Preparation of N-acetylchitooligosaccharides from lysozymic hydrolysates of partially N-acetylated chitosans

Abstract

The conditions for preparing partially N-acetylated chitosans (PNACs) by reaction of chitosan with acetic anhydride in homogeneous solution were investigated. The following conditions are recommended: chitosan, 1%; acetic acid, 0.4%; methanol, 50%; at room temperature. About 80% of the acetic anhydride reacted with amino groups and the degree of N-acetylation (da) of PNACs was easily controlled by the amount of acetic anhydride. PNACs were hydrolysed by lysozyme at 37°C for 6 days and hydrolysates were analysed by HPLC. 2-Acetamido-2-deoxy- d-glucose and N-acetylchitooligosaccharides [(GlcNAc) n ( n = 2–4)] together with heterooligosaccharides composed of 2-acetamido-2-deoxy- d-glucose and 2-amino-2-deoxy- d-glucose were detected. The latter were N-acetylated by acetic anhydride, to yield (GlcNAc) n . N-Acetylation of the hydrolysates doubled the amount of total (GlcNAc) n and (GlcNAc) 3, and increased the amount of (GlcNAc) 4 ninefold. The amount of (GlcNAc) n after the hydrolysis and the N-acetylation increased with the increase of da of PNACs. The optimum da for the formation of oligomers was ca. 70%. The yields of (GlcNAc) n ( n = 2–5) after gel filtration chromatography were 16.3, 10.3, 18.0, and 1.7 mg, respectively, from the hydrolysates of 72% N-acetylated chitosan (100 mg). This procedure is useful for the production of (GlcNAc) 2, (GlcNAc) 3, and (GlcNAc) 4.