Study On The Enzymatic Degradation Of Chitosan

Chitooligosaccharides (COS), the degradation products of chitosan, not only have some features of chitosan macromolecules, but also have lots of special physiological activities and functional properties.This paper aimed at probing into the non-specific enzymatic hydrolysis technology of chitosan,and molecular weight distribution of chitooligosaccharides.1.Five kinds of enzymes including cellulase, pectinase, papain, pepsin and subtilisin were used for the degradation of chitosan,and the conditions were discussed by single-factor analysis based on the release dose of reducing sugar. The results showed that the optimum time of cellulose, papain, pepsin was 4 h, pectinase is 3 h,and subtilisin2 h to hydrolysis chitosan .The optimum temperature of pectinase, subtilisin, cellulase and papain was 50℃,and 55℃for pepsin to hydrolysis chitosan ; the optimum content of enzyme for pectinase cellulase,papain and pepsin was 5%,and 7% for subtilisin;In addition, the optimal substrate concentration for the degradation was 1%. In this paper pH5.0 was the best choice for the degradation of chitosan to facilitate the degradation products of the appropriate pH adjustment.By the comparation of enzyme activity, the release amount of reducing sugar in enzymolysis liquid and the droop rate of the viscosity of these degradation products, the degradation ability sequence was:cellulase> papain> pepsin> subtilisin> pectinase.2. The orthogonal test showed that the optimum conditions on cellulase were as follows: the optimum enzyme dosage was 5%, substrate concentration was 1.25%, enzymolysis time was 6 h, enzymolysis temperature was 55℃, pH5.0;and the the optimum conditions on papain were as follows: the optimum enzyme dosage was 7%, substrate concentration was 1.25%, enzymolysis time was 6 h, enzymolysis temperature was 55℃, pH5.0.The Sephadex G-100 was selected as the separation medium to separete raw materials of colloidal chitosan and chitosan hydrolysis liquid respectively by gel chromatography. For the raw materials, the main distribution range of chitosan molecular weight was 3.5×10~5-1.2×10~5,and the study indicated that the range was mainly between 8.4×10~3 and 2.2×10~3 after degradation with cellulase in the optimal conditions(the elution volume was 104～130 mL ),and subsection was3.2×10~5～1.7×10~5(the elution volume was 34～46 mL) ; Besides, the main ranges were 3.2×10~5～1.7×10~5 (the elution volume was 34～56 mL) and 1.2×10~4～2.7×10~3(the elution volume was 98～126 mL)after degradation with papain in the optimal conditions.3.The composite enzymatic hydrolysis technology was applicated to discuss the effects on the degradation of chitosan for different enzymes by adjusting the ratio of cellulase and papain.The result showed that the concentration of reducing sugar was highest when the ratio was 1:2.Moreover,by rotation composite design of quadratic regression, a dynamic mathematic model that showed the connection between the enzyme dosage (X1) , hydrolysis temperature (X2), hydrolysis temperature (X3)and the droop rate of viscosity (Y1), the release amount of reducing sugar (Y2).The results of variance analysis showed that the correlation coefficient R2 of equation Y1 was 97.63%, the value of F was 59.5919, and significant coefficient was 0.0001, the conformity of the model was 97.63%; At the same time , the correlation coefficient R2 of equation Y2 was 97.69%, the value of F was 61.0413, and significant coefficient was 0.0001.As the result,these two regression equations were highly significant, and could be good used to predict the degradation results.In addition ,the method of response surface was used to optimize the process conditions,and the optimum conditions was as follows: the content of enzyne was 7.70%, enzymolysis temperature was 55℃,the concentration of chitosan was 1.25%, enzymolysis time was 5 h, pH5.0. Under these conditions, the degradation of chitosan, the droop rate of viscosity of chitosan was 97.21%, the concentration of reducing sugar reached 2.47 mmol/L,and the effect was better than the single enzyme.The Sephadex G-100 was selected as the separation medium to separete hydrolysis liquid with the method of gel chromatography. Elution curve shows that the distribution of relative molecular weight of degradation products was 2.3×10~5～1.1×10~5 and 1.0×10~4～2.7×10~3. Compared with the elution curve of raw materials , the degradation effect was significant,and the relative molecular weight of majority products were under 1×10~4.