Identifi cation of Inducers for Chitinase B (ChiB) Production in Bacillus

Chitin is the main component of exoskeleton (cuticle) of crabs. We consume large amounts of crabs and shrimps for food and as a result, enormous amounts of their shell wastes are produced every year. Although chitin is utilized in environmental technology and food medical industries, most of shell wastes are discarded by incineration and burying. Thus, reutilization of shell wastes is an important issue in ecological concern. Cuticles are biopolymers composed of rigid complex of N-acetylglucosamine polymers, proteins, and Ca and highly insoluble in water and organic solvents. Chitinolytic and non-chitinolytic microorganisms cooperatively degrade cuticles. To understand chitin degradation system, we isolated chitinolytic bacterium Bacillus cereus CH and investigated its chitinolytic enzyme system. B. cereus CH produces two chitinases, chitinase A (ChiA) and chitinase B (ChiB) and ChiB is responsible for the major extracellular chitinase activity. ChiA and ChiB production is inducible and colloidal chitin promotes transcription of the chiA and chiB genes. In this study, we report that the extent of acetylation of chitooligosaccharides affects induction of chiB transcription, and that there is the possibility that a two-component regulatory system is involved in the control of chiB transcription. B. cereus CH was cultivated in medium containing 0.2% triptone, 0.1% yeast extract, 0.2% NaCl, 0.025% KH2PO4, 0.025% K2HPO4, 0.01% calcium acetate, and 0.01% magnesium acetate (pH 7.2) at 33°C. At 12 h after the initiation of cultivation, 0.025% of test compounds were added to induce ChiB production. Extracellular and cytoplasmic ChiB was quantified by enzyme-linked immunosorbent assays (ELISA) with anti-ChiB serum solution as described in the previous report. Quantification of chiB mRNA was performed by reverse transcription PCR (RT-PCR) as described previously. In the previous study, we demonstrated that ChiA and ChiB production in B. cereus CH is induced by colloidal chitin and N-acetylglucosamine oligomers, but not by deacetylated chitin (chitosan 10B). This result suggests that the extent of acetylation of chitin affects the ability of chitin to induce chitinase genes in B. cereus CH. To investigate an effect of the extent of acetylation of chitin, we examined chitin products with different deacetylation ratio, i.e. chitosan 7B, 9B, and 10B for their ability to induce extracellular ChiB production. The deacetylation ration of chitosan 7B, 9B, and 10B is 70, 90, and 100%, respectively. As shown in Fig. 1, colloidal chitin showed the best induction activity and the induction activity decreased with increasing of the deacetylation ratio of chitin. To confirm an effect of acetylation of chitin on induction of ChiB production, we examined the activity of oligomers (dimer to hexamer, [(GlcN)2, (GlcN)4, (GlcN)5, and (GlcN)6]) of glucosamine (GlcN) to induce ChiB production and compared it with that of N-acetylglucosamine (GlcNAc) oligomers ((GlcNAc)2 to (GlcNAc)6). As reported in the previous study, GlcNAc oligomers showed remarkable induction activity which increased with residue numbers of the oligomers (Fig. 2). Identifi cation of Inducers for Chitinase B (ChiB) Production in Bacillus cereus CH and Estimation of Its Induction Mechanism