[Transaminases: high-throughput screening via a ketone-fluorescent probe and applications]

Area of Science:

• Biochemistry and enzymology
• Protein engineering
• Biocatalysis

Context:

• Transaminases are crucial enzymes in amino acid metabolism but often exhibit limited activity with non-natural substrates.
• Enzyme engineering is vital for enhancing transaminase catalytic efficiency and broadening their applications.
• Developing efficient screening methods is key to identifying improved enzyme variants.

Purpose:

• To establish a high-throughput screening method for transaminases using a fluorescent color reaction.
• To engineer a transaminase from *Actinobacteria* sp. with improved activity.
• To enhance the production of (R)-3-aminobutanol via enzyme-catalyzed synthesis.

Summary:

• A high-throughput screening method was developed utilizing the fluorescent reaction between methoxy-2-aminobenzoxime (PMA) and ketones to monitor substrate concentration changes.
• The screening system was optimized for improved efficiency, sensitivity, and accuracy.
• A mutant transaminase library was constructed using 4-hydroxy-2-butanone as a substrate, leading to the successful isolation of a variant with increased activity.

Impact:

• The developed screening method facilitates efficient identification and modification of transaminases.
• The engineered transaminase enhances the biocatalytic production of (R)-3-aminobutanol.
• This work provides a foundation for advancing transaminase engineering and applications in biocatalysis.