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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.

Sphingobium sp. strain TCM1 can degrade tris(2-chloroethyl) phosphate (TCEP) to inorganic phosphate and 2-chloroethanol. A phosphotriesterase (PTE), phosphodiesterase (PDE) and phosphomonoesterase (PME) are believed to be involved in the degradation of TCEP. The PTE and PME that respectively catalyze the first and third steps of TCEP degradation in TCM1 have been identified. However, no information has been reported on a PDE catalyzing the second step. In this study, we identified, purified, and characterized a PDE capable of hydrolyzing haloalkyl phosphate diesters. The final preparation of the enzyme had a specific activity of 29 µmol min -1  mg -1 with bis(p-nitrophenyl) phosphate (BpNPP) as the substrate. It also possessed low PME activity with p-nitrophenyl phosphate (pNPP) as substrate. The catalytic efficiency (k cat /K m ) with BpNPP was significantly higher than that with pNPP, indicating that the enzyme prefers the organophosphorus diester to the monoester. The enzyme degraded bis(2,3-dibromopropyl) phosphate, bis(1,3-dichloro-2-propyl) phosphate and bis(2-chloroethyl) phosphate, suggesting that it is involved in the metabolism of haloalkyl organophosphorus triesters. The primary structure of the PDE from TCM1 is distinct from those of typical PDE family members and the enzyme belongs to the polymerase and histidinol phosphatase superfamily.

Tris(2-chloroethyl) phosphate (TCEP) is a haloalkyl phosphate flame retardant and plasticizer that has been recognized as a global environmental contaminant. Sphingobium sp. strain TCM1 can utilize TCEP as a phosphorus source. To identify the phosphomonoesterase involved in TCEP utilization, we identified four putative alkaline phosphatase (APase) genes, named SbphoA, SbphoD1, SbphoD2, and SbphoX-II, in the genome sequence. Following expression of these genes in Escherichia coli, APase activity was confirmed for the SbphoA and SbphoX-II gene products but was not clearly observed for the SbphoD1 and SbphoD2 gene products, owing to their accumulation in inclusion bodies. The single deletion of either SbphoA or SbphoX-II retarded the growth and reduced the APase activity of strain TCM1 cells on medium containing TCEP as the sole phosphorus source; these changes were more marked in cells with the SbphoX-II gene deletion. In contrast, the deletion of either SbphoD1 or SbphoD2 had no effect on cell growth or APase activity. The double deletion of SbphoA and SbphoX-II resulted in the complete loss of cell growth on TCEP. These results show that SbPhoA and SbPhoX-II are involved in the utilization of TCEP as a phosphorus source and that SbPhoX-II is the major phosphomonoesterase involved in TCEP utilization.

