【摘要】 用Tris-HCl缓冲液抽提,硫酸铵分级沉淀,DEAE-纤维素和葡聚糖凝胶层析,从草鱼肠中提取出碱性磷酸酶(ALP).提取倍数为6.74,比活为684 U/mg蛋白.酶液经PAGE呈现单一带,该酶催化对硝基苯磷酸二钠(pNPP)水解反应的最适pH值为10.41,最适温度为27℃,Km值为1.01 mmol/L,酶的热稳定性与pH稳定性研究表明:该酶在pH 6.6~11.5区间和在温度低于45℃下稳定.研究金属离子对酶活力影响的结果表明:一价金属离子Li+、Na+和K+对酶活力没有影响;二价金属离子Mg2+、Mn2+和Co2+对酶有不同程度的激活作用;Zn2+对酶有抑制作用.更多还原

【Abstract】 An alkaline phosphatase was purified from Grass Carps（Ctenopharyngodon idellus） by following procedures:Tris-HCl buffer（pH 7.5） extraction,ammonium sulfate precipitation,ion-exchange chromatography on DEAE-32 cellulose,followed by gel filtration through SephadexG-200 and ion-exchange chromatography on DEAE-32.The final preparation was homogeneous on PAGE.The specific activity of the enzyme was 684 U/mg.The optimum pH is 10.41,and the optimum temperature is 27℃ for the hydrolysis of pNPP.Michaelis-Menten constant（K_m） is 1.01 mmol/L and the maximun velocity（V_m） is 5.49 mmol/L/min at pH 10.0 and 27℃.The enzyme is stable in the range of pH from 6.6 to 11.5 and in temperature below 45℃.A survey of effects of metal ions on the enzyme showed that the positive monovalent metal ions（Li⁺,Na⁺ and K⁺） have no effect on the enzyme;positive bivalent metal ions(Mg²⁺,Mn²⁺ and Co²⁺) activate the enzyme,Zn²⁺inhibit the enzyme.The result indicates that different metal（ions） have different effect on the ALP.The study of ALP from Grass Carps（Ctenopharyngodon idellus） may give some help to Grass Carps culture.更多还原