Sphingobium sp. strain TCM1 can significantly degrade chlorinated organophosphorus flame retardants, such as tris(2-chloroethyl) phosphate. The PhoK of strain TCM1 (Sb-PhoK) is the main alkaline phosphatase (APase) that catalyzes the last step in the degradation pathway. Here, we purified and characterized Sb-PhoK produced in E. coli, and analyzed the regulation of Sb-phoK gene expression in strain TCM1. The recombinant Sb-PhoK was produced in the mature form, lacking a putative signal peptide, and formed a homodimer. Purified Sb-PhoK exhibited 384 U/mg of specific activity at 37 °C. The optimum temperature was 50 °C, and Sb-PhoK was completely inactivated when incubated at 60 °C for 10 min. The optimum pH was 10, with stability observed at pH 6.0-10.5. Sb-PhoK was suggested to contain two Ca2+ and one Zn2+ per subunit, but excess addition of Zn2+ into the reaction mixture markedly inhibited the enzyme activity. Sb-PhoK showed phosphatase activity against various phosphorylated compounds, except for bis(p-nitrophenyl) phosphate, indicating that it is a phosphomonoesterase with broad substrate specificity. The Km and kcat for p-nitrophenyl phosphate were 2.31 mM and 1270 s-1, respectively, under optimal conditions. The enzyme was strongly inhibited by vanadate, dithiothreitol, and SDS, but was highly resistant to urea and Triton X-100. Sb-phoK gene expression was regulated by the inorganic phosphate concentration in culture medium, and was induced at a low inorganic phosphate concentration. The deletion of Sb-phoB gene resulted in no induction of Sb-phoK gene even at a low inorganic phosphate concentration, confirming that Sb-PhoK is a member of Pho regulon.

中文翻译：

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鞘氨醇单胞菌中PhoK碱性磷酸酶基因表达的酶学表征和调控。菌株TCM1。

鞘氨醇单胞菌 TCM1菌株可显着降解氯化有机磷阻燃剂，例如磷酸三（2-氯乙基）酯。菌株TCM1的PhoK（Sb-PhoK）是主要的碱性磷酸酶（APase），可催化降解途径的最后一步。在这里，我们纯化和表征了在大肠杆菌中产生的Sb-PhoK，并分析了菌株TCM1中Sb-phoK基因表达的调控。重组Sb-PhoK以成熟形式生产，缺少推定的信号肽，并形成了同源二聚体。纯化的Sb-PhoK在37°C下的比活性为384 U / mg。最佳温度为50°C，并且在60°C孵育10分钟时Sb-PhoK完全失活。最佳pH为10，在pH 6.0-10.5处观察到稳定性。建议Sb-PhoK每个亚基包含2个Ca2 +和1个Zn2 +，但是过量加入Zn2 +会明显抑制酶的活性。除双（对硝基苯基）磷酸酯外，Sb-PhoK表现出针对各种磷酸化化合物的磷酸酶活性，表明它是具有广泛底物特异性的磷酸单酯酶。在最佳条件下，对硝基苯基磷酸酯的Km和kcat分别为2.31 mM和1270 s-1。该酶被钒酸盐，二硫苏糖醇和SDS强烈抑制，但对尿素和Triton X-100具有高度抗性。Sb-phoK基因表达受培养基中无机磷酸盐浓度的调节，并在较低的无机磷酸盐浓度下被诱导。即使在低无机磷酸盐浓度下，Sb-phoB基因的缺失也不会导致Sb-phoK基因的诱导，这证实了Sb-PhoK是Pho regulon的成员。