Continuous Spectrophotometric Assay for Defluorinase and Dechlorinase Activities With α-Halocarboxylic Acids.

Area of Science:

• Environmental Chemistry
• Biochemistry
• Enzymology

Background:

• * Many environmental pollutants, including per- and polyfluorinated substances (PFAS), are α-halocarboxylic acids.
• * The environmental persistence of PFAS necessitates understanding their biodegradation pathways.
• * Defluorinase enzymes initiate biodegradation by removing fluorine or chlorine atoms from α-halocarboxylic acids.

Purpose of the Study:

• * To develop a continuous assay for monitoring defluorinase activity.
• * To characterize substrate specificity of different defluorinase enzymes.
• * To identify and optimize dehydrogenases for use in a coupled enzyme assay.

Main Methods:

• * Identification, purification, and optimization of dehydrogenases from Limosilactobacillus fermentum JN248 and Enterococcus faecium IAM10071.
• * Development of a spectrophotometric assay measuring NADH production/consumption at 340 nm.
• * Application of the coupled assay to compare substrate specificity of purified defluorinases from Delftia sp. and Dechloromonas sp.

Main Results:

• * Optimized dehydrogenases enabled continuous spectrophotometric monitoring of defluorinase activity.
• * The coupled assay successfully measured defluorinase activity with α-hydroxy and α-ketocarboxylic acid products.
• * Delftia sp. defluorinase showed superior activity against most tested substrates, including difluoroacetate.

Conclusions:

• * A novel coupled-enzyme continuous assay for α-halocarboxylic acid hydrolysis was established.
• * This assay facilitates efficient monitoring and characterization of defluorinase enzymes.
• * The findings contribute to understanding PFAS biodegradation and developing bioremediation strategies.