僧帽牡蛎碱性磷酸酶性质的研究

Studies on properties of alkaline phosphatase from Ostrea cucullate

从僧帽牡蛎分离纯化出一种碱性磷酸酶,采用Sephadex G-200凝胶过滤柱层析法测得该酶分子量为1.57×10~5.以对硝基苯磷酸为底物,该酶的最适温度为43℃,最适pH值为10.0.在37℃和pH值为10.0的条件下,酶催化反应的米氏常数(K_m)为9.68×10^(-4)mol/dm^3,活化能(E_a)为45.85kJ/mol,温度系数(Q_(10))为1.80(30～40℃).对该酶的热稳定性研究表明:其在45℃以下较为稳定,50℃以上明显失活,并测定了该酶的热失活速度常数.金属离子对酶活力的效应研究结果表明:Mg^(2+)、Mn^(2+)、Ca^(2+)、Co^(2+)对酶有激活作用,Ag^+、Hg^(2+)、Zn^(2+)表现为抑制作用,Cd^(2+)几乎没有影响.Mg^(2+)是较有效的激活剂,表现为部分非竞争性效应,激活常数(K_a)为2.48×10^(-4)mol/dm^3,有Mg^(2+)存在时该酶活性中心的转换数量是无Mg^(2+)时的4.0倍.几种效应物对酶的抑制机理研究表明:HPO_4^(2-)、HAsO_4^(2-)、Cys为竞争性,其抑制常数分别为1.61、1.18和0.15mmol/dm^3;L-Phe为反竞争性,抑制常数为3.26mmol/dm^3.

A kind of alkaline phosphatase (ALP) was isolated and purified from Ostrea cucul-late. The molecular weight of the enzyme was measured to be about 1.57 × 105 by means of gel filtration on Sephadex G-200 column. The optimum temperature of the enzyme for the hydrolysis of disodium p-nitrophenyl phosphate is 43℃, and the optimum pH is 10.0, the Michaelis-con-stant (Km) is 0.968 mmol/dm3(at 37℃ , pH 10.0), the activation energy (Ea)is45.85 kJ/mol, the temperature coefficient (Q10) is 1.80 (30 - 40℃). The enzyme is stable when temperature below 45℃, and is inactivated by heat above 50℃ rapidly, and then the inactivation velocity constant by was been determined. A survey of effect of metal ions on the enzymed showed that Mg2+ , Mn2+ , Ca2+ and Co2+ activate the enzyme; Ag+, Hg2+ and Zn2+ inhibit the enzyme; and Cd2 + has almost not any effect. The magnesium ion is a more efficacious activator and the effect is classified as partly noncompetitive type, and the activation constant is 2.48 × 10-4 mol/dm3. The turnover number of the enzyme active center in the presence of Mg ion is 4.0 times as much as that is in the absence. The inhibition of HPO42- , HAsO42- and Cys on the enzyme is found to be of competitive type, and the inhibition constant is 1.61, 1.18 and 0.15 mmol/dm3 respectively. L-Phe is of uncompetitive type, and the inhibition constant is 3.26 mmol/dm .