WO2024163931A1

WO2024163931A1 - Enzymatic degradation of poly and perfluorinated compounds

Info

Publication number: WO2024163931A1
Application number: PCT/US2024/014299
Authority: WO
Inventors: Fatemeh Aileen MASTOURI; Miloš MILČIĆ; Karla MILČIĆ
Original assignee: Revivbio, Inc.
Priority date: 2023-02-03
Filing date: 2024-02-02
Publication date: 2024-08-08

Abstract

The present disclosure relates processes for degrading poly- and perfluorinated compounds using bio- or chemo-enzymatic processes.

Description

ENZYMATIC DEGRADATION OF POLY AND PERFLUORINATED COMPOUNDS

RELATED APPLICATIONS

[0001] This application claims the benefit of priority under 35 U.S.C. § 119(e) to U.S. Provisional Application No: 63/443,252, filed February 3, 2023, which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

[0002] The present disclosure relates to chemo-enzymatic and bio-enzymatic processes for degrading poly- and perfluorinated compounds.

BACKGROUND

[0003] Poly- and perfluorinated compounds (e.g., perfluoropolyethers and perfluorinated carboxylic acids) are organic compounds characterized by a partially or fully fluorinated hydrophobic linear carbon chain attached to one or more hydrophilic head groups. Poly- and perfluorinated compounds repel both water and oil, making their presence highly advantageous in numerous industrial and consumer products. The use of perfluorinated compounds as stain repelling agents, non-stick coatings, and additives in paint, waxes, polishes, electronics, adhesives, agrochemicals, pharmaceuticals, and food packaging has been widespread for decades.

[0004] Perfluorinated compounds have exceptional stability, which is desirable from an applications perspective, but problematic from environmental and health standpoints. Poly- and perfluorinated compounds are persistent, bioaccumulative, and toxic. The abbreviation “PFAS” is used to refer to per- and polyfluoroalkyl substances (PFAS). Common poly- and perfluorinated compounds include perfluorinated carboxylic acids (PFCAs) and perfluorocarbonsulfonic acids (PFSAs), of which perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) are the most well-known. Other perfluorinated compounds include fluorotelomer alcohols (FTOHs), fluorotelomer methacrylates (FTMACs), fluorotelomer acrylates (FTACs), perfluorooctane sulfonamides (FOSAs), perfluorooctane sulfonamidoethanols (FOSEs), polyfluoroalkyl phosphoric acid diesters (diPAPs), and perfluorinated phosphonic acids (PFPAs). [0005] Accordingly, a need exists for remitting perfluorinated compounds from the environment following disposal of such industrial and consumer products

SUMMARY

[0006] The present disclosure describes bio- or chemo-enzymatic mechanisms, methods, and compounds for degrading perfluorinated compounds (also referred to herein as “substrates”). It overcomes a disadvantage associated with naturally occurring fluoroacetate dehalogenases, namely that they are only able to remove the fluorine atoms on an a-carbon present in a chain of fluorinated carbons (relative to a hydrophilic head group such as a carboxylic acid, sulfonic acid, or phorphonic acid group). They are unable to partially or fully defluorinate small or bulky substrates with fluorine atoms on carbons other than the a-carbon. Applicant’s disclosed processes produces a modified substrate that is one fluorinated carbon shorter than the starting substrate. In so doing, an adjacent fluorinated carbon becomes situated as the a-carbon, which enable re-use of the dehalogenase. The process may be repeated one or more times corresponding to the number of fluorinated carbons remaining in the modified substrate, which may result in removal of fluorine atoms on other carbons in the compounds, and in some embodiments, all the fluorine atoms in the poly- or perfluorinated substrate.

[0007] A first aspect of the present disclosure is directed to a process for degrading a poly- or perfluorinated substrate that has a terminal carboxylic acid group, and a linear or branched hydrophobic tail comprising a plurality of fluorinated carbon atoms. The process comprises:

(1) contacting the poly- or perfluorinated substrate that has a terminal carboxylic acid group, and a linear or branched hydrophobic tail comprising a plurality of fluorinated carbon atoms, with a fluoroacetate dehalogenase, to produce a first modified poly- or perfluorinated substrate having an a-keto group;

(2) decarboxylating the first modified poly- or perfluorinated substrate, to produce a second modified poly- or perfluorinated substrate having a terminal aldehyde group and a hydrophobic tail that is one carbon shorter in length than the poly- or perfluorinated substrate; and

(3) functionalizing the second modified poly- or perfluorinated substrate by contacting the second modified poly- or perfluorinated substrate with an aldehyde dehydrogenase, to produce a third modified poly- or perfluorinated substrate that has a terminal carboxylic acid and wherein the hydrophobic tail is one fluorinated carbon shorter in length than the poly- or perfluorinated substrate.

