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Related Experiment Videos

A versatile high throughput screen for dioxygenase activity using solid-phase digital imaging.

J M Joern1, T Sakamoto, A Arisawa

  • 1California Institute of Technology, Pasadena, CA 91125, USA.

Journal of Biomolecular Screening
|November 2, 2001
PubMed
Summary

We developed a high-throughput screen for dioxygenase activity using a colorimetric assay. This method enables rapid discovery and engineering of enzymes involved in aromatic oxidation.

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Area of Science:

  • Biochemistry
  • Enzymology
  • Molecular Biology

Background:

  • Dioxygenases are crucial enzymes for aromatic compound metabolism.
  • Discovering and engineering novel dioxygenases is essential for various biotechnological applications.
  • Existing screening methods for dioxygenase activity can be low-throughput and labor-intensive.

Purpose of the Study:

  • To develop a novel, high-throughput screening method for dioxygenase activity.
  • To enable efficient discovery and directed evolution of oxygenase enzymes.
  • To provide a quantitative and reproducible assay for aromatic oxidation.

Main Methods:

  • A solid-phase, high-throughput screen (10,000 clones/day) was developed.
  • Dioxygenase bioconversion products (cis-dihydrodiols) were converted to phenols or catechols.

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  • Gibbs reagent was used to detect oxygenated aromatics, generating quantifiable colored products.

    • Main Results:

    • The assay is reproducible and quantitative down to 30 microM product concentrations.
    • The method demonstrates minimal background interference from media components.
    • The screen effectively detects aromatic oxidation by dioxygenases.

    Conclusions:

    • The developed method is an effective general screen for aromatic oxidation.
    • This assay is a valuable tool for the discovery and directed evolution of oxygenases.
    • The high-throughput nature facilitates rapid enzyme screening and engineering.