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Function-based isolation of novel enzymes from a large library.

M J Olsen¹, D Stephens, D Griffiths

¹Department of Chemistry and Biochemistry, The University of Texas at Austin, Austin, TX 78712, USA.

Nature Biotechnology

|October 4, 2000

Summary

We developed a new method to find enzymes with specific functions using engineered microorganisms and fluorescence. This technique rapidly isolates active enzyme variants from large protein libraries for biotechnological applications.

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Area of Science:

Biochemistry
Molecular Biology
Biotechnology

Background:

Enzyme discovery is crucial for various applications.
Isolating enzymes with specific substrate specificities from large libraries is challenging.
Current methods often lack high-throughput capabilities.

Purpose of the Study:

To develop a high-throughput, quantitative method for isolating enzymes with novel substrate specificities.
To enable the screening of large libraries of protein variants based on catalytic activity.

Main Methods:

Displaying protein variants on the surface of microorganisms.
Incubating variants with a synthetic substrate containing a fluorescent dye and a FRET quenching partner.
Utilizing fluorescence-activated cell sorting (FACS) to isolate catalytically active clones.

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Main Results:

Enriched Escherichia coli expressing serine protease OmpT over 5,000-fold in a single round.
Isolated variant proteases with up to 60-fold enhanced catalytic activity from a library of 6 x 10^5 variants.
Demonstrated a 5,000-fold enrichment of active OmpT enzyme variants.

Conclusions:

The described method is a powerful tool for high-throughput screening of enzyme libraries.
This approach facilitates the discovery of enzymes with tailored substrate specificities.
The technique has broad applicability in enzyme engineering and biotechnology.