A high-throughput assay for enzymatic polyester hydrolysis activity by fluorimetric detection

Area of Science:

• Biocatalysis and Enzyme Engineering
• Analytical Chemistry
• Polymer Science

Background:

• Enzymatic hydrolysis of polyethylene terephthalate (PET) produces terephthalic acid (TPA).
• Accurate and rapid quantification of TPA is crucial for evaluating polyester hydrolase activity.
• Existing methods like HPLC are time-consuming and not suitable for high-throughput screening.

Purpose of the Study:

• To develop a fast and robust fluorimetric assay for determining polyester hydrolase activity.
• To enable high-throughput screening of enzymes for synthetic polymer degradation.
• To quantify TPA formation from PET hydrolysis.

Main Methods:

• Developed a fluorimetric assay based on converting terephthalate to fluorescent 2-hydroxyterephthalate via iron autoxidation.
• Validated the assay using polyester hydrolase from Thermobifida fusca KW3 acting on PET films and nanoparticles.
• Performed analysis in a 96-well microplate format for high-throughput screening.

Main Results:

• The fluorimetric assay demonstrated robustness across various conditions (pH, buffer concentration, time, protein presence).
• Results showed a high correlation (R² = 0.99) with the HPLC method.
• The assay accurately quantified TPA formation from PET hydrolysis.

Conclusions:

• The developed fluorimetric assay is a rapid, robust, and high-throughput method for quantifying polyester hydrolase activity.
• This assay facilitates the detection and development of biocatalysts for synthetic polymer modification and degradation.
• The microplate format is suitable for screening enzymes involved in PET biodegradation.