High yield production of monomer-free chitosan oligosaccharides by pepsin catalyzed hydrolysis of a high deacetylation degree chitosan

The high molecular weight of chitosan, which results in a poor solubility at neutral pH values and high viscosity aqueous solutions, limits its potential uses in the fields of food, health and agriculture. However, most of these limitations are overcome by chitosan oligosaccharides obtained by enzymatic hydrolysis of the polymer. Several commercial enzymes with different original specificities were assayed for their ability to hydrolyze a 93% deacetylation degree chitosan and compared with a chitosanase. According to the patterns of viscosity decrease and reducing end formation, three enzymes-cellulase, pepsin and lipase A-were found to be particularly suitable for hydrolyzing chitosan at a level comparable to that achieved by chitosanase. Unlike the appreciable levels of both 2-amino-2-deoxy-d-glucose and 2-acetamido-2-deoxy-d-glucose monomers released from chitosan by the other enzymes after a 20 h-hydrolysis (4.6-9.1% of the total product weight), no monomer could be detected following pepsin cleavage. As a result, pepsin produced a higher yield of chitosan oligosaccharides than the other enzymes: 52% versus as much as 46%, respectively. Low molecular weight chitosans accounted for the remaining 48% of hydrolysis products. The calculated average polymerization degree of the products released by pepsin was around 16 units after 20 h of hydrolysis. This product pattern and yield are proposed to be related to the bond cleavage specificity of pepsin and the high deacetylation degree of chitosan used as substrate. The optimal reaction conditions for hydrolysis of chitosan by pepsin were 40 °C and pH 4.5, and an enzyme/substrate ratio of 1:100 (w/w) for reactions longer than 1 h.