FIG. 4.

Hydrolysis of various β-disaccharides (GlcNAc₂, GlcN₂, lactose [Lac], cellobiose [Glc₂], and laminaribiose [β-1,3-Glc₂]) (A), chitooligosaccharides (GlcN_3-6) (B), and reduced chitooligosaccharides (GlcN_2-5OH) (C) with various chain lengths by GlmA_Tk. The reaction mixture (25 μl) containing 0.2 mg of the substrate and 25 pmol of GlmA_Tk in 50 mM MES-NaOH (pH 5.0) was incubated at 70°C. The reaction products were analyzed at the indicated times. TLC plates were visualized with aniline-diphenylamine reagent (A and B) and with ninhydrin reagent (C). The spots undetectable with the aniline-diphenylamine reagent in C are boxed. The spots pointed to by arrowheads are probable reduced GlcN. Lanes C, control without GlmA_Tk after each reaction; lanes S, various chain lengths of saccharides corresponding to the substrates used in each reaction; lanes rS, standard reduced chitooligosaccharides from GlcN₂OH to GlcN₅OH.