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

Abstract

ABSTRACT Many environmental pollutants have a fluorine or chlorine atom on a carbon atom adjacent to a carboxylic acid. These α‐halocarboxylic acids include heavily regulated compounds such as per‐ and polyfluorinated substances (PFAS). Due to PFAS persistence in the environment, there is intense interest in characterising the biodegradation of α‐halocarboxylic acids. Their initial biodegradation often proceeds via defluorinase enzymes that catalyse hydrolytic removal of alpha fluorine or chlorine atoms. These enzymes can dehalogenate both mono‐halocarboxylate and dihalocarboxylate substrates, generating α‐hydroxy and α‐ketocarboxylic acid products, respectively. To enable continuous monitoring of defluorinase activity, we identified, purified and optimised dehydrogenases from Limosilactobacillus fermentum JN248 and Enterococcus faecium IAM10071 that reacted with the specific α‐hydroxy and α‐ketocarboxylic acid products of the defluorinases. The dehydrogenases make or consume NADH, measured by absorbance readings at 340 nm, thus allowing continuous measurement of defluorinase activity using a spectrophotometer. Using the coupled assay, purified defluorinases from a Delftia sp. and a Dechloromonas sp. were compared with respect to substrate specificity. The Delftia defluorinase demonstrated superior activity with most substrates, including difluoroacetate. To our knowledge, this is the first report of a coupled‐enzyme continuous assay method for enzymes that catalyse the hydrolysis of α‐halocarboxylic acids.

Keywords

• activity determination
• bacteria
• coupled
• dehalogenase
• dehydrogenase
• enzyme