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Applied and Environmental Microbiology, September 2008, p. 5516-5523, Vol. 74, No. 17
0099-2240/08/$08.00+0     doi:10.1128/AEM.00107-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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

Kayzad S. Nilgiriwala,1 Anuradha Alahari,2 Amara Sambasiva Rao,1 and Shree Kumar Apte1*

Molecular Biology Division, Bhabha Atomic Research Centre, Trombay, Mumbai 400 085, India,1 UMR5235, Universite de Montpellier, Place Eugene Bataillon, 34095 Montpellier, France2

Received 14 January 2008/ Accepted 9 July 2008

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.

* Corresponding author. Mailing address: Molecular Biology Division, Bhabha Atomic Research Centre, Trombay, Mumbai 400 085, India. Phone: 91-22-25595342. Fax: 91-22-25505189. E-mail: aptesk{at}barc.gov.in

{triangledown} Published ahead of print on 18 July 2008.

Applied and Environmental Microbiology, September 2008, p. 5516-5523, Vol. 74, No. 17
0099-2240/08/$08.00+0     doi:10.1128/AEM.00107-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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