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Appl. Environ. Microbiol. doi:10.1128/AEM.00107-08
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Cloning and overexpression of an alkaline phosphatase PhoK from Sphingomonas sp. BSAR-1 for uranium bioprecipitation from alkaline solutions

Molecular Biology Division, Bhabha Atomic Research Centre, Trombay, Mumbai - 400 085, India

^* To whom correspondence should be addressed. Email: aptesk{at}barc.gov.in.

	Abstract

Cells of Sphingomonas species 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. The resultant E. coli strain EK4 overexpressed 55 times higher cellular activity and 13 times higher secreted extracellular PhoK activity, compared to 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 – 5 mM of uranyl carbonate compared to BSAR-1 (>7 h). In both strains BSAR-1 and EK4, precipitated uranium remained cell bound. The EK4 cells exhibited 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 genetic engineering PhoK for bioprecipitation of uranium from alkaline solutions.