A high-throughput pH-based colorimetric assay: application focus on alpha/beta hydrolases

Area of Science:

• Biochemistry
• Enzymology

Background:

• Research on alpha/beta hydrolases is hindered by the absence of high throughput screening assays.
• Existing methods like HPLC are time-consuming and less efficient for broad screening.

Purpose of the Study:

• To develop a general framework for pH-based colorimetric assays for alpha/beta hydrolase activity.
• To provide mathematical considerations for hit/miss assignment without standard curves.
• To enable efficient screening of hydrolysis and synthesis activities.

Main Methods:

• Development of a general framework for pH-based colorimetric assays.
• Mathematical modeling for de novo estimation of experimental response.
• Application to screening alpha/beta hydrolase activity on various substrates.

Main Results:

• The assay effectively screens both hydrolysis and synthesis activity of alpha/beta hydrolases.
• Data generated is comparable to High Performance Liquid Chromatography (HPLC) standards.
• The assay significantly improves screening efficiency compared to HPLC.

Conclusions:

• The developed assay provides an efficient and accurate method for high throughput screening of alpha/beta hydrolases.
• This framework facilitates broader scientific inquiry into enzyme function and application.
• The assay offers a valuable alternative to traditional methods, accelerating enzyme research.