The Kinetics And Influeneing Factors In Enzymatic Hydrolysis Of Chitosan

Chitosan is a polysaccharide widely used in the fields of medicine, food, paper,printing, dyeing, and daily chemical and other fields due to its good adsorptioncapacity, permeability, fiberizability and retention of moisture and attracts theattention of researchers as the new source of biopolysaccharides in the last decade.Papain is of good stability, high efficient ability of degrading chitosan and low-costand easy to be industrialized. Therefore, the study of enzymatic hydrolysis of chitosanwith papain can further understand the chemical properties of enzymes and chitosan,It is helpful to further explore the mechanism of non-specific enzymatic hydrolysis ofchitosan and provided the theoretical direction on industrial production ofwate-soluble low-molecular weight chitosan.Papain was used to hydrolyze chitosan with moisture content8.49%and degreeof deacetylation91.94%, The hydrolysis conditions were optimized as follows:substrate concentration of0.6mg/mL, enzyme-substrate ratio of10.0%, pH4.5and45oC.With the consideration of the effect of substrate concentration on the kinetics ofhydrolysis of chitosan with papain, the substrate inhibition kinetic model of enzymatichydrolysis of chitosan with papain was established on the basis of the theory ofintermediate product. The nonlinear fitting method was used for the model fitting andthe kinetics parameters were obtained. The result indicated that chitosan hydrolyzedby papain showed no substrate inhibition in the initial substrate concentration range of0.2~0.6mg/mL, however, excess substrate (0.6~1.2mg/mL) caused inhibition ofenzyme activity. It conformed to the substrate inhibition kinetic model. The modelcalculations showed good agreement with the experimental results and theexperimental fitting relative error was3.74%(<10%).pH value can affect the secondary structure of the papain, the content of theordered structure increase with the increase of pH value and the disordered structuredecrease accordingly. β-turn and random coil are closely associated with the enzymeactivity, the enzyme active center maybe mainly β-turn and random coil. β-turncontent decrease then random coil content increase, and this contribute to increaseenzyme activity, whereas papain activity decreased and the maximum reaction rate isobtained at pH4.5. The pK of amino acid side-chain group under different pH were analyzed and it revealed that histidine residue was essential for papain active site toexhibit catalytic activity. reaction mechanism model was proposed with theconsideration of substrate hydrolysis and product inhibition and kinetic equation ofthe system was build. The fitted results of kinetics model agree well with that ofexperiment data basically and the optimal pH calculated from the fitting curve wasconsistent with experimental results.The secondary structure of papain under different temperature was analyzed byFT-IR and the second derivatives result showed that, the content of the orderedstructure reduced with increase of temperature while the disordered structureincreased and the maximum reaction rate was obtained at45oC. The result suggestedthat random coil content increased and β-turn content decreased under45oC lead tothe activity center structure was more flexible and reasonable and enhanced itscatalytic activity. The thermodynamic parameters obtained were showed as follows:A=0.8421, Ea=84.9393KJ/mol,△H-19.7575KJ/mol,△S-16.2252KJ/(mol·K), A2.3354. Based on the analysis of chitosan enzymatic hydrolysiscurves under different experimental conditions and the kinetic equation underdifferent reaction temperature was created, the kinetic model was fitted and thepredicted result accords with the experiment data well. The optimum temperatureobtained from the fitting curve was consistent with the experimental result and thisshowed that the model had a high fitting precision and a good predicting ability.