海参肠碱性磷酸酶的提取及粗酶的特性研究

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+(1～30mmol/L)、Zn2+(<10mmol/L)对海参肠ALP粗酶具有显著的激活作用;Fe3+、Fe2+、Cu2+和Mn2+(10mmol/L)可明显抑制该酶活力。EDTA-Na2、DTT、Na2WO4及Na2HPO4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>Na2WO4>Na2HPO4。

Extraction and characterization of crude alkaline phosphatase from the gut of sea cucumber Stichopus japonicus

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 n-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+(1-30 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.