MODE OF ACTION OF CHITIN DEACETYLASE FROM MUCOR-ROUXII ON PARTIALITY N-ACETYLATED CHITOSANS

The mode of action of chitin deacetylase (CDA) from Mucor rouxii on fu lly water-soluble partially N-acetylated chitosans was investigated. T he Michaelis-Menten constants of three high-molecular-weight chitosans with initial fraction of acetylated units (F-A) of 0.08, 0.35, and 0. 62 were determined to 2.1 +/- 0.4, 1.7 +/- 0.7, and 2.1 +/- 0.3 mg/mL, respectively. The relative rate of enzymatic deacetylation increased linearly with increasing F-A On the chitosans, indicating that CDA doe s not preferentially attack any sequences in the chitosan molecules. A water-soluble and highly N acetylated chitosan with F-A of 0.681, hav ing a Bernoullian distribution of acetylated (A) and deacetylated (D) units, and a number-average degree of polymerization (dp(n)) of 30, wa s selected as substrate for CDA for more detailed studies of the mode of action. The chitosan was enzymatically deacetylated to decreasing F -A-values ( F-A of 0.582, 0.400, and 0.188), and the nearest neighbour frequencies (F-AA, F-AD, F-DA and F-DD) were determined by NMR spectr oscopy, showing that the transition frequencies F-AD and F-DA were low er than expected from a random (Bernoullian) distribution in the furth er enzymatically deacetylated chitosans, while F-AA and F-DD were high er compared to a random distribution. The experimental results were co mpared with model data, assuming an endo-type mechanism with no prefer ential attack at any sequences in the chitosan chain. The comparison s uggested that CDA hydrolysed acetyl-groups according to a multiple att ack mechanism, with a degree of multiple attack of at least three. No deacetylation could be detected at the non-reducing end of the enzymat ically deacetylated chitosans. (C) 1998 Elsevier Science Ltd. All righ ts reserved.