The First Identification of Carbohydrate Binding Modules Specific to Chitosan

Background: Carbohydrate binding modules (CBMs) specific to chitosan have yet to be identified. Results: Two CBMs located at the C terminus of a chitosanase from Paenibacillus sp. IK-5 specifically bound chitosan oligosaccharides. Conclusion: Individual CBMs can accommodate at least two glucosamine units at loops extruded from the core -sandwich. Significance: The synergistic action of the two CBMs appears to facilitate chitosan hydrolysis. Two carbohydrate binding modules (DD1 and DD2) belonging to CBM32 are located at the C terminus of a chitosanase from Paenibacillus sp. IK-5. We produced three proteins, DD1, DD2, and tandem DD1/DD2 (DD1+DD2), and characterized their binding ability. Transition temperature of thermal unfolding (T-m) of each protein was elevated by the addition of cello-, laminari-, chitin-, or chitosan-hexamer (GlcN)(6). The T-m elevation (T-m) in DD1 was the highest (10.3 degrees C) upon the addition of (GlcN)(6) and was markedly higher than that in DD2 (1.0 degrees C). A synergistic effect was observed (T-m = 13.6 degrees C), when (GlcN)(6) was added to DD1+DD2. From isothermal titration calorimetry experiments, affinities to DD1 were not clearly dependent upon chain length of (GlcN)(n); G(r)degrees values were -7.8 (n = 6), -7.6 (n = 5), -7.6 (n = 4), -7.6 (n = 3), and -7.1 (n = 2) kcal/mol, and the value was not obtained for GlcN due to the lowest affinity. DD2 bound (GlcN)(n) with the lower affinities (G(r)degrees = -5.0 (n = 3) approximate to -5.2 (n = 6) kcal/mol). Isothermal titration calorimetry profiles obtained for DD1+DD2 exhibited a better fit when the two-site model was used for analysis and provided greater affinities to (GlcN)(6) for individual DD1 and DD2 sites (G(r)degrees = -8.6 and -6.4 kcal/mol, respectively). From NMR titration experiments, (GlcN)(n) (n = 2 approximate to 6) were found to bind to loops extruded from the core -sandwich of individual DD1 and DD2, and the interaction sites were similar to each other. Taken together, DD1+DD2 is specific to chitosan, and individual modules synergistically interact with at least two GlcN units, facilitating chitosan hydrolysis.