The first identification of carbohydrate binding modules specific to chitosan.

Abstract 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 (Tm) of each protein was elevated by the addition of cello-, laminari-, chitin-, or chitosan-hexamer (GlcN)6. The Tm elevation (ΔTm) in DD1 was the highest (10.3 ˚C) upon addition of (GlcN)6, and was markedly higher than that in DD2 (1.0 ˚C). A synergistic effect was observed (ΔTm=13.6 ˚C), when (GlcN)6 was added to DD1+DD2. From ITC experiments, affinities to DD1 were not clearly dependent upon chain length of (GlcN)n; ΔGr˚ 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 (ΔGr˚ = -5.0 (n=3) ~ -5.2 (n=6) kcal/mol). ITC 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 (ΔGr˚ = -8.6 and -6.4 kcal/mol, respectively). From NMR titration experiments, (GlcN)n (n=2~6) were found to bind to loops extruded from the core β-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.