Elucidation of sulfate-dependent acid phosphatase from Thiobacillus thiooxidans and determination of sulfate in waters using whole cells

我们开发了一种微生物方法，以全细胞硫酸依赖酸性磷酸酶（APase）为基础，测定水中硫酸盐浓度。以对硝基苯基磷酸为底物，比色法测定了APase的活性。硫酸对APase进行了活化。磷酸酶活性与硫酸盐浓度在0.6mM ~ 0.6mM范围内呈线性关系。因此，硫酸盐的浓度是由吸光度所代表的APase活性来估计的。采用微生物法测定水中硫酸根的含量。微生物法测定的结果与传统氯苯甲酸钡法测定的结果基本一致。建立了以硫代氧化霉细胞为填料的微生物柱和氧电极固定化葡萄糖氧化酶（GOD）组成的硫酸盐连续测定系统。在本系统中，采用热处理后的细胞作为APase，因为当细胞经过70℃，30min的热处理后，硫酸盐对APase的活化作用增强，并且以葡萄糖-6-磷酸（G-6-P）作为APase的底物。经热处理的细胞中APase由G-6-P产生葡萄糖，其活性被硫酸盐激活。用氧电极测定G-6-P产葡萄糖的浓度。葡萄糖浓度对应的氧电极响应值与硫酸浓度在0 ~ 1.0mM范围内呈线性关系。因此，通过氧电极的响应值来估计硫酸盐的浓度。该系统对5个样品的测量值具有较高的重现性。

We developed a microbial method to determine sulfate concentration in water based on a sulfate-dependent acid phosphatase (APase) in whole cells of Thiobacillus thiooxidans.The activity of APase was colorimetrically determined by using p-nitrophenylphosphate as substrate. The APase was activated by sulfate. A linear relationship was obtained between the activity of APase and the concentration of sulfate in the range of to 0.6mM.Therefore, the concentration of sulfate was estimated from the activity of APase represented byabsorbance. The microbial method was applied to the determination of sulfate in water. The values measured by the microbial method almost agreed with those by a conventional barium chloranilate method.A system composed of a microbial column packing the cells of T.thiooxidans and an oxygen electrode immobilizing glucose oxidase (GOD) was developed to perform a continuous determination of sulfate. In this system, the heat-treated cells were employed as APase because the activation effect by sulfate was increased when the cells were exposed to the heat-treatment (70ﾟC,30min), and glucose-6-phosphate (G-6-P) was used as substrate for APase. The APase in the heat-treated cells produced glucose from G-6-P and its activity was activated by sulfate. The concentration of glucose produced from G-6-P was determined by the oxygen electrode with GOD.The linear relationship was obtained between the response value of oxygen electrode corresponding to glucose concentration and the concentration of sulfate in the range of 0 to 1.0mM.Therefore, the concentration of sulfate was estimated from the response value of the oxygen electrode. The values measured by this system expressed a high reproducibility for 5 measurements of sample.