High-Throughput Solid-Phase Assay for Substrate Profiling and Directed Evolution of Transketolase.

Area of Science:

• Biocatalysis and enzyme engineering
• Organic chemistry
• Biotechnology

Background:

• Thiamine diphosphate-dependent enzymes, particularly transketolases, are crucial for enantioselective carbon-carbon bond formation.
• Developing efficient methods for substrate profiling is essential for directed evolution of these enzymes.

Purpose of the Study:

• To develop a simple, direct, high-throughput colorimetric assay for transketolase activity.
• To enable efficient substrate profiling of transketolases for directed evolution.

Main Methods:

• A solid-phase assay was designed utilizing pH reaction variation.
• Transketolase activity was detected by monitoring bicarbonate anion release from α-ketoacid donors.
• Bromothymol blue pH indicator was used for naked-eye detection of active variants.

Main Results:

• The assay successfully detected transketolase activity through a color change from yellow to blue.
• This method allows for direct screening of active variants without enzyme extraction.
• The assay facilitates high-throughput screening, reducing effort in directed evolution.

Conclusions:

• A novel colorimetric assay provides a simple and efficient method for assessing transketolase activity.
• This assay supports high-throughput screening and directed evolution of transketolases for biocatalytic applications.
• The method obviates the need for enzyme extraction, streamlining the screening process.