As a means of making chitosan more useful in biotechnological applications, it was hydrolyzed using pepsin, chitosanase and alpha-amylase. The enzymolysis behavior of these enzymes was further systematically studied for its effectiveness in the production of low-molecular-weight chitosans (LMWCs) and other derivatives. The study showed that these enzymes depend on ion hydronium (H3O+), thus on pH with a pH dependence fitting R-2 value of 0.99. In y = 1.484 [H+] + 0.114, the equation of pH dependence, when [H+] increases by one, y (k(0)/k(m)) increases by 1.484. From the temperature dependence study, the activation energy (E-a) and pre-exponential factor (A) were almost identical for two of the enzymes, but a considerable difference was observed in comparison with the third enzyme. Chitosanase and pepsin had nearly identical Ea, but alpha-amylase was significantly lower. This serves as evidence that the hydrolysis reaction of alpha-amylase relies on low-barrier hydrogen bonds (LBHBs), which explains its low Ea in actual conditions. The confirmation of this phenomenon was further derived from a similarly considerable difference in the order magnitudes of A between alpha-amylase and the other two enzymes, which was more than five. Variation of the rate constants of the enzymatic hydrolysis of chitosan with temperature follows the Arrhenius equation.
