Purification to homogeneity and characterization of a novel Pseudomonas putida chromate reductase

Journal Article · Mon May 01 04:00:00 EDT 2000 · Applied and Environmental Microbiology

DOI:https://doi.org/10.1128/AEM.66.5.1788-1795.2000· OSTI ID:20075754

Park, C H; Keyhan, M; Wielinga, B; Fendorf, S; Matin, A

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: US Department of Energy
• DOE Contract Number: FG03-97ER62494
• OSTI ID: 20075754
• Journal Information: Applied and Environmental Microbiology, Journal Name: Applied and Environmental Microbiology Journal Issue: 5 Vol. 66; ISSN AEMIDF; ISSN 0099-2240
• Country of Publication: United States
• Language: English

Related Subjects

54 ENVIRONMENTAL SCIENCES
BIODEGRADATION
CHROMIUM
ENZYME ACTIVITY
ION EXCHANGE CHROMATOGRAPHY
OXIDOREDUCTASES
PSEUDOMONAS
PURIFICATION
REMEDIAL ACTION