Purification And Characterization Of Chitosanase From Snail

Chitin is the second most abundant polysaccharide less than cellulose but similar in structure.N-acetylchitooligosaccharides (NCOS) and chitooligosaccharides (COS), which are soluble pruducts hydrolyzed from chitin and chitosan respectively, are of special interest for a variety of uses, such as foodstuff, agriculture, medicine, and so on.Among the methods for preparing COS including H2O2 degradation, acid degradation, enzymatic degradation, and physical degradation, the method of enzymatic degradation has certain advantage over others due to less pollution, fewer side-product, and simple and convenient operation. There are many polysacchride degradation enzymes in snail digestion system including the enzyme hydrolyzing chitosan, and the enzyme mixture certainly can degrade chitosan and produce COS efficiently. Also the enzyme is abundant in nature and relatively inexpensive, therefore the cost for manufacture of COS could be reduced. The ultimate goal of this research is to prepare purified enzyme to provide genetic information for cloning the gene coding for the enzyme by molecular biology techniques.The following conclusion have been drawn from this experiment:1.The p-hydroxybenzonic acide hydrazidde (PAHBAH) assay was tested and confirmed for measurement of reducing sugar as the detection of enzyme activity 2.Diferent types of purification columns were run for screening suitable chromatography methods, and hydrophobic interaction, salt precipatation,ion exchange, gel filtration, hydroxylapatite, DEAE-Sepharose, Sephacryl S-300, and hydroxylapatite chromatography media are decided to employ.3.To complete purify the enzyme protein, a series of columns was determined as hydrophobic interaction followed by DEAE-Sepharose ion exchange, and Sephacryl S-300 gel filtration. The purity of purified enzyme was checked by SDS-PAGE, and the enzyme showed a single band. The specific activity was increased by 24 folds during purification.This study provided a scheme for Chitosanase purification. The purified protein could be used for generating protein sequence, designing PCR primer, and finally cloning the gene.