Matrix-independent screening of defluorination <i>in vitro</i> and <i>in vivo</i>

There is intense interest in biodegrading fluorinated pesticides and other commercial products, some of which are per- and poly-fluorinated alkyl substances, or PFAS. Enzymatic carbon-fluorine bond cleavage via hydrolytic, reductive, and eliminative mechanisms generates an organic product, fluoride anion, and a proton. Biodegradation is typically determined by tracking the organic product using liquid chromatography-mass spectrometry (LC-MS) or the anion determined by a fluoride-specific electrode. Here, we monitored the protons that are produced. A pH indicator method was developed using a hydrolytic defluorinase from <i>Delftia acidovorans</i> strain B in purified form or expressed recombinantly in <i>Pseudomonas putida</i> ATCC 12633. The method was also shown to be effective with <i>P. putida</i> F1 catalyzing oxygenative defluorination with α,α,α-trifluorotoluene. <i>P. putida</i> ATCC 12633 strains expressing different recombinant defluorinases showed differential growth and coloration on agar plates containing bromothymol blue and a fluorinated substrate. A purified defluorinase with a high pH optimum was assayed using the pH indicator <i>m</i>-cresol purple to identify six new substrates, one of which is a PFAS. LC-MS and fluoride electrode determinations require a single sample work-up and milliliter volumes. The proton monitoring methods described here can be performed in a microliter high-throughput format. It can also be used in solid matrices such as hydrogels. Although less rigorously quantitative than the single determination methods, rapid screening methods as described here are currently needed by researchers seeking to identify and characterize new microbes and enzymes able to biodegrade commercially relevant PFAS.IMPORTANCEFluorinated compounds are widespread as pesticides, pharmaceuticals, and legacy chemicals. Human health and ecosystem health problems arise from exposure to these chemicals. Currently, there is great interest in reducing exposure via bioremediation, and this is spurring efforts in screening for C-F bond-cleaving microbes and enzymes. C-F bond cleavage produces fluoride and a proton. Fluoride determination is difficult in many matrices and involves milliliter volumes and single-sample determinations. Here, proton release by enzymes and microbes was monitored on agar, in hydrogels, and in a microliter liquid high-throughput screening format. Six new substrates were identified for one microbial defluorinase enzyme.