Mode of action of chitin deacetylase from Mucor rouxii on partially N-acetylated chitosans

Abstract

The mode of action of chitin deacetylase (CDA) from Mucor rouxii on fully water–soluble partially N-acetylated chitosans was investigated. The 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 does not preferentially attack any sequences in the chitosan molecules. A water-soluble and highly N-acetylated chitosan with F A of 0.681, having a Bernoullian distribution of acetylated ( A) and deacetylated ( D) units, and a number-average degree of polymerization ( dp n ) of 30, was 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 spectroscopy, showing that the transition frequencies F AD and F DA were lower than expected from a random (Bernoullian) distribution in the further enzymatically deacetylated chitosans, while F AA and F DD were higher compared to a random distribution. The experimental results were compared with model data, assuming an endo-type mechanism with no preferential attack at any sequences in the chitosan chain. The comparison suggested that CDA hydrolysed acetyl-groups according to a multiple attack mechanism, with a degree of multiple attack of at least three. No deacetylation could be detected at the non-reducing end of the enzymatically deacetylated chitosans.