长毛对虾磷酸酯酶的研究

Studies of Phosphatase from Penaeus penicillatus

从长毛对虾 (Penaeuspenicillatus)头胸部、肌肉分别提取纯化碱性磷酸酶ALPase(EC 3 1 3 1)和酸性磷酸酶ACPase (EC 3 1 3 2 ) ,经快速蛋白液相色谱 (FPLC)检测AL Pase ,聚丙烯酰胺凝胶电泳以及等电聚焦电泳检测ACPase ,得到单一纯酶 .ALPase紫外特征吸收峰在 2 78nm处 ,荧光激发峰在 2 82nm处 ,发射光谱特征峰在 340nm处 .ACPase在 2 80nm处有紫外吸收特征峰 ,荧光激发峰在 2 84nm处 ,发射峰在 347nm处 .分别测定了酶的相对分子质量、亚基数、氨基酸组成、等电点、酶水解对硝基苯磷酸二钠 (pNPP)的最适温度 ,最适pH ,米氏常数Km 值 ,金属离子Mg2 +、Cu2 +、Hg2 +,有机溶剂甲醇、乙醇、乙二醇 ,化学修饰剂BrAe、NBS、pCMB等以及蛋白质变性剂胍、脲等对酶活力的影响 .比较研究健康虾和病虾ACPase酶活力及酶动力学特征常数的变化 (健康虾酶Km=0 0 8mmol/L ,病虾酶Km=0 143mmol/L) .病虾酶Km 值明显增大 ,表现对底物亲和力下降 .活化能明显增大 (健康虾酶 4 1 0 3kJ·mol- 1 ·L- 1 ,病虾酶 4 5 7kJ·mol- 1 ·L- 1 )表明酶的催化能力下降 .

ALPase (DC 3 1 3 1) and ACPase (EC 3 1 3 2) from Penaeus penicillatus were prepared and purified The preparation was shown to be homogenous by FPLC and polyacrylamied gel electrophoresis ALPase characteristic peak of UV absorption spectrum of the enzyme is at 278 nm, and that of the fluorescence excitaton spectrum at 282 nm and the fluorescence emission spectrum at 340 nm,ACPase characteristic peak of UV absorption spectrum presents at 280 nm, the fluorescence excitation spectrum at 284 nm and the fluorescence emission band at 347 nm.The molecular weight,enzyme subunit,amino acid compositions,isoelectric point,optimum pH,optimum temperature and the Michaelis constant of the enzymes for the hydrolysis of p nitrophenyl phosphate (pNPP) was messured respectively.The enzyme activity is markedly inhibited by copper ion, mercury ion, methanol,ethanol and ethylene glycol. Denaturation and inactivaion kinetics of the enzyme from the healthy shrimp and the sick are 41 03 kJ·mol -1 ·L -1 and 45 78 kJ·mol -1 ·L -1 ,espectively at pH 4 5, 37 ℃,the Michaelis constant of each is 0 08 mmol/L and 0 143 mmol/L, respectively.