PREPARATION OF N-ACETYLCHITOOLIGOSACCHARIDES FROM LYSOZYMIC HYDROLYSATES OF PARTIALLY N-ACETYLATED CHITOSANS

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 degrees C for 6 days and hydrolysates were analysed by HPLC. 2-Acetamido-2-deoxy-D-glucase 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).