Developing a cell-bound detection system for the screening of oxidase activity using the fluorescent peroxide sensor...

Area of Science:

• Biotechnology
• Enzyme Engineering
• Synthetic Biology

Background:

• High-throughput screening is crucial for enzyme engineering via directed evolution.
• Current methods for oxidoreductase screening often use surface display and emulsification, limiting applicability.
• There is a need for more versatile and efficient screening platforms.

Purpose of the Study:

• To develop a novel whole-cell biosensor for detecting enzymatic hydrogen peroxide (H2O2) formation.
• To optimize the roGFP2-Orp1 fluorescent system for H2O2 detection in a cellular context.
• To enable efficient screening of enzyme variants for H2O2 production.

Main Methods:

• Utilized the roGFP2-Orp1 fluorescent protein as a one-component detection system for H2O2.
• Determined the kinetic parameters of the roGFP2-Orp1 reaction with H2O2.
• Developed an immobilization technique using Concanavalin A to attach the sensor to Saccharomyces cerevisiae cells.

Main Results:

• Successfully established a peroxide-sensing whole-cell system by immobilizing roGFP2-Orp1 on yeast cells.
• Demonstrated the system's effectiveness in screening for H2O2 formation in enzyme variants.
• Validated the sensor's performance in a whole-cell setting for directed evolution applications.

Conclusions:

• The developed whole-cell biosensor provides an accurate and efficient platform for screening oxidoreductases.
• This approach overcomes limitations of traditional screening methods, expanding applicability in enzyme engineering.
• The roGFP2-Orp1 based system offers a promising tool for advancing directed evolution of H2O2-producing enzymes.