Products Cited in Publications Worldwide Continuous Spectrophotometric Assay for Defluorinase and Dechlorinase Activities With α‐Halocarboxylic Acids

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

Ronnander, Marie; Dodge, Anthony G; O'Neal, Erin; Pauls, Caroline; Hanson, Jack; Christenson, James K; Wackett, Lawrence P

DOI: PMID:

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 ; organofluorine ; PFAS

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