[0008] A second aspect of the present disclosure is directed to a process for degrading a poly- or perfluorinated substrate that has a terminal sulfonic acid or phosphonic acid group, and a linear or branched hydrophobic tail comprising a plurality of fluorinated carbon atoms. The process comprises:

(1) converting a poly- or perfluorinated substrate that has a terminal sulfonic or phosphonic acid group, and a linear or branched hydrophobic tail comprising a plurality of fluorinated carbon atoms, to produce a first modified poly- or perfluorinated substrate having a terminal carboxyl group in place of the terminal sulfonic or phosphonic acid group, and an a-keto group, by (la) reacting the poly- or perfluorinated substrate with a fluoroacetate dehalogenase, followed by enzymatic or microbial biotransformation to convert the terminal sulfonic or phosphonic acid group to a carboxylic acid group, or (lb) converting the terminal sulfonic or phosphonic acid group of the poly- or perfluorinated substrate to a carboxylic acid group, followed by use of the fluoroacetate dehydrogenase, to produce the first modified poly- or perfluorinated substrate;

(2) decarboxylating the first modified poly- or perfluorinated substrate to produce a second modified poly- or perfluorinated substrate having a terminal aldehyde group and hydrophobic tail that is one carbon shorter in length than the second modified poly- or perfluorinated substrate; and

(3) functionalizing the second modified poly- or perfluorinated substrate by contacting the third modified poly- or perfluorinated substrate with an aldehyde dehydrogenase, to produce a third modified poly- or perfluorinated substrate that has a terminal carboxylic acid and wherein the hydrophobic tail is one fluorinated carbon shorter in length than the poly- or perfluorinated substrate.

[0009] A related aspect is directed to a chemoenzymatic process for degrading a poly- or perfluorinated substrate, the chemoenzymatic process comprising:

(1) contacting a poly- or perfluorinated substrate that has a terminal carboxylic acid group, and a linear or branched hydrophobic tail comprising a plurality of fluorinated carbon atoms, with a fluoroacetate dehalogenase, to produce a first modified poly- or perfluorinated substrate having an a-keto group;

(2) reacting the first modified poly- or perfluorinated substrate with a radical, to produce a second modified poly- or perfluorinated substrate having a terminal carboxylic acid and wherein the hydrophobic tail is one fluorinated carbon shorter in length than the poly- or perfluorinated substrate.

[0010] A further aspect of the disclosure is directed to a process for degrading a modified poly- or perfluorinated substrate that has a terminal carboxylic acid and a hydrophobic tail, and an a- keto group. The process comprises:

(1) conducting a decarboxylation reaction on a poly- or perfluorinated substrate that has a terminal carboxylic acid and a hydrophobic tail, and an a-keto group, to produce a first modified poly- or perfluorinated substrate having a terminal aldehyde group; and

(2) converting the terminal aldehyde group of the first modified poly- or perfluorinated substrate to produce a second modified poly- or perfluorinated substrate having a terminal carboxylic acid, wherein the second modified poly- or perfluorinated substrate contains a hydrophobic tail that is one fluorinated carbon shorter in length than the poly- or perfluorinated substrate.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011] FIG. 1 shows the chemical structures of various perfluorinated compounds that may be degraded according to the disclosed methods.

[0012] FIG. 2 shows a postulated chemical reaction mechanism of PFOA degradation in accordance with some example embodiments.

[0013] FIG. 3 shows an exemplary pathway for the full degradation (or defluorination) of PFBA. [0014] FIG. 4 shows an alternative pathway for regeneration of the terminal carboxyl group.

[0015] FIG. 5 shows defluorination activity and substrate specificity of a model fluoroacetate dehalogenase, RPA1163, from Rhodopseudomonas palustris.

[0016] FIG. 6 shows principal component analysis and differences in conformations in fluoroacetate dehalogenase.

[0017] FIG. 7 shows energies needed for decarboxylation of 3,3,3-trifluoro-2-oxo-propionic acid and pyruvate using Gaussian program package.

[0018] FIG. 8 shows a decarboxylation reaction according to some embodiments of the disclosure. [0019] FIG. 9 shows decarboxylation activity of pyruvate oxidase on pyruvate and 3,3,3- trifluoro-2-oxo-propionic acid (generated by defluorination of pentafluoro propionic acid) as substrates.

[0020] FIG. 10 shows an oxidation reaction according to some embodiments of the disclosure.

[0021] FIG. 11 shows a sample pathway for single carbon removal according to some embodiments of the disclosure.

[0022] FIG. 12 is a series of bar graphs for indicated perfluorinated compounds with various fluoroacetate dehalogenases showing fluoride release from perfluorinated substrates with different hydrophobic chain lengths.

DETAILED DESCRIPTION

[0023] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the subject matter herein belongs. As used in the specification and the appended claims, unless specified to the contrary, the following terms have the meaning indicated in order to facilitate the understanding of the present disclosure.

[0024] In the following description, like components have the same reference numerals, regardless of different illustrated embodiments. To illustrate embodiments clearly and concisely, the drawings may not necessarily reflect appropriate scale and may have certain structures shown in somewhat schematic form. The disclosure may describe and/or illustrate structures in one embodiment, and in the same way or in a similar way in one or more other embodiments, and/or combined with or instead of the structures of the other embodiments.

