The tryptic glycopeptides of horseradish peroxidase isozyme c (HRPc) were studied by methylation linkage analysis, exoglycosidase degradation, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDITOFMS). Over 90% of the p

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 (FA) 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 FA 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 FA of 0.681, having a Bernoullian distribution of acetylated (A) and deacetylated (D) units, and a number-average degree of polymerization (dpn) of 30, was selected as substrate for CDA for more detailed studies of the mode of action. The chitosan was enzymatically deacetylated to decreasing FA-values (FA of 0.582, 0.400, and 0.188), and the nearest neighbour frequencies (FAA, FAD, FDA and FDD) were determined by NMR spectroscopy, showing that the transition frequencies FAD and FDA were lower than expected from a random (Bernoullian) distribution in the further enzymatically deacetylated chitosans, while FAA and FDD 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.