Related Experiment Video
Updated: Aug 4, 2026
10:21
A High Throughput Screen for Biomining Cellulase Activity from Metagenomic Libraries
Published on: February 1, 2011
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
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.
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.
- 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.
