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

Alkaline phosphatase (ALP) is one of the most important phosphatase, widely existing in the microorganism and animal kingdom. It directly participates in the transform and metabolism of phosphate groups. It also plays an important role in calcium absorption, bone formation and calcium phosphate sedimentation. It’s also a high valuable tool enzyme which can be used in studying nucleic acid, analyzing nucleic sequences and the isolation, recombination of genes, blotting and ELISA.The purification and property of alkaline phosphatase obtained from the gut of sea cucumber were studied firstly in this paper. ALP was extracted by Tris-HCl (pH 8.9) buffer and further isolated by n-butanol, ammonium sulfate precipitation (70%), ultrafiltration, chromatography with DEAE-52, Sephacryl S-100 and Native-PAGE. The purified ALP had two protein bands with estimated Mw of 97.2kDa and 34.7kDa on SDS-PAGE.The ALP was 20.55-fold. The specific activity was 58.6U/mg.With p-NPP being the substrate of alkaline phosphatase, the enzyme displayed maximum activity at pH 10.5 and 40℃, and showed high stability in the range of 20～30℃. The enzyme activity was markedly activated by Mg2+, whereas inhibited by Zn2+, Ba2+, Sn2+ and Ca2+. Some inhibitors such as EDTA, L-Phe, Na2WO4, Na2HPO4 and levomisole significantly inhibited the ALP activity. EDTA and Na2WO4 inhibited the ALP activity more obviously than others. The product HPO42- and the product-analog WO42- could inhibit the activity of the ALP, which is only with about 43.7% and 41.8% activity left at concentrations of 30 mmol/L. The Michaelis constant (Km) and maximum velocity (Vm) of alkaline phosphatase was Km=5.76 mmol/L, Vm=24.45 μmol/L·min.The alkaline phosphatase was selectively modified by chloramines-T, PMSF, IAc and DTT, and changes in the activities of the enzyme have been detected. The results showed that Met and His residues couldn’t be the essential groups of ALP, Ser residues maybe considered as essential groups of ALP, and partial disulfide bonds was essential for the function of the enzyme.