[0025] In the specification and claims, for the purposes of describing and defining the invention, the terms “about” and “substantially” represent the inherent degree of uncertainty attributed to any quantitative comparison, value, measurement, or other representation. The terms “about” and “substantially” moreover represent the degree by which a quantitative representation may vary from a stated reference without resulting in a change in the basic function of the subject matter at issue. Open-ended terms, such as “comprise,” “include,” and/or plural forms of each, include the listed parts and can include additional parts not listed, while terms such as “and/or” include one or more of the listed parts and combinations of the listed parts. [0026] As used herein, the terms “degrading” (and variations thereof) embrace complete degradation of the substrate such that all the fluorine atoms are cleaved from the poly or perfluorinated substrate , e.g., in the case of poly and perfluorinated substrates having carboxylic acid heads, producing CO2, defluorinated organic acids, F', and in the case of poly and perfluorinated substrates having sulfonic acid heads, producing CO2, defluorinated organic acids, F’ and SC ²", and in the case of poly and perfluorinated substrates having phosphonic acid heads, producing CO2, defluorinated organic acids, F' and PO4²’. The term also embraces less than complete degradation such that not all fluorine atoms are cleaved from the substrate, which still may be beneficial to the environment, e.g., less toxic, or less bioaccumulating.

[0027] Processes of the present disclosure utilize poly- or perfluorinated substrates. These compounds are known in the art. Poly- or perfluorinated substrates are organofluorine chemical compounds that contain a fully (per) or partially (poly) fluorinated carbon chain connected to a terminal hydrophilic group, e.g, a carboxylic acid, sulfonic acid or phosphonic acid group. In some embodiments, the fluorinated carbon chain is a single chain. In some embodiments, the fluorinated carbon chain is a branched chain.

[0028] In some embodiments, the linear or branched chain of the poly- or perfluorinated substrate is saturated or unsaturated and the linear or branched chain may be interrupted by one or more heteroatoms and/or functional groups.

[0029] The linear or branched chain of the poly- or perfluorinated substrate may be interrupted by at least one of -O-, -S-, -N(R’)-, -C=C- -C(O)-, -C(O)O-, -OC(O)-, -C(O)N(R’)-,- N(R’)C(O)-, -S(O)-, -OS(O)-, -S(O)O-, -S(O)₂- -OS(O)₂-, -S(O)₂O-, -N(R’)S(O)-, - S(O)2N(R’)-, -N(R’)S(O)2-, -S(O)2N(R’)-, wherein R’ is H or Ci-Ce alkyl, wherein the one or more interrupting groups may be the same or different.

[0030] In some embodiments, the perfluorinated substrate has a structure represented by formula (I) or (II):

wherein,

X is absent or O; and each n is independently an integer 0-15.

[0031] In some embodiments, n is an integer of 1-10. In some embodiments, n is an integer of 1-8. In some embodiments, n is an integer of 1-6. In some embodiments, n is an integer of 1-4. These ranges also constitute disclosre of each and every integer in the range, e.g., 1-10 effectively discloses n representing 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0032] In some embodiments, the substrate is perfluorinated. Representative examples of perperfluorinated substrates include perfluorobutanoic acid (PFBA), perfluoropentanoic acid (PFPeA), perfluorohexanoic acid (PFHxA), perfluoroheptanoic acid (PFHpA), perfluorooctanoic acid (PFOA), hexafluor opropylene oxide dimer acid (HFPO-DA), perfluorononancanoic acid (PFNA), perfluorodecanoic acid (PFDA), perfluoropropionic acid (PFPrA), perfluoroundecanoic acid (PFUnDA), perfluorododecanoic acid (PFDoA), perfluorotridecanoic acid (PFTrDA), or perfluorotetradecanoic acid (PFTeDA).

[0033] In some embodiments, the perfluorinated substrate is perfluorobutane sulfonic acid (PFBS), perfluorohexane sulfonic acid (PFHxS), perfluorooctane sulfonic acid (PFOS), or perfluorooctanesulfonamide (PF O SA) .

[0034] FIG. 1 illustrates the chemical structures of representative perfluorinated compounds that may be degraded in accordance with the disclosed processes.

[0035] In some embodiments, the substrate is polyfluorinated, representative examples of which include 8H-perfluorooctanoic acid (CAS No. 13973-14-3) and octafluoroadipic acid (CAS No. 336-08-3).

[0036] All aspects of the disclosed processes may include one or more cycles. The cycles may be repeated for a number of times from once to a number corresponding to up to a number of cycles equal to the number of fluorinated carbons in the hydrophobic tail of the first modified poly- or perfluorinated substrate (and numbers of cycles therebetween). In some embodiments, the cycle of reactions is repeated a number of times corresponding to (or provided that) the chain shortened, modified substrate produced by each cycle contains an a-fluorinated carbon. In some embodiments, e.g., in the case of perfluorinated substrates, the number of cycles corresponds to the number of fluorinated carbons in the chain. Also, the enzymes used in each successive cycle may be same or different provided that the same modified substrate is produced, e.g., the fluoroacetate dehydrogenase used in a first cycle may be same as or different from the fluoroacetate used in one or more subsequent cycles. In some embodiments, the enzymes are the same.

[0037] In some embodiments, a multi-cycle process enables the full degradation of perfluorinated compounds (e.g., one or more compounds shown in FIG. 1), such as PFOA to CO2, defluorinated organic acids, and F’ and poly- and perfluorosulfonic acids (PFSA) such as PFOS to CO2, defluorinated organic acids, F' and SC ²'. As described in additional detail below, degradation may be achieved through a series of enzymatic reactions involving desulfonation, defluorination, decarboxylation, and/or oxidation/hydrolysis, wherein each cycle results in the removal of one carbon atom from PFCA/PFSA/PFPA compound. In these or other embodiments, a combination of enzymatic defluorination and decarboxylation may be used to achieve full or partial degradation of the poly or perfluorinated compound(s).

