ChBD(ChiA1) specifically bound to various forms of insoluble chitin but not to other polysaccharides, including chitosan, cellulose, and starch. Interaction of soluble chitinous substrates with ChBD(ChiA1) 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 ChBD(ChiA1) to regenerated chitin. These observations suggest that ChBD(ChiA1) recognizes a structure which is present in insoluble or crystalline chitin but not in chito-oligosaccharides or in soluble derivatives of chitin. ChBD(ChiA1) 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 ChBD(ChiA1) 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.
Hashimoto
M
M
Department of Biosystem Science, Graduate School of Science and Technology, Niigata University, 8050 Ikarashi-2, Niigata 950-2181, Japan.
Ikegami
T
T
Seino
S
S
Ohuchi
N
N
Fukada
H
H
Sugiyama
J
J
Shirakawa
M
M
Watanabe
T
T
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology*; D001665:Binding Sites; D002686:Chitin / Q000031:analogs & derivatives / Q000378:metabolism* / Q000648:ultrastructure; D002688:Chitinases / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000378:metabolism; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D011134:Polysaccharides / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012965:Sodium Chloride; D012995:Solubility
D009844:Oligosaccharides; D010446:Peptide Fragments; D011134:Polysaccharides; D011994:Recombinant Proteins; D002686:Chitin; D012965:Sodium Chloride; C044872:O-(carboxymethyl)chitin; C031610:glycolchitin; D002688:Chitinases; C094590:chitinase ChiA