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

Abstract

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.

Authors:
; ; ; ;
Publication Date:
Research Org.:
Stanford Univ., CA (US)
Sponsoring Org.:
USDOE
OSTI Identifier:
20075754
DOE Contract Number:  
FG03-97ER62494
Resource Type:
Journal Article
Journal Name:
Applied and Environmental Microbiology
Additional Journal Information:
Journal Volume: 66; Journal Issue: 5; Other Information: PBD: May 2000; Journal ID: ISSN 0099-2240
Country of Publication:
United States
Language:
English
Subject:
54 ENVIRONMENTAL SCIENCES; BIODEGRADATION; CHROMIUM; REMEDIAL ACTION; OXIDOREDUCTASES; PSEUDOMONAS; PURIFICATION; ION EXCHANGE CHROMATOGRAPHY; ENZYME ACTIVITY

Citation Formats

Park, C.H., Keyhan, M., Wielinga, B., Fendorf, S., and Matin, A. Purification to homogeneity and characterization of a novel Pseudomonas putida chromate reductase. United States: N. p., 2000. Web. doi:10.1128/AEM.66.5.1788-1795.2000.
Park, C.H., Keyhan, M., Wielinga, B., Fendorf, S., & Matin, A. Purification to homogeneity and characterization of a novel Pseudomonas putida chromate reductase. United States. doi:10.1128/AEM.66.5.1788-1795.2000.
Park, C.H., Keyhan, M., Wielinga, B., Fendorf, S., and Matin, A. Mon . "Purification to homogeneity and characterization of a novel Pseudomonas putida chromate reductase". United States. doi:10.1128/AEM.66.5.1788-1795.2000.
@article{osti_20075754,
title = {Purification to homogeneity and characterization of a novel Pseudomonas putida chromate reductase},
author = {Park, C.H. and Keyhan, M. and Wielinga, B. and Fendorf, S. and Matin, A.},
abstractNote = {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.},
doi = {10.1128/AEM.66.5.1788-1795.2000},
journal = {Applied and Environmental Microbiology},
issn = {0099-2240},
number = 5,
volume = 66,
place = {United States},
year = {2000},
month = {5}
}