Purification to homogeneity and characterization of a novel Pseudomonas putida chromate reductase
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, the authors purified to homogeneity and characterized a novel soluble chromate reductase from Pseudomonas putida, using ammonium sulfate precipitation, anion-exchange chromatography, chromatofocusing, and gel filtration. 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 K{sub m} was 374 {micro}M, with a V{sub max} of 1.72 {micro}mol/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.
- Research Organization:
- Stanford Univ., CA (US)
- Sponsoring Organization:
- USDOE
- DOE Contract Number:
- FG03-97ER62494
- OSTI ID:
- 20075754
- Journal Information:
- Applied and Environmental Microbiology, Vol. 66, Issue 5; Other Information: PBD: May 2000; ISSN 0099-2240
- Country of Publication:
- United States
- Language:
- English
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