[0038] In one embodiment, a fluoroacetate dehalogenase may be used to degrade a PFCA compound, e g., TFA, PFPrA, PFBA, PFPnA, PFHA, or PFOA. FIG. 2 shows a representative method of PFOA degradation in accordance with some embodiments. PFOA is a substrate with carboxylic acid on one terminus of the molecule and 7 additional carbon atoms, each with 2 fluorides. As shown in FIG. 2, a fluoroacetate dehalogenase may be used to remove two F’ from the carbon atom closest to the carboxyl group. This leaves a geminal diol that may undergo dehydration to yield a-keto acid with 6 fluorinated carbon atoms, as illustrated in FIG. 2. FIG. 3 shows the cascade and all subsequent steps that result in full degradation of PFBA as an example. This exemplary chain-shortening cascade will fully degrade other PFCAs with varying fluorinated chain lengths.

[0039] This and FIG. 4 shows an alternative pathway that regenerates terminal carboxyl group. [0040] Aspects of the present disclosure are directed to processes for degrading a poly- or perfluorinated substrate.

[0041] In one aspect, the process comprises:

[0042] In some embodiments, the decarboxylating the first modified poly- or perfluorinated substrate is conducted using an oxidase followed by an acid phosphatase.

[0043] In some embodiments, the decarboxylating the first modified poly- or perfluorinated substrate is conducted using an a -keto acid decarboxylase.

[0044] In some embodiments, steps 1-3 constitute a cycle, and the cycle is repeated, beginning with the poly- or perfluorinated substrate produced by the prior cycle, at least once and in some embodiments, up to a number of cycles equal to the number of fluorinated carbons in the hydrophobic tail of the first modified poly- or perfluorinated substrate.

[0045] In another aspect, the process comprises:

(2) decarboxylating the first modified poly- or perfluorinated substrate to produce a second modified poly- or perfluorinated substrate having a terminal aldehyde group and hydrophobic tail that is one carbon shorter in length than the second modified poly- or perfluorinated substrate; and (3) functionalizing the second modified poly- or perfluorinated substrate by contacting the third modified poly- or perfluorinated substrate with an aldehyde dehydrogenase, to produce a third modified poly- or perfluorinated substrate that has a terminal carboxylic acid and wherein the hydrophobic tail is one fluorinated carbon shorter in length than the poly- or perfluorinated substrate.

[0046] In some embodiments, the decarboxylating the first modified poly- or perfluorinated substrate is conducted using an oxidase followed by an acid phosphatase.

[0047] In some embodiments, the decarboxylating the first modified poly- or perfluorinated substrate is conducted using an a-keto acid decarboxylase.

[0048] In some embodiments, the process further comprises:

(4) contacting the third modified poly- or perfluorinated substrate with a fluoroacetate dehalogenase, to produce a fourth modified poly- or perfluorinated substrate having an a-keto group;

(5) decarboxylating the fourth modified poly- or perfluorinated substrate, to produce a sixth modified poly- or perfluorinated substrate having a terminal aldehyde group and hydrophobic tail that is one carbon shorter in length than the fourth modified poly- or perfluorinated substrate; and

(6) functionalizing the fifth modified poly- or perfluorinated substrate by contacting the fifth modified poly- or perfluorinated substrate with an aldehyde dehydrogenase, to produce a sixth modified poly- or perfluorinated substrate that has a terminal carboxylic acid and wherein the hydrophobic tail is one fluorinated carbon shorter in length than the third modified poly- or perfluorinated substrate.

[0049] In some embodiments, steps 4-6 constitute a cycle, and the cycle is repeated, beginning with the poly- or perfluorinated substrate produced by the prior cycle, at least once and up to a number of cycles equal to the number of fluorinated carbons in the hydrophobic tail of the poly- or perfluorinated substrate produced by the prior cycle.

[0050] In a further aspect, the process comprises a subset of the forementioned processes, as follows:

(1) conducting a decarboxylation reaction on a poly- or perfluorinated substrate that has a terminal carboxylic acid and a hydrophobic tail, and an a-keto group, to produce a first modified poly- or perfluorinated substrate having a terminal aldehyde group; and (2) converting the terminal aldehyde group of the first modified poly- or perfluorinated substrate to produce a second modified poly- or perfluorinated substrate having a terminal carboxylic acid, wherein the second modified poly- or perfluorinated substrate contains a hydrophobic tail that is one fluorinated carbon shorter in length than the poly- or perfluorinated substrate.

[0051] As will be appreciated by those skilled in the art upon consideration of the present disclosure, the disclosed techniques may be applicable to all PFAS species with a carboxyl terminus. As described in additional detail herein, for PFAS species such as PFOS that have an SO4 terminus, additional enzymes (or microorganisms) known in the art (for example, Yang et al., Journal of Hazardous Materials, 2022, 423(Part A .127052), may also be used.

