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Updated: Aug 8, 2025

Multi-enzyme Screening Using a High-throughput Genetic Enzyme Screening System

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A Flow Cytometry-Based Ultrahigh-Throughput Screening Method for Directed Evolution of Oxidases.

Lilin Feng^1,2, Liang Gao¹, Volkan Besirlioglu¹

¹Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 3, 52074, Aachen, Germany.

Angewandte Chemie (International Ed. in English)

|March 2, 2023

Summary

We developed FlOxi, a flow cytometry platform for directed evolution of oxidases. This method enhances enzyme performance for industrial applications by screening hydrogen peroxide-producing oxidase variants.

Keywords:

Directed Evolution Fe3+His6-Tagged eGFP Oxidase Ultrahigh-Throughput Screening

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Screening Assay for Oxidative Stress in a Feline Astrocyte Cell Line, G355-5

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Published on: July 13, 2011

Area of Science:

Biocatalysis and enzyme engineering
Synthetic biology applications
Protein evolution and directed evolution

Background:

Oxidases are crucial industrial catalysts but often require re-engineering for synthetic use.
Directed evolution is key to optimizing enzyme function for specific applications.

Purpose of the Study:

To develop a versatile and robust screening platform for directed oxidase evolution.
To enable the engineering of hydrogen peroxide-producing oxidases for industrial applications.

Main Methods:

A flow cytometry-based screening platform, FlOxi, was developed.
FlOxi uses hydrogen peroxide (H2O2) produced by engineered E. coli to catalyze the Fenton reaction, immobilizing His6-tagged eGFP on cell surfaces.
Beneficial oxidase variants are identified via flow cytometry based on cell surface immobilization.

Main Results:

FlOxi was validated using galactose oxidase (GalOx) and D-amino acid oxidase (D-AAO).
A GalOx variant (T521A) showed a 4.4-fold lower K_m.
A D-AAO variant (L86M/G14/A48/T205) exhibited a 4.2-fold higher k_cat compared to wildtypes.

Conclusions:

The FlOxi platform is effective for the directed evolution of hydrogen peroxide-producing oxidases.
This method is applicable to enzymes with non-fluorescent substrates, broadening its utility in biocatalysis.