Stdudies On Relativity Between α-Glucosidic Bond Hydrolase From Sea Cucumber And Autolysis Of Sea Cucumber Body Wall

The sea cucumber was rich in nutrition, but tend to autolyze. Even the cooked body wall of sea cucumber turned melting during its processing, storage and marketing, resulting in the loss of nutrition from its body wall. This thesis had studied a-glucosidic bond hydrolase (focusing on pectinase, a polygalacturonic acid hydrolysing enzyme) and its correlation to the unstablizing of body wall of sea cucumber.First of all, three reducing end assay methods employed for polysaccharide hydrolase activity were compared, such as DNS method, PAHBAH method and MBTH method. The results showed that the curves of the three methods had good linear relationship. MBTH method was the most sensitive, and DNS method was the least sensitive that it can not be used in the activity detection in sea cucumber. PAHBAH method was not fit for measuring in large scale owing to its poor stability of the colored product. Therefore, MBTH method was used for the trace activity of polysaacharide hydrolase assay.Secondly, crude enzymes were primarily isolated from sea cucumber (Apostichopus japonicus) body wall through mechanical homogenization, buffer extraction, ammonium sulfate precipitation, dialysis. The activities of pectinase, amylase and mannase had been detected in the crude enzyme, in which pectinase had the highest activity. Alkaline pH was efficient for the pectinase extraction. The enzymatic properties were studied using pectin from citrus peel as a substrate, and the results showed that the optimum pH was 7.0. The residual activities, after incubation in corresponding pH (3.0-10.0) solution for 4 hr, increased slightly, indicating that certain ionic strength could acitivate pectinase’s activity and the enzyme (or enzymes were) was acid and alkali resistant. The activity of pectinase was highest at 90℃. Incubation at 90℃ for 20 min could hardly decrease the pectinase activity, suggesting that the enzyme was thermal stable, which was of the important significance for studying on the degradation mechanism of cooked-processed sea cucumber.Thirdly, the charge nature of crude enzyme was studied for further purification, and the isoelectric point of most protein was concentrated on pH4.40. Therefore, anion exchange chromatography was used and the weak DEAE-52 and strong Q-Fast Flow were selected. The specific activity of the ion exchange chromatography peaks from 0.1 and 0.2 mol/L NaCl were higher than other peaks. To collect more pectinase, strong anion resin Q-Fast Flow was used nstead of DEAE-52, mobile phase pH increased from 7.0 to 8.0, but significant change was not obtained, indicating that both methods could be used to isolate pectinase. Compared the results of electrophoresis, the molecular weight of pectinase derived from sea cucumber body wall may be 280-288kDa.Besides, the pectinase isolated from the ion exchange chromatography peaks in 0.1 (peak 1) and 0.2 (peak 2) mol/L NaCl fractions were characterized. In peak 1, the activity of pectinase was highest when at 90℃, pH7.0. Its residual activity still remained more than 40% after incubation at 100℃ for 20 min. Ba2+、K+ could activate the pectinase significantly, Fe3+, Mg2+, Zn2+ could inhibit it significantly. Ca2+, Mn2+ had little inhibition to the enzyme. EDTA had the significant inhibition, indicating it was a metalloenzyme. In peak 2, the activity of pectinase was highest when at 80℃, pH7.0. The pectinase was obtained 150% of its activity after incubation at 100℃ for 20 min, suggesting that it was the intensed heat induced enzyme. Ba2+, Fe3+, Ca2+ could activate the pectinase significantly, Mg2+, Mn2+, K+ had the opposite effect significantly. Zn2+ had little inhibition. EDTA had the significant inhibition, indicating it was also a metal loenzyme. The reason why intensed heat could activate the pectinase requires further intensive research for the near future.Finally, the correlation of pectinase existed in the body wall of sea cucumber to the autolysis was studied using inactivated body wall as substrates and dialytic solution and elution from Q-Fast Folw as pectinase for the susceptibility research. The results showed that the enzyme solutions described above having the activity of pectinase released reducing substance. Most importantly, intense-heat processing could increase both the pectinase activity and the reducing substance from the body wall of the sea cucumber, implying that the activity of the enzyme hydrolyzing sea cucumber body wall was most positively correlated with pectinase that existed in the body wall of sea cucumber, regardless of protease activity.