Extraction, Purification And Characterization Of Acetylcholinesterase From The Gut Of Sea Cucumber Stichopus Japonicus

The crude AChE was extracted from the gut or body wall of sea cucumber with PBS (0.1 mol/L, pH 7.4) containing 0.1% Triton X-100. The results indicated that AChEs from the gut and body wall of sea cucumber have the similar enzymatic characteristics. The crude AChE displayed maximum activity at pH 8.0 and over 35℃, and showed high stability in the range of pH 6.0～9.0 and 25～40℃. AChE from sea cucumber hydrolyzed acetylthiocholine iodide (AcSChI) effectively, but showed little effect on butyrylthiocholine iodide (BuSChI). At a concentration higher than 50 mmol/L, AcSChI inhibited the enzyme activity obviously. The activity of AChE was inhibited by Sn2+, Zn2+, Hg2+, Ag+, Cr6+, Cu2+ and some inhibitors such as Eserine and BW284c51.The changes of AChE activity were investigated after the gut of the sea cucumber was aulyzed by UV irradiation for 30 min and placing for different period. AChE did not have remarkable change exactly during auto lysis. After the gut of the sea cucumber was irradiated for 30 min by UV and placed for 4 h, the effect of AChE inhibitor eserine on the dissolution rate of TCA soluble protein was inspected. The results demonstrated that eserine promote the autolysis of sea cucumber. It showed that AChE may inhibit the autolysis of sea cucumber.It was purified to homogeneity by DEAE-52 and Sepharose CL-6B. The enzyme was purified 35.49-fold through the consecutive separation. The enzyme displayed maximum activity at pH 7.5 and over 35℃, and showed high stability in the range of pH 6.0～8.0 and 25～40℃. AcSChI was a specific substrate and Km was 0.62 mmol/L. At a concentration higher than 0.8 mmol/L, AcSChI inhibited the enzyme activity obviously. The activity of AChE was greatly inhibited by Eserine and BW284c51 and scarcely inhibited by iso-OMPA.The elution conditions for affinity chromatography of acetylcholinesterase from the gut of sea cucumber were optimized by using 3-carboxyphenyl ethyldimethyl ammonium (CEA) coupled with CNBr-Sepharose-4B colum. The results indicated that the equilibrium system was PBS (0.05 mol/L, pH 7.4) containing 0.3 mol/L NaCl and the elution system was PBS (0.05 mol/L, pH 7.4) containing 0.2 mol/L tetraethylammonium iodide. Under this condition,AChE was well adsorbed and separated. The AChE purified preliminarily by affinity chromatography had three enzyme activity bands on Native-PAGE, suggesting AChE in the gut of sea cucumber may have different forms and display the polymorphism.