Purification and characterization of a Bacillus cereus exochitinase

Five extracellular chitinases of Bacillus cereus 6E1 were detected by a nov el in-gel chitinase assay using carboxymethyl-chitin-remazol brilliant viol et 5R (CM-chitin-RBV) as a substrate. The major chitinase activity was asso ciated with a 36-kDa (Chi36) gel band. Chi36 was purified by a one-step, na tive gel purification procedure derived from the new in-gel chitinase assay . The purified Chi36 has optimal activity at pH 5.8 and retains some enzyma tic activity between pH 2.5-8. The temperature optimum for Chi36 was 35 deg reesC, but the enzyme was active between 4-70 degreesC. Based on its abilit y to hydrolyze mainly p-nitrophenyl-(N-acetyl-beta -D-glucosaminide)(2), Ch i36 is characterized as a chitobiosidase, a type of exochitinase. The N-ter minal amino acid sequence of mature Chi36 was determined (25 amino acids). Alanine is the first N-terminal amino acid residue indicating the cleavage of a signal peptide from a Chi36 precursor to form the mature extracellular Chi36. The N-terminal sequence of Chi36 demonstrated highest similarity wi th Bacillus circulans WL-12 chitinase D and significant similarity with sev eral other bacterial chitinases. (C) 2001 Elsevier Science Inc. All rights reserved.