Purification to Homogeneity and Characterization of a Novel Pseudomonas putida Chromate Reductase
AUTOR(ES)
Park, C. H.
FONTE
American Society for Microbiology
RESUMO
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.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=101413Documentos Relacionados
- Purification and characterization of a heme-containing amine dehydrogenase from Pseudomonas putida.
- Degradation of Chloroaromatics: Purification and Characterization of a Novel Type of Chlorocatechol 2,3-Dioxygenase of Pseudomonas putida GJ31
- Purification and properties of a novel ferricyanide-linked xanthine dehydrogenase from Pseudomonas putida 40.
- Purification and Characterization of a Novel Erythrose Reductase from Candida magnoliae
- Chromate-Reducing Properties of Soluble Flavoproteins from Pseudomonas putida and Escherichia coli