pH Dependence of Chitosan Enzymolysis.

As a means of making chitosan more useful in biotechnological applications, it was hydrolyzed using pepsin, chitosanase and α-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 R2 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 (Ea) 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 α-amylase was significantly lower. This serves as evidence that the hydrolysis reaction of α-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 α-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.

Heat-pretreatment and enzymolysis behavior of the lotus seed protein.

Lotus seed protein (LSP) was heat-pretreated before enzymolysis in order to seek a greater degree of hydrolysis (DH) during enzymatic hydrolysis. The parameters including substrate concentration, temperature, pH, and papain concentration were optimized by response surface methodology in the enzymolysis of the heat-pretreated LSP. The influence of substrate concentration on the non-pretreated LSP enzymolysis was assessed, and the enzymolysis was found to obey the Haldane model with inhibition by LSP substrate. The initial concentration of non-pretreated LSP was inferred theoretically to be 11.07 g/L in order to avoid substrate inhibition. On the other hand, Chrastil model was fitted and the diffusion resistance constant values were in the range of 0.5-0.6 for the diffusion-controlled encounter of enzyme and substrate, implying that diffusion was a rate-limiting step. The heat-pretreatment at 60 °C for 60 min could increase the DH of the LSP, which enhanced the efficiency of the enzymolysis by papain.

Enzymolysis of chitosan by papain and its kinetics.

Low molecular weight chitosan (LMWC) was obtained by the enzymolysis of chitosan by papain. Enzymolysis conditions (initial chitosan concentration, temperature, pH and ratio of papain to chitosan) were optimized by conducting experiments at three different levels using the response surface methodology (RSM) to obtain high soluble reducing sugars (SRSs) concentrations. Meanwhile, the influence of chitosan substrate concentration on the activity of papain was assessed in the experiments. The enzymolysis process was analyzed using pseudo-first-order and pseudo-second-order kinetic models and the experiment data were found to be more consistent with the pseudo-second-order kinetic model. In addition, the kinetic behavior of the enzymolysis was also investigated by using Haldane model, and chitosan exhibited substrate inhibition. It was clear that the Haldane kinetic model adequately described the dynamic behavior of the chitosan enzymolysis by papain. When the initial chitosan concentration was above 8.0g/L, the papain was overloaded and exhibited significant inhibition.

Optimization and Characterization of Chitosan Enzymolysis by Pepsin.

Pepsin was used to effectively degrade chitosan in order to make it more useful in biotechnological applications. The optimal conditions of enzymolysis were investigated on the basis of the response surface methodology (RSM). The structure of the degraded product was characterized by degree of depolymerization (DD), viscosity, molecular weight, FTIR, UV-VIS, SEM and polydispersity index analyses. The mechanism of chitosan degradation was correlated with cleavage of the glycosidic bond, whereby the chain of chitosan macromolecules was broken into smaller units, resulting in decreasing viscosity. The enzymolysis by pepsin was therefore a potentially applicable technique for the production of low molecular chitosan. Additionally, the substrate degradation kinetics of chitosan were also studied over a range of initial chitosan concentrations (3.0~18.0 g/L) in order to study the characteristics of chitosan degradation. The dependence of the rate of chitosan degradation on the concentration of the chitosan can be described by Haldane's model. In this model, the initial chitosan concentration above which the pepsin undergoes inhibition is inferred theoretically to be about 10.5 g/L.