Chitinase (EC 3.2.1.14) and lysozyme (EC 3.2.1.17) hydrolyse chitin [(l-4)-2acetamido-2-deoxy$-D-glucan],
partially N-deacetylated chitinlm3, and N-acyl ana-logues4 prepared from lower fatty acids. Because of the insolubility of these derivatives in water, both the structure and physical form have significant effects on the enzymic hydrolysis5. We now describe the preparation of novel water-soluble ZV-acyl derivatives of O-carboxymethylchitosan', and their hydrolyses by chitinase and lysozyme under homogeneous conditions.
O-Carboxymethylchitin' (d.s. 0.59) was N-deacetylated by heating in aqueous 10% sodium hydroxide containing sodium borohydride for 9 h at 80" to give Ocarboxymethylchitosan (d.s. 0.59). These conditions are milder than those for the N-deacetylation of chitin (e.g., aqueous 45% sodium hydroxyde, 5 h, 100")"'". The d.s. was calculated from the elemental analyses for C and N (see Experimental).
The 0-carboxymethylchitosan was treated with a series of carboxylic anhydrides (5 equiv. per GlcN) in aqueous acetic acid-methanol" to give N-acyl derivatives in yields of 59-97%. The lower N-acyl (C,-C,, and benzoyl) and hihger N-acyl (C,,-C,,) derivatives formed gels and precipitates, respectively, but the N-succinyl and N-phthaloloyl derivatives did not. The ds. for ZV-acyl, calculated from the elemental analyses for C and N, varied from 0.25 to 0.75 (Table I), which probably reflects steric effects. The distribution of the 0-carboxymethyl and N-acyl groups in the derivatives is not symmetrical", but is not known in detail. The lower A'-acyl ( <C, for fatty acyl), iV-succinyl, and N-phthalolyl derivatives (Na salts) were soluble in water, aqueous 0.05% hydrochloric acid, and aqueous 0.05% sodium hydroxide (Table I). The lower N-acyl derivatives had isoelectric points in the pH range 3-5, at which a precipitate was produced.
Chitinase and lysozyrne hydrolysed the water-soluble lower N-acyl derivatives (Table II). The results indicate that the susceptibility to enzymic degradation decreased as the length of the N-acyl group increased. However, in the absence of values for J& and VA,,,, the data in Table II should be regarded as qualitative.