MULTIPLE CHITINASE ENZYMES FROM A SINGLE-GENE OF BACILLUS-LICHENIFORMIS TP-1

Three chitinase activity bands in a culture supernatant of Bacillus li cheniformis TP-1 were detected by non-denaturing PAGE. They were desig nated chitinase bands 1, 2, and 3 in order from the gel origin. Analys is by immunodiffusion and immunoelectrophoresis using polyclonal antib ody raised against chitinase band 3 indicated that these chitinases we re antigenically similar. B. licheniformis TP-I and Escherichia coli h arboring the cloned chitinase gene (pCHIL3) from strain TP-1 expressed 3 chitinase bands by non-denaturing PAGE and SDS-PAGE which gave esti mated molecular masses of 68, 62, and 50 kDa (named Chi68, Chi62, and Chi50, respectively). All three chitinases had the same N-terminal ami no acid sequences, strongly suggesting that Chi62 and ChiSO were deriv ed from Chi68 by a processing step(s) at the C-terminus. The deduced C -terminal amino acid sequence of Chi68 showed homology to amino acid s equences of known chitin and cellulose binding domains. Chi62 and ChiS O lacked this domain (judging from their deduced amino acid sequences and calculated molecular masses) and they were unable to bind chitin, suggesting that they were generated from Chi68 by cleavage of the chit in binding domain at the C-terminus. Comparison of chitinase activitie s indicated that this binding domain was important for complete hydrol ytic activity towards colloidal chitin.