[0052] Fluoroacetate Dehalogenase

[0053] Fluoroacetate dehalogenase is an enzyme that catalyzes defluorination of fluoroacetate (its naturally occurring substrate) and difluoro acetate (a synthetic substrate) to either acetic acid or glycolic acid (each molecule having 2 carbon atoms). (See, Yue et al., Environ. Sci. Technol., 2021, 55(7 ):9817-9825 and Khusnutdinova, el al., FEBS J., 2023, 290:4966-4983). The naturally occurring enzymes are unable to efficiently catalyze defluorination of longer-chain fluorinated compounds, such as perfluoroalkyl and polyfluoroalkyl substances (FIG. 5). Fluoroacetate dehalogenases can be used in concert with a biological, chemical or bio-chemical chain-shortening mechanism to systematically remove all fluorine atoms from a poly- or perfluorinated compound. [0054] Representative examples of fluoroacetate dehalogenase enzymes that may be useful in the practice of any of the aspects of the disclosed processes include gene bank ID. 3R41A from Rhodopseudomonas palustris, gene bank ID. Q479B8 from Dechloromonas aromatica, gene bank ID. WP_010994216 from Nostoc sp., gene bank ID. WP_011485352.1 (Q123C8) from Polaromonas sp., gene bank ID. Q01399 and Q01398 from Moraxellales sp., gene bank ID. GJL83598.1 from an identified gammaprotebacteria, gene bank ID. SPJ18144 frm a Burkhulderiales bacteria, UniProt ID A0A1B5CV88 from Pseudomonas sp. , A0A5A4LHT2 from Psueodomonas auroginosa, A0A066PP34 from acidiphillum sp., A0A090F522 from Mesorhizobium sp., A0A098EGY6 from Pianococcus massiliensis, A0A0A1Q126 and A0A0A1Q4B0 from bacterium YEK0313, A0A0B6S6R7 from Burkholderia plantarii, A0A0B7G5F6 from Thanatephorus cucumeris, A0A0E4A2G3 from Rhodococcus erythropolis, A0A0H1R951 from Microvirga vignae, A0A0H5P8I6 from Nocardia farcinica, A0A0M7ABY7 from Roseibium album, A0A0N0GQ05 Amantichitinum ursilacus, A0A1L9P1Q7 from Planktotalea frisia, and A0A1J5PX08 from mine drainage metagenome.

[0055] Enzymatic Decarboxylation of Poly- and Perfluorinated a-Keto Acids

[0056] Different enzymes are capable of catalyzing decarboxylation of various a-keto acids following various oxidative and non-oxidative mechanisms (See, Li et al., Bioorg. Chem., 2012, 73:2-14). In the described PFAS degradation pathway, perfluorinated a-keto acid is formed when two fluorine ions are removed from the a-carbon atom of the poly- or perfluorinated molecule (FIG. 2).

[0057] In some embodiments, the enzyme is a decarboxylase. In some embodiments, the decarboxylase is oxaloacetate decarboxylase, pyruvate dehydrogenase multienzyme complex, a- ketoglutarate dehydrogenase, oxalate decarboxylase, branched chain a-keto acid decarboxylase, or pyruvate decarboxylase.

[0058] Branched-chain a-keto acid decarboxylase is an enzyme that catalyzes removal of a carboxyl group from a-keto acid to form CO2 and -1C aldehyde (See, Berthold et al., Acta Cryst., 2007, D63.1217-1224). Another enzyme that may additionally or alternatively be used in this step is pyruvate decarboxylase. (See, Buddrus et al., Acta Cryst., 2016, F72:700-706). All described enzyme classes result in the formation of a terminal aldehyde group with one carbon atom shorter product (FIG. 8). Representative examples of decarboxylases that may be useful in the practice of any aspects of the disclosed processes include branched-chain a-keto acid decarboxylases including gene bank ID No. BCDH BETA1 from Arabidopsis thaliana, NCBI reference sequence NC_007969. 1 from Psychrobacter cryohalolentis K5, gene bank ID No. AJ746364 and AY548760 from Lactobacillus lactis, transaminated amino acid decarboxylase from saccharomyces cerevisisae ARO10, pyruvate decarboxylases including gene bank ID No. L09727 from Kluyveromyces marxianus, gene bank ID No. L09125.1 from Neurospora crassa, gene bank ID No. M15393.2 from Zymomonas mobilis, Pyruvate decarboxylase isozymes 3, 1, 5 from saccharomyces cerevisiae, respectively PDC6, PDC1, and PDC5, NCU02193 from Neurospora crassa, A0A804LIK4, A0A804LIK5, Q8S4W8, A0A804UGT8, C4J495, B7ZX31, A0A804QR90, A0A1D6FS81, A0A1D6FS83, B8A1S0, A0A3L6DL94 from Zea mays, 082647, Q9FFT4, Q9M040, Q9M039 from Arabidopsis thaliana, alpha-keto-acid decarboxylase Rv0853c, MT0876 and MRA_0861 from Mycbacterium tuberculosis, ML2167 from Mycobacterium leprae, BQ2027 MB0876C from Mycbacterium bovis, MUL_0302 from Mycbacterium ulceras, SIRAN8386 from Streptomyces iranensis, SL103_21815 from Streptomyces lydicus, Aspartate 1- decarboxylase from heliobacter pylori, Pyruvate decarboxylase isozyme 3, and YGR087C from Saccharomyces cerevisiae.

