Purification and characterization of alkaline phosphatase from the gut of sea cucumber Stichopus japonicus |
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Authors: | Hai-Tao Wu Dong-Mei Li Bei-Wei Zhu Jing-Heng Cheng Jin-Jian Sun Feng-Lin Wang Yang Yang Yu-Kun Song Chen-Xu Yu |
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Institution: | 1. School of Food Science and Technology, Dalian Polytechnic University, Dalian, 116034, People’s Republic of China 2. Engineering Research Center of Seafood, Ministry of Education, Dalian, 116034, People’s Republic of China 3. Department of Agricultural and Biosystems Engineering, Iowa State University, Ames, IA, 50011, USA
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Abstract: | An alkaline phosphatase was purified from the gut of sea cucumber Stichopus japonicus by n-butyl alcohol extract, ammonium sulfate precipitation, ion exchange chromatography with diethylaminoethyl cellulose, gel filtration chromatography with Sephacryl S-200 and preparative electrophoresis with polyacrylamide gel electrophoresis. The native enzyme was estimated to be 166 ± 9 kDa and produced a single predominant band corresponding to active enzyme on nondenaturing electrophoresis, but showed 2 bands of 97 and 35 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting that the native enzyme is composed of two dissimilar subunits. The enzyme displayed maximum activity at pH 11 and 40 °C, showing narrow pH stability (pH 10–12) and thermal instability at temperature higher than 30 °C. The activity of the purified alkaline phosphatase was enhanced by Mg2+, whereas inhibited by Zn2+, Ca2+ and EDTA at 1 and 10 mM, suggesting its activity is in a magnesium ion-dependent manner. The product-analog WO4 2? and product HPO4 2? showed strong inhibitory effects on the enzyme activity. Using p-nitrophenyl phosphate as substrate, the V max and K m values were 24.45 μmol/L min and 5.76 mM, respectively. |
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