Fluorescent assay for directed evolution of perhydrolases.

Area of Science:

• Biochemistry
• Enzymology
• Directed Evolution

Background:

• Hydrolases possess promiscuous activities that can be harnessed and improved through directed evolution.
• High-throughput screening is crucial for identifying beneficial enzyme variants during directed evolution.

Purpose of the Study:

• To develop and validate a sensitive fluorescent screening platform for improving the perhydrolytic activity of proteases and other hydrolases.
• To enable continuous measurement of peroxycarboxylic acid formation for enzyme engineering.

Main Methods:

• Development of a highly sensitive fluorescent assay based on 3-carboxy-7-hydroxycoumarin (HCC) formation.
• Utilized hypobromite-mediated oxidation of 7-(4'-aminophenoxy)-3-carboxycoumarin (APCC) for HCC release.
• Applied site saturation mutagenesis at position G165 of subtilisin Carlsberg.

Main Results:

• Established a continuous fluorescent assay with µM sensitivity and 11% standard deviation in microtiter plates.
• The assay functions effectively across a wide pH range (5-9).
• A subtilisin Carlsberg variant (T58A/G165L/L216W) exhibited a 5.4-fold increase in k(cat) for perhydrolytic activity compared to wild type.

Conclusions:

• The developed screening platform is effective for directed evolution of hydrolases with improved perhydrolytic activity.
• The fluorescent assay provides a robust and sensitive method for enzyme engineering.
• This approach can be applied to optimize promiscuous enzyme activities for various applications.