Studies On Protease And Amylase Of Holothurian

Holothurian which is recognized as a kind of cate as well as nutritional food has highlynutritial value and pharmacological value. In order to provide reference for digestivephysiology, artificial propagation, the preparation of compound feed and potential utilizationof enzymes from Holothurian, the characteristics of digestive proteases, amylases in theintestines and proteases from the wall of Holothurian were investigated respectively by usingbiochemical methods and electrophoretic techniques in this study. The results were as follows:The effects of pH, temperature, metal irons on the activities of digestive proteases in theintestine of Holothurian were researched. According to the study, the favorable reacting pHand temperature for digestive proteases were5.0～6.0and60℃. The activities of digestiveproteases was stable at the temperatures of30℃～50℃. The effects of ten metal irons on theactivities of digestive proteases were studied by using enzyme analytical method. The resultsdemonstrated that: the proteases were potently inhibited by Ag⁺and Cu²⁺; partially activatedby K⁺, Ba²⁺, Zn²⁺, Pb²⁺, Fe²⁺, Al³⁺and Cr³⁺, and faintly influenced by Ca²⁺. The separationand analysis of digestive proteases were executed through native-PAGE and glutin digestionin situ. Two protease isoenzymes were separated from digestive proteases, in which therewere one bright band and one mid-bright band.The characteristics of proteases from the body wall of Holothurian were discussedsecondly. According to the results, the optimum pH value and temperature for the proteaseswere determined as9.0and50℃respectively. Incubated at different temperatures for sometime, activities of proteases from the body wall of Holothurian decreased quickly. Itdemonstrated that the enzymes were unstable to heat. The effects of ten metal irons on theactivities of the proteases were studied by using enzyme analytical method. The resultsdemonstrated that: the proteases were potently inhibited by Fe²⁺and Al³⁺, partially inhibitedby Pb²⁺, Ag⁺, Cr³⁺, Zn²⁺and Ca²⁺, activated by K⁺, Ba²⁺and Cu²⁺. Glutin digestion in situ wasalso applied to the proteases, one dim band was found.The characteristics of amylases in intestine of Holothurian were also researched. Theoptimum pH value and temperature of the enzymes were6.5and40℃. The amylases were stable below the temperature of40℃. The effects of ten metal irons on the activity ofdigestive amylases were also studied by using enzyme analytical method. The resultsdemonstrated that: the enzymes were inhibited by Cu²⁺, Cr³⁺, Al³⁺, Zn²⁺, Pb²⁺, Fe²⁺andpartially activated by K⁺, Ag⁺, Ba²⁺and Ca²⁺. Isozyme technology was applied to digestiveamylases. Two zymograms were detected. The digestive amylases from fresh Holothurianwere separated and purified by ammonium sulfate precipition and Sephadex G-100columnchromatography. The purity of purified amylase was8.37fold than the original crudeamylases. The experiment results indicated that the enzyme’s molecular weight was about37.8kDa.