Extraction And Characterization Of Polysaccharide Hydrolase From Intestinal Viscera Of Sea Cucumber

This thesis focuses on the polysacchsride hydrolases in sea cucumber intestinal wall in order to provide the theoretical support for its comprehensive utilization.Firstly, a-starch, sodium cellulose, chitosan, xylan and mannan were used as substrates to explore their existence in sea cucumber intestinal wall. The results show that the extracts could hydrolyze there substrates and break down α-1,4 and β-1,3 glycosidic bond, in which a-amylase activity was the highest.Secondly, amylase activity was used as a technical index to optimize the extraction process of amylase in sea cucumber intestinal wall and explore the physicochemical properties for the amylase extracts. The results show that the optimum extraction conditions for amylase:extraction pH was 7.0, extraction ionic strength was 0.05M and extraction time was 4h; the optimum pH for extracts was 6.0, which was close to sea cucumber intestinal physiological pH 6.2. The optimum temperature for extracts was 20℃ with bad thermal stability and 60℃, the thermal stability of which was better that 20℃. Therefore, there could be two enzymes in the extracts which could degrade α-starch.Thirdly, Optimization for the saturation of ammonium sulfate was made, and the crude amylase resulted from salting and dialysis was studied for the physicochemical properties. The results show that proteins were precipitated scarcely when the saturation of ammonium sulfate was less than 50%; when in 50%-60%, the precipitated proteins appeared quickly; and when at 90%, the amylase activity with the reaction temperature at 60℃ was up to maximum value, but the enzyme activity in every saturation with the reaction temperature at 20 ℃ was significantly lower than that with the reaction temperature at 60 ℃. There could be two reasons for this.① the enzyme with high activity at 20 ℃ could be a relatively low molecular enzyme, which was easy to be dialyzed out of the enzyme solution during the dialysis process; ② the enzyme could be a kind of metal-activated enzymes, which lost enzyme activity because of the loss of metal ions during the process of salt precipitation and dialysis. The optimum pH for crude enzyme was 6.0, and the optimum temperature for crude enzyme was 60℃. The thermal stability began to deteriorate from 60℃ and the the pH at 7.0 was the most stable for amylaseFinally, the amylase in the crude enzyme was purified 15.04-fold after QFF anion exchange chromatography and Sephacryl S-300 gel chromatography. We found that the amylase has two subunit chains by SDS-PAGE. The molecular weight of which was 35.5kDa and 33.2 kDa, Partial characterization of the amylase was studied. The optimim temperature and pH of the amylase is 60℃ and 6.0 respectively. The thermal stability began to deteriorate from 60℃. The amylase can be inhibited by Ca2+, Fe3+, Zn2+, Cu2+, Mn2+ and strongly activated by K+, EDTA could reduce the enzymatic activity of about 30%, therefore, the amylase could be a kind of metalloenzymes.