Characterization of chitinases excreted by Bacillus cereus CH

Bacillus cereus CH was shown to excrete chitinases into the culture superna tant when cultivated in a medium containing 0.2% colloidal chitin, whereas the removal of colloidal chitin resulted in a low activity. After concentra tion of the culture supernatant by precipitation with ammonium sulfate, the induced chitinases were purified by sequential chromatography. Four differ ent chitinases, A, B1, B2, and B3 with molecular masses of 35, 47, 58, and 64 kDa, respectively, were separated. All chitinases showed similarities in their kinetic parameters when observed with colloidal chitin, including an optimal pH of 5.0-7.5, and an optimal temperature between 50-60%C. Chitina se A hydrolyzed glycol chitin and p-nitrophenyl-di-N-acetyl-beta-chitobiosi de at similar rates to that of colloidal chitin, whereas group B chitinases hydrolyzed both substrates in much lower rates. From analyses of the react ion products, it is most likely that chitinase A and all group B chitinases hydrolyze the substrates tested in an endo-fashion. However, group B chiti nases were distinct from chitinase A in possessing high transglycosylation activity. From amino terminal sequencing, chitinases B1, B2, and B3 were sh own to have almost identical sequences, which differed from that of chitina se A. The similarities in the reaction modes and amino terminal sequences a mong chitinases B1, B2, and B3 suggest that these chitinases may be derived from a presumptive precursor protein through C-terminal processing.