3 CHITINASE GENES (CHIA, CHIC AND CHID) COMPRISE THE CHITINASE SYSTEMOF BACILLUS-CIRCULANS WL-12

This study was aimed at clarifying the origin and mechanism of process ing of multiple chitinases detected in the culture supernatant of Baci llus circulans WL-12 grown in the presence of chitin, Re-examination o f N-terminal amino acid sequences of chitinase B1 and B2 led us to pro pose that these chitinases are derived from chitinase D. Lysyl endopep tidase digestion of both chitinase B1 (39 kDa) and B2 (38 kDa) generat ed seven common peptide fragments which correspond to different portio ns of the catalytic domain of chitinase D (newly designated as chitina se D1). Peptide mapping of chitinase D1, B1 and B2 using CNBr digestio n indicated that chitinase B1 and B2 are truncated forms of chitinase D1 formed by cleavage of its binding domain and fibronectin type III-l ike domain. Therefore, it is concluded that chitinase B1 and B2 are de rived from chitinase D1 and correspond to the catalytic domain of this chitinase. Limited proteolysis of chitinase D1 with different endopro teases under non-denaturing conditions generated active molecules corr esponding to chitinase B1 and B2, providing evidence that chitinase B1 and B2 are produced by proteolytic cleavage at interdomain region of chitinase D1. Results of binding experiments of chitinase D1, B2 and t he proteolysed form of chitinase D1 to regenerated chitin demonstrated that the N-terminal portion of chitinase D1, lacking in chitinase B1 and B2, is important for binding activity. These overall findings sugg ested that chitinases of this bacterium detected to date in its cultur e supernatant are encoded by only three genes (chiA, chiC and chiD). A ll of these genes have already been cloned, sequenced and analyzed.