Enzymatic characterization and regulation of gene expression of PhoK alkaline phosphatase in Sphingobium sp. strain TCM1

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 Ca 2+ and one Zn 2+ per subunit, but excess addition of Zn 2+ 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 K m and k cat 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.