Phosphotriesterases catalyze the first step of organophosphorus triester degradation. The bacterial phosphotriesterases purified and characterized to date hydrolyze mainly aryl dialkyl phosphates, such as parathion, paraoxon, and chlorpyrifos. In this study, we purified and cloned two novel phosphotriesterases from Sphingomonas sp. strain TDK1 and Sphingobium sp. strain TCM1 that hydrolyze tri(haloalkyl)phosphates, and we named these enzymes haloalkylphosphorus hydrolases (TDK-HAD and TCM-HAD, respectively). Both HADs are monomeric proteins with molecular masses of 59.6 (TDK-HAD) and 58.4 kDa (TCM-HAD). The enzyme activities were affected by the addition of divalent cations, and inductively coupled plasma mass spectrometry analysis suggested that zinc is a native cofactor for HADs. These enzymes hydrolyzed not only chlorinated organophosphates but also a brominated organophosphate [tris(2,3-dibromopropyl) phosphate], as well as triaryl phosphates (tricresyl and triphenyl phosphates). Paraoxon-methyl and paraoxon were efficiently degraded by TCM-HAD, whereas TDK-HAD showed weak activity toward these substrates. Dichlorvos was degraded only by TCM-HAD. The enzymes displayed weak or no activity against trialkyl phosphates and organophosphorothioates. The TCM-HAD and TDK-HAD genes were cloned and found to encode proteins of 583 and 574 amino acid residues, respectively. The primary structures of TCM-HAD and TDK-HAD were very similar, and the enzymes also shared sequence similarity with fenitrothion hydrolase (FedA) of Burkholderia sp. strain NF100 and organophosphorus hydrolase (OphB) of Burkholderia sp. strain JBA3. However, the substrate specificities and quaternary structures of the HADs were largely different from those of FedA and OphB. These results show that HADs from sphingomonads are novel members of the bacterial phosphotriesterase family., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Tris(2-chloroethyl) phosphate (TCEP), a flame retardant, is recently regarded as a potentially toxic and persistent environmental contaminant. We previously isolated TCEP-degrading bacterium, Sphingobium sp. strain TCM1, which, however, produced a toxic metabolite: 2-chloroethanol (2-CE). This study was undertaken to develop a detoxification technique of TCEP using strain TCM1 with a 2-CE-degrading bacterium: Xanthobacter autotrophicus strain GJ10. TCEP degradation by strain TCM1-resting cells was thermally stable for 30 min at 30 °C. It was optimal at 30 °C and at pH 8.5. In the optimum condition, TCM1 cells up to a final cell density of 0.8 at OD(660) in the reaction mixture were unable to hydrolyze the phosphotriester bonds of 10 μM TCEP completely. The addition of 50 μM Co(2+) to reaction mixture enhanced the hydrolysis and caused the complete hydrolysis at the cell density of 0.8. Strain GJ10 resting cells degraded 2-CE only slightly, which might be attributable to lack of coenzyme regeneration of enzymes involved in the degradation. In contrast, the growing cells degraded approximately 180 μM of 2-CE within 24 h. Based on these results, we designed a two-step TCEP detoxification reaction consisting of TCEP hydrolysis to 2-CE by strain TCM1-resting cells and the following degradation of the resulting 2-CE by strain GJ10-growing cells. The combined reaction completely detoxified 10 μM TCEP, and thus opens a way to microbial detoxification of the potential toxic, persistent organophosphorus compound., (Copyright © 2012 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Tris(1,3-dichloro-2-propyl) phosphate (TDCPP), a flame retardant, is regarded as a potentially toxic and persistent environmental contaminant. We previously isolated a TDCPP-degrading bacterium, Sphingobium sp. strain TCM1, which, however, produced a toxic metabolite: 1,3-dichloro-2-propanol (1,3-DCP). This study was undertaken to develop a technique for complete TDCPP detoxification using strain TCM1 with a 1,3-DCP-degrading bacterium, Arthrobacter sp. strain PY1. For efficient detoxification, we designed a resting cell system and examined the effect of freezing and lyophilization treatments for preparation of their resting cells. Results show that treatments had no marked adverse effect on their activities. The TDCPP dephosphorylation by TCM1 resting cells was optimal at 30°C and pH 8.5. Also, 1,3-DCP dehalogenation by strain PY1 resting cells was optimal at 35°C and pH 9.5. Under those respective conditions, the activities were 2.48 μmol h⁻¹·OD₆₆₀⁻¹ for TDCPP and 0.95 μmol h⁻¹·OD₆₆₀⁻¹ for 1,3-DCP. Based on these results, we set the reaction temperature to 30°C and pH to 9.0. Then we examined the detoxification of 50 μM TDCPP using mixed resting cells at a final OD(660) of 0.05 for strain TCM1 and 0.2 for strain PY1. In these conditions, TDCPP was eliminated after 1h, but some of the resulting 1,3-DCP remained at a constant level. The increase in strain PY1 cells to a final OD₆₆₀ of 4.0 decreased the TDCPP dephosphorylation rate of strain TCM1 cells but achieved complete detoxification of TDCPP during 12 h of reaction., (Copyright © 2011 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)