Determination of enzymatic hydrolysis specificity of partially N-acetylated chitosans

Abstract

A new method for determining the specificity of hydrolysis of the linear binary heteropolysaccharide chitosan composed of (1 → 4)-linked 2-acetamido-2-deoxy-β- d-glucopyranose (GlcNAc; A-unit) and 2-amino-2-deoxy-β- d-glucopyranose (GlcN; D-unit) residues is described. The method is based on the assignments of the 13C chemical shifts of the identity (A- or D-units) of the new reducing and non-reducing ends and the variation in their nearest neighbours, using low molecular weight chitosans with known random distribution of A- and D-units as substrate. A highly N-acetylated chitosan with fraction of acetylated units ( F A) of 0.68 and a number-average degree of polymerization ( DP n) of 30 was hydrolysed with hen egg-white lysozyme, showing that both the new reducing and non-reducing ends consisted exclusively of A-units, indicating a high specificity for A-units in subsites D L and E L on lysozyme. Our data suggests that the preceding unit of the reducing A-units is invariable, and based on earlier studies, most probably an A-unit, while the unit following the non-reducing A-units can be either an A- or a D-unit. A more detailed study of the specificity of lysozyme at subsite D L was performed by hydrolyzing a more deacetylated chitosan ( F A = 0.35 and DP n of 20), to a DP n of 9, showing that even for this chitosan more than 90% of the new reducing ends were acetylated units. Thus, lysozyme depolymerizes partially N-acetylated chitosans by preferentially hydrolyzing sequences of acetylated units bound to site C L, D L and E L of the active cleft, while there is no specificity between acetylated and deacetylated units to site F L. In addition, a moderately N-acetylated chitosan with fraction of acetylated units ( F A) of 0.35 and a DP n of 20 was hydrolysed with Bacillus sp. No. 7-M chitosanase, showing that both the new reducing and non-reducing ends consisted exclusively of D-units. Our data suggests that the nearest neigbour to the D-unit at the reducing end is invariable, and based on earlier studies, most probably a D-unit, while the unit following the non-reducing D-units can be either an A- or a D-unit. We conclude that the Bacillus chitosanase hydrolyzes partially N-acetylated chitosan by preferentially attacking sequences of three consecutive deacetylated units, hypothetical subsites C C, D C and E C, where the cleavage occur between sugar units bound to subsites D C and E C. A hypothetical subsite F C on the chitosanase show no specificity with respect to A- and D-units. The new NMR method described herein offers a time and labour-saving alternative to the procedure of extensive hydrolysis of the binary heteropolysaccharide chitosan and subsequent isolation and characterization of the oligosaccharides.