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

基于硫氧硫杆菌全细胞内的硫酸盐依赖酸性磷酸酶(APase)，以对硝基苯磷酸为底物，用比色法测定水中硫酸盐的含量。APase被硫酸盐激活。APase活性与硫酸盐浓度在0.6 mM范围内呈线性关系。因此，可以用吸光度表示的APase活性来估算硫酸盐浓度。将微生物法应用于水中硫酸盐的测定。微生物法测定硫酸盐的结果与常规氯苯胺酸钾法的测定值基本一致。建立了一种由填充硫代氧化亚硫杆菌细胞的微生物柱和固定葡萄糖氧化酶(GOD)的氧电极组成的连续测定硫酸盐的系统。该体系以葡萄糖-6-磷酸(G-6-P)为底物，在热处理(70ﾟC，30min)条件下，硫酸盐对细胞的激活作用增强，因此采用热处理细胞作为酶的底物。热处理细胞中的APase从G-6-P产生葡萄糖，其活性被硫酸盐激活。用带有GOD的氧电极测定G-6-P产生的葡萄糖的浓度，氧电极的响应值与葡萄糖浓度在0~1.0 mM范围内呈线性关系，从而根据氧电极的响应值来估算硫酸盐的浓度。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.