Chitin

The reaction mixtures (500 μl each) were prepared with 0.01 mg/ml purified protein, 1% (w/v) soluble or insoluble polysaccharides, such as chitin (Nacalai Tesque Inc.), chitosan (from crab shell; Nacalai Tesque Inc.), curdlan, cellulose (α-cellulose; Nacalai Tesque Inc.), carboxymethyl cellulose sodium salt (Nacalai Tesque Inc.), xylan (from beechwood; Nacalai Tesque Inc.), laminarin (Nacalai Tesque Inc.), sodium alginate (500 cps), pectin (from citrus; Nacalai Tesque Inc.), heparin sodium, xanthan gum (Nacalai Tesque Inc.), or gellan gum (Nacalai Tesque Inc.), and 50 mM Tris buffer pH 7.5. These reaction mixtures were incubated at 37 °C for 12 h. Then, the reaction mixtures were centrifuged at 15,000 g for 10 min and the clear fractions were transferred to clean tubes for assays. The degradation of the polysaccharides was determined using the DNS assay^{43 (link)}. The DNS reagent consisted of 1% (w/v) DNS, 30% (w/v) potassium sodium tartrate, and 0.4M NaOH. The aliquots (100 μl) were mixed with equal volumes (100 μl) of DNS reagent, boiled for 5 min, cooled to 20 °C, and then observed for a red colour or measured at an absorbance of 525 nm^{43 (link)}.

Alg-Na (300 cps) was obtained from Nacalai Tesque Inc. (Kyoto, Japan). Low-molecular-weight Alg-Na (80–120 cps), ALG-S (swelling type), ALG-NS (nonswelling type), and the model drug VST were obtained from Wako Pure Chemicals (Osaka, Japan). Chitosan (CS; degree of deacetylation (DA) 75–85%), CS-F, was obtained from Kimitsu Chemical Industries Co. Ltd. (Tokyo, Japan). CS-K (DA 85%) was obtained from Koyo Chemical Co. (Osaka, Japan) and β-CS (DA 96%) from Yaegaki Bio-Industry Inc. (Himeji, Japan). Chitin was obtained from Nacalai Tesque Inc. Pullulan was supplied by Hayashibara Biochemical Laboratories (Okayama, Japan). All other chemicals were of reagent grade.