[0059] In some embodiments, the enzyme is an oxidase. In some embodiments, the oxidase is pyruvate oxidase or oxalate oxidase.

[0060] Pyruvate oxidase is an enzyme that catalyzes FAD and TPP-dependent oxidative decarboxylation of pyruvate to generate hydrogen peroxide and acetyl phosphate (See, Juan et al., Acta Cryst., 2007, F63: 900-907). Our computational analysis showed that the substitution of hydrogens in pyruvate molecule with fluorine atoms does not result in steric interference. Our experimental results showed that a mixture of natural fluoroacetate dehalogenase and pyruvate oxidase with pentafluoropropionic acid does result in the formation of hydrogen peroxide (FIG. 9). This suggests that both defluorination and decarboxylation starting from perfluorinated substrate ccurs. Pyruvate oxidase reaction would result in formation of phosphorylated, one carbon atom shorter substrate that needs to be hydrolyzed to regenerate the carboxyl group (FIG. 4). Representative examples of pyruvates oxidases that may be useful in the practice any aspects of the disclosed processes include pyruvate oxidases including Uniprot Entry No. P37063 from Lactiplantibacillus plantarum and Uniprot Entry No. Q54970 from Streptococcus pneumoniae.

[0061] Oxalate oxidase is an enzyme that catalyzes oxidative decarboxylation of oxalate and results in the formation of two CO2 molecules and H2O2 using manganese ion and dioxygen (See, Svedruzic et al., Arch. Biochem. Biophys., 2005, 433(1) '.176-192). It has been shown that natural oxalate oxidase enzymes with varying substrate promiscuity exists. The naturally occurring enzymes could be engineered to also catalyze decarboxylation of poly- and perfluorinated molecules and result in one atom shorter carboxylic acid, oxalate decarboxylase is an enzyme that uses a similar mechanism for decarboxylation as oxalate oxidase but does not result in formation of H2O2 (See, Burrell et al., Biochemistry, 2007, 46 43): 12327-12336). Representative examples of oxalate oxidases that may be useful in the practice of any aspects of the disclosed processes include oxalate oxidases including Uniprot Entry No. P45850, and P45851 from hordeum vulgare, Uniprot Entry No. P26759 and Uniprot Entry No. Pl 5290 from triticum aestivum, Uniprot Entry No. Q10CE4 from oryza sativa, and Uniprot Entry No. Q44467 from aerococus viridans.

[0062] In some embodiments, enzymes are used to generate a hydroxyl radical or a hydroperoxyl radical. [0063] The enyzmes that may be utilized in the practice that may be useful in the practice of any of the aspects of the disclosed processes may be in substantially isolated and purified form (e.g., in the form of recombinant enzymes) or not. In the latter case, they may be used in the form of lysed cells or an extract from a heterologous host or natural source such as a microbe or plant (e.g., a microbial extract or a plant extract). Live microbes expressing the enzymes, micrbial consortia expressing the enzymes, and plants expressing the enzymes may be used. Enzymes may also be immobilized on a solid support e.g., cellulose, starch, agarose, agar, calcium alginate, carragenans, chitosan, activated carbon, porous ceramic, diatomeous earth or modified polymers derived from described natural polymers.

[0064] Therefore, in some embodiments of any of the aspects of the processes, the processes utilize purified enzymes that are in free or immobilized form.

[0065] Therefore, in some embodiments of any of the aspects of the processes, the processes utilize crude microbial extracts containing the enzymes.

[0066] Therefore, in some embodiments of any of the aspects of the processes, the processes utilize enzymes that are expressed in live microbes or plants.

[0067] Nonenzymatic Decarboxylation

[0068] Decarboxylation may be conducted with the use of a hydroxyl radical or a hydroperoxyl radical. Accordingly, a further aspect of the disclosure is directed to a chemoenzymatic process, the chemoenzymatic process comprises:

[0069] In some embodiments, steps 1-2 constitute a cycle, and the cycle is repeated, beginning with the modified poly- or perfluorinated substrate produced by the prior cycle, at least once and up to a number of cycles equal to the number of fluorinated carbons of the first modified poly- or perfluorinated substrate. [0070] In some embodiments, the hydroxyl or hydroperoxyl radical is generated using hydrogen peroxide or an organic peroxide. See, e.g., Lopalco et al., J. Pharm. Sci., 2016, 105(2) :705-712 and Wardman et al., Radiation Research, 1996, 7- : 523-531.

[0071] In some embodiments, the radical is an organic radical. In some embodiments, the organic radical is a peroxide radical. In some embodiments, the peroxide radical is generated using Zc/7-buryl hydroperoxide, dicumyl peroxide, /cvV-butylperoxybenzoate, or dibenzyl peroxide.

