| 摘要: |
| 于1995年2月在厦门海域采集锯缘青蟹，取其内脏经正丁醇抽提、硫酸铵分级分离、DEAE-52离子交换柱层析及Sephadex G-200凝胶过滤柱层析纯化，获得碱性磷酸酶制剂，以快速蛋白液相色谱及聚丙烯酰胺凝胶电泳检验其纯度，获得单一蛋白纯的酶制剂。研究酶的物理性质得出该酶的紫外特征吸收峰在278nm处，荧光激发光谱特征峰在282nm处，荧光发射光谱特征峰在343nm处，全酶分子量为78kD。研究酶催化对－硝基苯磷酸二钠水解的动力学性质，金属离子及有机溶剂对其活力的影响，得知该酶水解对－硝基苯磷酸二的最适温度为52℃，最适pH为9.2，米氏常数为6.67×10-4 mol/L，酶活性中心的转换常数为460 min-1；Mg2+对酶有显著的激活作用；Cn2+，Hg2+和甲醇、乙醇、乙二醇对酶有不同程度的抑制，说明在一定条件下，效应物引起酶分子构象发生了不同程度的变化。 |
| 关键词: 锯缘青蟹 碱性磷酸酶 分离 纯化 理化性质 |
| DOI： |
| 分类号: |
| 基金项目:国家自然科学基金资助项目，39470561号 |
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| STUDIES ON ISOLATION, PURIFICATION AND SOME PHYSICAL AND CHEIVHCAL PROPERTIES OF ALKALINE PHOSPHATASEFROM GREENCRAB (SCYLLA SERRATA) |
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CHEN Qing-xi,ZHANG Zhe,ZHUANG Zong-lai,CHEN Xiang-ren
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Department of Biology Xiamen University, Xiamen, 361005
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| Abstract: |
| An alkaline phosphatase from green crab (Scylla serrata) was collected from Xiamen seawater in February 1995. It was prepared and purified by means of the following techniques: n-butanol extraction, ammonium sulfate precipitation, ion exchange chromatography on DEAE cellulose column (DEAE-52) and gel filtration chromatography on Sephadex G-200. The preparation was shown to be homogenous on FPLC and polyacrylarnide gel electrophoresis. The characteristic peak of UV-absorption spectrum of the enzyme was found to be at 278 nm, the fluorescence excitation spectrum at 282 nm, and the fluorescence emission spectrum at 343 nm. The molecular weight of the enzyme is 78 kD. At pH = 9.5, the optimum pH is 9.2, at 37°C. At pH = 9.0, 37°C, the Michaelis constant (KM) is 6.67×10-4 mol/L; Kcat, 460 min-1. Magnesium ion activates the enzyme significantly. Copper ion, mercury ion, methanol, ethanol and ethylene glycol inhibit the enzyme activity in varying degrees, which indicates that the conformation of the enzyme caused by the effectors changes at different levels. The inhibition mechanism had been preliminarily studied. |
| Key words: Scylla serrata, Alkaline phosphatase, Isolation, Purification Physical and chemical properties |
