Cloning and Overexpression of Alkaline Phosphatase PhoK from Sphingomonas sp. Strain BSAR-1 for Bioprecipitation of Uranium from Alkaline Solutions

Abstract

Cells of Sphingomonas sp. strain BSAR-1 constitutively expressed an alkaline phosphatase, which was also secreted in the extracellular medium. A null mutant lacking this alkaline phosphatase activity was isolated by Tn5 random mutagenesis. The corresponding gene, designated phoK, was cloned and overexpressed in Escherichia coli strain BL21(DE3). The resultant E. coli strain EK4 overexpressed cellular activity 55 times higher and secreted extracellular PhoK activity 13 times higher than did BSAR-1. The recombinant strain very rapidly precipitated >90% of input uranium in less than 2 h from alkaline solutions (pH, 9 ؎ 0.2) containing 0.5 to 5 mM of uranyl carbonate, compared to BSAR-1, which precipitated uranium in >7 h. In both strains BSAR-1 and EK4, precipitated uranium remained cell bound. The EK4 cells exhibited a much higher loading capacity of 3.8 g U/g dry weight in <2 h compared to only 1.5 g U/g dry weight in >7 h in BSAR-1. The data demonstrate the potential utility of genetically engineering PhoK for the bioprecipitation of uranium from alkaline solutions.Environmental metal pollution is a serious problem, and treatment/recovery of desired metals from such wastes is a major challenge. Effective immobilization of radionuclides of metals is critical in order to prevent groundwater contamination (17). Bioremediation of the toxic metal wastes by microbes offers a relatively inexpensive and ecofriendly alternative to commonly used physical and chemical methods (7,19,26). In particular, enzymatic bioprecipitation of heavy metals as metal phosphates is very attractive, since it can recover metals from very low concentrations not amenable to chemical techniques (18). Successful bioprecipitation of metals, such as uranium and cadmium, using acid phosphatase from naturally occurring bacteria, such as Citrobacter sp. (19), has been reported. The uranium bioprecipitation potentials of Bacillus sp., Rahnella sp. (5, 20), Pseudomonas sp. (22), and Salmonella sp. (27) in an acidic-to-neutral pH range have also been explored. Genetic engineering of the radio-resistant bacterium Deinococcus radiodurans R1 by using a nonspecific acid phosphatase, PhoN, for the biorecovery of uranium from dilute acidic/neutral wastes was reported by our laboratory recently (2).Based on the process used, uranium mining and processing generate large quantities of dilute acidic and alkaline nuclear waste containing uranium, which are dumped as mill tailings. Alkaline wastes containing traces of uranium also arise from nuclear reactors and power plants using uranium as fuel. In nature, uranium (VI) forms highly soluble carbonate complexes, such as [UO 2 (CO 3 ) 2 ] Ϫ2 and [UO 2 (CO 3 ) 3 ] Ϫ4 , at alkaline pH levels (9). This leads to increase in mobility and availability of uranium to groundwater and soil from the dumped nuclear wastes, leading to health hazards. Nearly 130 million liters of alkaline nuclear wastes containing uranium carbonate awaits disposition at the Savannah River Site, Aiken, SC, alone (9). In order to extend microbial ...

Novel Organophosphate Pesticide Utilizing Alkaline Phosphatase Producing Polyextremophile Bacillus Flexus from Lake Ecosystem of North Gujarat, India

“…This medium and dye has been earlier utilized by Satta et al, 1979 [15]; Riccio et al, 1997 [16] and Nilgiriwala et al, 2008 [17]. The other techniques for selecting phosphatase producers are Yellow colonies through p-NPP [18], indigo blue colonies through indolyl phosphate [19] and precipitating fluorescence using 2-(5"-chloro-2"-phosphoryloxyphenyl)-4-[3H]-quinazolinone [20].…”

Interaction of Uranium with Bacterial Cell Surfaces: Inferences from Phosphatase-Mediated Uranium Precipitation

“…PhoK-expressing recombinants were shown to efficiently bioprecipitate U under carbonate-abundant conditions from alkaline solutions (11,32). The crystalline precipitate formed by PhoN-/PhoKexpressing cells was identified as uranyl hydrogen phosphate by X-ray diffraction (XRD) analysis (11,29,32). Such crystalline aggregates of uranyl phosphate can be easily visualized by electron microscopy (11,31).…”

“…PhoN and PhoK show good activity between pH 5 and 7 and between pH 7 and 9, respectively. PhoK-expressing recombinants were shown to efficiently bioprecipitate U under carbonate-abundant conditions from alkaline solutions (11,32). The crystalline precipitate formed by PhoN-/PhoKexpressing cells was identified as uranyl hydrogen phosphate by X-ray diffraction (XRD) analysis (11,29,32).…”

Evaluation of Natural and Synthetic Phosphate Donors for the Improved Enzymatic Synthesis of Phosphate Monoesters

“…BSAR‐1,47 PhoC−Mm wild‐type and G92D/I171T from Morganella morganii 35 and NSAP−Eb 11‐variant from Escherichia blattae 35 were expressed as reported. Phytase from Aspergillus niger was from BASF.…”