[0072] Regenaration of Terminal Carboxyl Group

[0073] In some embodiments in which a pyruvate decarboxylase is used to decarboxylate the a- keto acid, the resulting product will be an aldehyde. To regenerate the terminal carboxyl group another enzyme would need to be employed. Aldehyde dehydrogenase enzymes catalyze oxidation of aldehydes to carboxylic acid (FIG. 10). This enzyme group is divided based on the substrate length and a combination of long and medium chain aldehyde dehydrogenases may be used to oxidize all substrates. Representative examples of aldehyde dehydrogenases that may be useful in the practice of any aspects of the disclosed processes include A0A7L4W7A1 from Lactococcus cat nosus, A0A7L4WFD2 from Lactococcus paracar nosus, A0A387BIQ9 from Lactococcus allomyrinae, Q8GAK7 from Paenarthrobacter nicotinovorans, and P37685 from Escherichia coli. [0074] In some embodiments in which a pyruvate oxidase is used to decarboxylate a-keto acid, the resulting product will be an acetyl phosphate. To regenerate the terminal carboxyl group another enzyme is needed (FIG. 4). Phosphatases are enzymes that catalyze hydrolysis of phosphomonoesters, by removing a phosphate moiety from the substrate, and can utilize different mechanisms and be divided into alkaline and acid phosphatases (See, Holtz et al., FEBS Lett., 1999,

and Chini et al., Biochem. Biophys. Acta., 1990, 1030(l .152- 156). Representative examples of phosphatases that may be useful in the practice of any of the aspects of the disclosed processes include alkaline phosphatase Uniprot Entry No. P00634 from Escharichia coir, Alkaline phosphatase H Uniprot Entry No. P35483 from Pseudomonas aeruginosa, Alkaline phosphatase L Uniprot Entry No. P35482 from Pseudomonas aeruginosa, Pyridoxal phosphate phosphatase Uniprot Entry No. P27848 from Escharichia coir, Class B acid phosphatase Uniprot Entry No. P0AE22 from Escharichia coli.

[0075] Alkanesulfonate Monooxygenase

[0076] Alkanesulfonate monooxygenase may be used to remove a sulfate group from a PFAS compound and form an aldehyde (See, Liew et al., J. Biol. Chem., 2021, 297(7):100823). In other embodiments, an alternative chemoenzymatic route may be used that involves the removal of the fluorine atoms from the C2 (first fluorinated carbon next to carboxyl group), followed by spontaneous formation of a-keto acid, then addition of H2O2 to remove the keto group as follows: R-COCOOH+H2O2 ->R-COOH + H2O + CO2.

[0077] Additional Embodiments and Examples

[0078] Although some particular embodiments of the disclosed mechanisms, methods, and compounds have already been described in detail, additional and alternative embodiments are also possible. A few particular examples are discussed below.

[0079] These and other aspects of the present disclosure will be further appreciated upon consideration of the following Examples, which are intended to illustrate certain particular embodiments of the disclosure but are not intended to limit its scope, as defined by the claims.

[0080] Example 1

[0081] In a first example, after the perfluorinated a-keto acid is formed, branched-chain a-keto acid decarboxylase, a pyruvate decarboxylase, an oxalate oxidase, an oxalate decarboxylase, pyruvate dehydrogenase multienzyme complex, or a-ketoglutarate dehydrogenase catalyzes a decarboxylation reaction, resulting in the formation of a CO2 molecule and - 1C perfluoro aldehyde (FIG. 2). To form a new carboxyl group from the aldehyde group, an aldehyde dehydrogenase may be used (FIG. 2 and FIG. 10).

[0082] An exemplary pathway of one carbon removal is shown in FIG. 8. Newly formed perfluoro heptanoic acid may start a new cycle that results in the formation of perfluoro hexenoic acid, 2F‘ and CO2. These cycles may be repeated until all fluorine atoms are removed from the substrate (FIG. 3).

[0083] Example 2

[0084] In a second example, pyruvate oxidase is used to remove CO2 and form a phospho-ester (Chornacchione et al., BMC Microbiol., 2020, 20(7 128). Subsequently, an acid phosphatase is used to hydrolyze the phospho-ester bond to form a carboxyl group (Margalef et al., Sci. Rep., 2017, 7(1)'.1337) (FIG. 2).

[0085] Example 3

[0086] In a third example, a reactive species may be used to form a-keto acid radicals that lead to spontaneous decarboxylation. [0087] In each of the examples described herein, some or all enzymes may be optimized to function in industrial setups. Additionally, all reactions discussed herein may be performed with substrates having varying lengths (i.e., differing numbers of carbon atoms).

[0088] Example 4

[0089] Defluorination activity was determined using fluoride-specific aptamer sensor and fluorescent dye TOl-biotin as described in Husser et al., Small, 2022, 19(13) :2205232 (FIG. 5). [0090] Example 5

[0091] Molecular dynamics data was generated using AMBER program package and principal component analysis was performed using Bio3D program package (FIG. 6).

[0092] Example 6

[0093] Decarboxylation activity was measured using pyruvate detection kit (VWR catalog number: ABNOKA1674) following the instruction manual (FIG. 9).

[0094] Example 7

[0095] Defluorination activity was determined using fluoride-specific aptamer sensor and fluorescent dye TOl-biotin as described in Husser et al., Small, 2022, 79(73/2205232 (FIG. 12).

[0096] All patent publications and non-patent publications are indicative of the level of skill of those skilled in the art to which this disclosure pertains. All these publications (including any specific portions thereof that are referenced) are herein incorporated by reference to the same extent as if each individual publication were specifically and individually indicated as being incorporated by reference.

