海参肠碱性磷酸酶的提取及粗酶的特性研究

摘要

The extraction and characterization of crude alkaline phosphatase (ALP) from the gut of the sea cucumber were studied in this paper. The crude ALP was extracted with Tris-HCl (pH 8. 9) buffer containing 0. 2% of Triton X-100 and isolated by w-butanol treatment, ammonium sulfate (70%) precipitation and ultrafiltration. The procedure resulted in a purification fold of 2.74 and a yield of 63. 32%. The crude ALP displayed maximum activity at pH 11.0 and 45 ℃ , and showed high stability in the range of pH 10.0-12.0 and 20-45 ℃. The activity of crude ALP was markedly activated by Mg2+(l-30 mmol/L) and Zn2+(<10 mmol/L), whereas strongly inhibited by Fe3+ , Fe2+ , Cu2+ and Mn2+(10 mmol/L). The activity of ALP significantly inhibited by some inhibitors such'as EDTA-Na2, DTT, Na2WO4 and Na2HPO4 following order of EDTA-Na2 > DTT > Na2WO4 > Na2HPO4.%碱性磷酸酶(Alkaline phosphatase,ALP)是最重要的磷酸酶之一,在生物体内直接参与磷酸基团的转移和代谢过程.本文研究了海参肠碱性磷酸酶粗酶的提取,并分析了其酶学特性.经含有0.2％Triton X-100的Tris-HCl缓冲液(pH 8.9)浸提、正丁醇处理、70％硫酸铵沉淀和超滤等步骤获得海参肠ALP粗酶,纯化倍数达到2.74倍,得率为63.32％.ALP粗酶的最适反应pH为11.0,在pH 10.0～12.0稳定性较好；最适反应温度为45℃,在20～45℃具有很高的稳定性.金属离子Mg2+(1～30 mmol/L)、Zn2+ (＜10 mmol/L)对海参肠ALP粗酶具有显著的激活作用；Fe3+、Fe2+、Cu2+和Mn2+ (10 mmol/L)可明显抑制该酶活力.EDTA-Na2、DTT、Na2 WO4及Na2 HPO4对海参肠ALP粗酶有明显的抑制作用,抑制程度依次为:EDTA-Na2＞DTT＞Na2 WO4＞Na2 HPO4.