Characterization of chitinases excreted by Bacillus cereus CH.
Bacillus cereus CH was shown to excrete chitinases into the culture supernatant when cultivated in a medium containing 0.2% colloidal chitin, whereas the removal of colloidal chitin resulted in a low activity. After concentration of the culture supernatant by precipitation with ammonium sulfate, the induced chitinases were purified by sequential chromatography. Four different 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 degrees C. Chitinase A hydrolyzed glycol chitin and p-nitrophenyl-di-N-acetyl-beta-chitobioside at similar rates to that of colloidal chitin, whereas group B chitinases hydrolyzed both substrates in much lower rates. From analyses of the reaction products, it is most likely that chitinase A and all group B chitinases hydrolyze the substrates tested in an endo-fashion. However, group B chitinases were distinct from chitinase A in possessing high transglycosylation activity. From amino terminal sequencing, chitinases B1, B2, and B3 were shown to have almost identical sequences, which differed from that of chitinase A. The similarities in the reaction modes and amino terminal sequences among chitinases B1, B2, and B3 suggest that these chitinases may be derived from a presumptive precursor protein through C-terminal processing.
Mabuchi
N
N
Laboratory of Environmental Molecular Biology, Graduate School of Environmental Earth Science, Hokkaido University, Japan. araki@ees.hokudai.ac.jp
Hashizume
I
I
Araki
Y
Y
D000595:Amino Acid Sequence; D001409:Bacillus cereus / Q000201:enzymology* / Q000254:growth & development; D002688:Chitinases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D003470:Culture Media; D006868:Hydrolysis; D008969:Molecular Sequence Data
D003470:Culture Media; D002688:Chitinases