CLONING AND SEQUENCING OF CHIC GENE OF BACILLUS-CIRCULANS WL-12 AND RELATIONSHIP OF ITS PRODUCT TO SOME OTHER CHITINASES AND CHITINASE-LIKEPROTEINS

To clarify the roles of individual chitinases comprising in the chitin ase system of Bacillus circulans WL-12, the gene (chiC) encoding chiti nase C, a minor constituent of the chitinase system, was cloned and ex pressed in Escherichia coli. The predicted product of the chiC gene is 491 amino acids long with a calculated molecular mass of 53,447 Da. T he N-terminal portion of the deduced polypeptide exhibited 46.8% amino acid homology with the catalytic domain of chitinase Al of this bacte rium, suggesting that the chitinase encoded by the chiC gene is compri sed of an N-terminal catalytic domain and a C-terminal domain with unk nown function. Two bands of chitinases with estimated molecular masses of 55 kDa and 40 kDa were detected on SDS-PAGE of the periplasmic fra ction of E. coli carrying the cloned chiC gene. Chitinase C of B. circ ulans WL-12 is practically identical to the 40 kDa chitinase detected in E. coli based on their N-terminal amino acid sequences, isoelectric points and estimated sizes, and corresponds to the catalytic domain o f the initial product of the chiC gene (55 kDa chitinase, designated a s chitinase C1). Comparison of domain organizations of chitinases, of this bacterium, sequenced so far suggested that they represent two typ es of catalytic domains and that domain shuffling occurred relatively recently giving rise to three chitinases. Sequence comparison and the evolutionary relationship of chitinase C1 with other chitinases and ch itinase-like proteins are also discussed.