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Applied and Environmental Microbiology, May 2000, p. 1788-1795, Vol. 66, No. 5
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Purification to Homogeneity and Characterization of a Novel Pseudomonas putida Chromate Reductase

C. H. Park,1 M. Keyhan,1 B. Wielinga,2 S. Fendorf,2 and A. Matin1,*

Department of Microbiology & Immunology1 and Department of Geological & Environmental Sciences,2 Stanford University, Stanford, California 94305

Received 14 December 1999/Accepted 6 February 2000

Cr(VI) (chromate) is a widespread environmental contaminant. Bacterial chromate reductases can convert soluble and toxic chromate to the insoluble and less toxic Cr(III). Bioremediation can therefore be effective in removing chromate from the environment, especially if the bacterial propensity for such removal is enhanced by genetic and biochemical engineering. To clone the chromate reductase-encoding gene, we purified to homogeneity (>600-fold purification) and characterized a novel soluble chromate reductase from Pseudomonas putida, using ammonium sulfate precipitation (55 to 70%), anion-exchange chromatography (DEAE Sepharose CL-6B), chromatofocusing (Polybuffer exchanger 94), and gel filtration (Superose 12 HR 10/30). The enzyme activity was dependent on NADH or NADPH; the temperature and pH optima for chromate reduction were 80°C and 5, respectively; and the Km was 374 µM, with a Vmax of 1.72 µmol/min/mg of protein. Sulfate inhibited the enzyme activity noncompetitively. The reductase activity remained virtually unaltered after 30 min of exposure to 50°C; even exposure to higher temperatures did not immediately inactivate the enzyme. X-ray absorption near-edge-structure spectra showed quantitative conversion of chromate to Cr(III) during the enzyme reaction.

* Corresponding author. Mailing address: Department of Microbiology & Immunology, Sherman Fairchild Science Building, 299 Campus Dr., Stanford University, Stanford, CA 94305-5124. Phone: (650) 725-4745. Fax: (650) 725-6757. E-mail: A.Matin{at}Forsythe.Stanford.edu.

Applied and Environmental Microbiology, May 2000, p. 1788-1795, Vol. 66, No. 5
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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