Applied Biochemistry and Biotechnology

, Volume 144, Issue 3, pp 293–300 | Cite as

An Improved Enzyme Assay for Molybdenum-Reducing Activity in Bacteria

  • M. Y. Shukor
  • M. F. A. Rahman
  • N. A. Shamaan
  • C. H. Lee
  • M. I. A. Karim
  • M. A. Syed
Article

Abstract

Molybdenum-reducing activity in the heterotrophic bacteria is a phenomenon that has been reported for more than 100 years. In the presence of molybdenum in the growth media, bacterial colonies turn to blue. The enzyme(s) responsible for the reduction of molybdenum to molybdenum blue in these bacteria has never been purified. In our quest to purify the molybdenum-reducing enzyme, we have devised a better substrate for the enzyme activity using laboratory-prepared phosphomolybdate instead of the commercial 12-phosphomolybdate we developed previously. Using laboratory-prepared phosphomolybdate, the highest activity is given by 10:4-phosphomolybdate. The apparent Michaelis constant, Km for the laboratory-prepared 10:4-phosphomolybdate is 2.56 ± 0.25 mM (arbitrary concentration), whereas the apparent Vmax is 99.4 ± 2.85 nmol Mo-blue min−1 mg−1 protein. The apparent Michaelis constant or Km for NADH as the electron donor is 1.38 ± 0.09 mM, whereas the apparent Vmax is 102.6 ± 1.73 nmol Mo-blue min−1 mg−1 protein. The apparent Km and Vmax for another electron donor, NADPH, is 1.43 ± 0.10 mM and 57.16 ± 1.01 nmol Mo-blue min−1 mg−1 protein, respectively, using the same batch of molybdenum-reducing enzyme. The apparent Vmax obtained for NADH and 10:4-phosphomolybdate is approximately 13 times better than 12-phoshomolybdate using the same batch of enzyme, and hence, the laboratory-prepared phosphomolybdate is a much better substrate than 12-phoshomolybdate. In addition, 10:4-phosphomolybdate can be routinely prepared from phosphate and molybdate, two common chemicals in the laboratory.

Keywords

Assay Mo-reducing enzyme Molybdate Phosphomolybdate Molybdenum blue 

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Copyright information

© Humana Press Inc. 2007

Authors and Affiliations

  • M. Y. Shukor
    • 1
  • M. F. A. Rahman
    • 1
  • N. A. Shamaan
    • 1
  • C. H. Lee
    • 1
  • M. I. A. Karim
    • 2
  • M. A. Syed
    • 1
  1. 1.Department of Biochemistry, Faculty of Biotechnology and Biomolecular SciencesUniversiti Putra MalaysiaSelangorMalaysia
  2. 2.Department of Biotechnology Engineering, Faculty of EngineeringInternational Islamic University, MalaysiaKuala LumpurMalaysia

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