Di-N-acetylchitobiase (Ctbs) degrades β-1,4 glycoside bonds of the chitobiose core of free asparagine-linked glycan. This study examined whether Ctbs degrades chitin-oligosaccharides to GlcNAc in mammals. We analyzed Ctbs mRNA and protein expression in mouse tissues and characterized enzymatic activity using recombinant mouse Ctbs expressed in Escherichia coli. Ctbs mRNA and protein were expressed in various tissues of mouse, including the stomach. Optimal conditions for recombinant Ctbs were pH 3.0 and 45°C, and the recombinant enzyme was retained more than 94% activity after incubation at pH 3.0-7.0 and below 37°C. The recombinant Ctbs hydrolyzed (GlcNAc)3 and (GlcNAc)6 at pH 3.0 and produced GlcNAc. The K m of Ctbs was lowest with (GlcNAc)3 as a substrate. k cat/K m was fourfold as high with (GlcNAc)3 and (GlcNAc)4 as substrates than with (GlcNAc)2. These results suggest that Ctbs digests chitin-oligosaccharides or (GlcNAc)2 of reducing-end residues of oligosaccharides and produces GlcNAc in mouse tissues.
Keywords: n-acetyl-D-glucosamine; Di-N-acetylchitobiase; chitin-oligosaccharide.