[0097] Although the disclosure herein has been described with reference to particular embodiments, it is to be understood that these embodiments are merely illustrative of the principles and applications of the present disclosure. It is therefore to be understood that numerous modifications may be made to the illustrative embodiments and that other arrangements may be devised without departing from the spirit and scope of the present disclosure as defined by the appended claims.

Claims

What is claimed is:

1. A process for degrading a poly- or perfluorinated substrate, the process comprising:

2. The process of claim 1, wherein the decarboxylating the first modified poly- or perfluorinated substrate is conducted using an oxidase followed by an acid phosphatase.

3. The process of claim 1, wherein the decarboxylating the first modified poly- or perfluorinated substrate is conducted using an a-keto acid decarboxylase.

4. The process of claim 1, wherein steps 1-3 constitute a cycle, and the cycle is repeated, beginning with the poly- or perfluorinated substrate produced by the prior cycle, at least once and up to a number of cycles equal to the number of fluorinated carbons in the hydrophobic tail of the first modified poly- or perfluorinated substrate.

5. A process for degrading a poly- or perfluorinated substrate, the process comprising:

(1) converting a poly- or perfluorinated substrate that has a terminal sulfonic or phosphonic acid group, and a linear or branched hydrophobic tail comprising a plurality of fluorinated carbon atoms, to produce a first modified poly- or perfluorinated substrate having a terminal carboxyl Attorney Docket No. 59599-001PCT group in place of the terminal sulfonic or phosphonic acid group, and an a-keto group, by (la) reacting the poly- or perfluorinated substrate with a fluoroacetate dehalogenase, followed by enzymatic or microbial biotransformation to convert the terminal sulfonic or phosphonic acid group to a carboxylic acid group, or (lb) converting the terminal sulfonic or phosphonic acid group of the poly- or perfluorinated substrate to a carboxylic acid group, followed by use of the fluoroacetate dehydrogenase, to produce the first modified poly- or perfluorinated substrate;

6. The process of claim 5, wherein the decarboxylating the first modified poly- or perfluorinated substrate is conducted using an oxidase followed by an acid phosphatase.

7. The process of claim 5, wherein the decarboxylating the first modified poly- or perfluorinated substrate is conducted using an a-keto acid decarboxylase.

8. The process of claim 5, further comprising:

(6) functionalizing the fifth modified poly- or perfluorinated substrate by contacting the fifth modified poly- or perfluorinated substrate with an aldehyde dehydrogenase, to produce a sixth modified poly- or perfluorinated substrate that has a terminal carboxylic acid and wherein the Attorney Docket No. 59599-001PCT hydrophobic tail is one fluorinated carbon shorter in length than the third modified poly- or perfluorinated substrate.

9. The process of claim 8, wherein steps 4-6 constitute a cycle, and the cycle is repeated, beginning with the poly- or perfluorinated substrate produced by the prior cycle, at least once and up to a number of cycles equal to the number of fluorinated carbons in the hydrophobic tail of the poly- or perfluorinated substrate produced by the prior cycle.

10. A chemoenzymatic process for degrading a poly- or perfluorinated substrate, the chemoenzymatic process comprising:

11. The process of claim 10, wherein steps 1-2 constitute a cycle, and the cycle is repeated, beginning with the modified poly- or perfluorinated substrate produced by the prior cycle, at least once and up to a number of cycles equal to the number of fluorinated carbons of the first modified poly- or perfluorinated substrate.

12. The process of claim 10, wherein the radical is a peroxide radical.

13. A process for degrading a poly- or perfluorinated substrate, the process comprising:

(1) conducting a decarboxylation reaction on a poly- or perfluorinated substrate that has a terminal carboxylic acid and a hydrophobic tail, and an a-keto group, to produce a first modified poly- or perfluorinated substrate having a terminal aldehyde group; and Attorney Docket No. 59599-001PCT

14. The process of any one of claims 1, 5, and 10, wherein the linear or branched chain of the poly- or perfluorinated substrate is saturated or unsaturated and wherein the linear or branched chain may be interrupted by one or more heteroatoms and/or functional groups.

15. The process of claim 14, wherein the poly- perfluorinated substrate is of formula (I) or (II):

X is absent or O; and each n is independently an integer 0-15.

16. The process of claim 15, wherein n is an integer of 1-10.

17. The process of any one of claims 1-4, wherein the poly- or perfluorinated substrate is perfluorobutanoic acid (PFBA), perfluoropentanoic acid (PFPeA), perfluorohexanoic acid (PFHxA), perfluoroheptanoic acid (PFHpA), peril uorooctanoic acid (PFOA), hexafluoropropylene oxide dimer acid (HFPO-DA), perfluorononancanoic acid (PFNA), perfluorodecanoic acid (PFDA), perfluoropropionic acid (PFPrA), perfluoroundecanoic acid (PFUnDA), perfluorododecanoic acid (PFDoA), perfluorotridecanoic acid (PFTrDA), or perfluorotetradecanoic acid (PFTeDA). Attorney Docket No. 59599-001PCT

18. The process of any one of claims 5-9, wherein the poly- or perfluorinated substrate is perfluorobutane sulfonic acid (PFBS), perfluorohexane sulfonic acid (PFHxS), perfluorooctane sulfonic acid (PFOS), or perfluorooctanesulfonamide (PFOSA).