Determination of enzymatic hydrolysis specificity of partially N-acetylated
chitosans
Biochimica et Biophysica Acta-General Subjects, 1291 (1):
5 -- 15 (August 1996)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-beta-D-glucopyranose (GlcNAc; A-unit) and 2-amino-2-deoxy-beta-D-glucopyranose
(GlcN; D-unit) residues is described. The method is based on the
assignments of the C-13 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 (DPn) 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 DPn of 10) to a DPn of 9, showing that even for this chitosan
more than 905 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 DPn 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 tither 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.