Nitroguanidine bioremediation

Abstract

A composition useful for nitroguanidine bioremediation includes a nitroguanidinase enzyme and an enzyme stabilizer. In one or more embodiments, the composition further includes a nitrourease. In one or more embodiments, the composition is formulated as a solid. In one or more embodiments, the composition is formulated as a liquid. In one or more embodiments, the composition is formulated as a foam. In one or more embodiments, the composition further includes a buffering agent, a desiccant, or structural material. The composition may be used in methods of bioremediation that include contacting the composition with an area contaminated with nitroguanidine.

Description

[0001] PCT Patent Application Attorney Docket No. 0110.000773WO01

[0002] NITROGUANIDINE BIOREMEDIATION

[0003] CROSS REFERENCE TO RELATED APPLICATION

[0004] This application claims the benefit of U. S. Provisional Patent Application No.

[0005] 63 / 790,902, filed April 18, 2025, which is incorporated herein by reference in its entirety.

[0006] GOVERNMENT FUNDING

[0007] This invention was made with government support under CHE2203750 awarded by the National Science Foundation. The government has certain rights in the invention.

[0008] SEQUENCE LISTING

[0009] This application contains a Sequence Listing electronically submitted via Patent Center to the United States Patent and Trademark Office as an XML file entitled “0110000773WO01.xml” having a size of 6,507 bytes and created on April 17, 2026. The information contained in the Sequence Listing is incorporated by reference herein.

[0010] SUMMARY

[0011] This disclosure describes, in one aspect, a composition that includes a nitroguanidinase enzyme and an enzyme stabilizer.

[0012] In one or more embodiments, the composition further includes a nitrourease.

[0013] In one or more embodiments, the composition further includes a nitrile hydratase, a metforminase, or both.

[0014] In one or more embodiments, the composition is formulated as a solid.

[0015] In one or more embodiments, the composition further is formulated as a liquid.

[0016] In one or more embodiments, the composition further is formulated as a foam.

[0017] In one or more embodiments, the nitroguanidinase enzyme includes a first subunit and a second subunit. The first subunit includes the amino acid sequence of SEQ ID NO:2, a fragment of SEQ ID NO:2 effective to convert nitroguanidine to nitrourea, or a polypeptide having a degree of structurally similarity to SEQ ID NO:2 effective to convert nitroguanidine to nitrourea.The second subunit includes the amino acid sequence of SEQ ID NO:4, a fragment of SEQ ID NO:4 effective to convert nitroguanidine to nitrourea, or a polypeptide having a degree of structurally similarity to SEQ ID NO:4 effective to convert nitroguanidine to nitrourea. In one or more embodiments, the nitroguanidinase enzyme is a heterodimer of the first subunit and the second subunit.

[0018] In one or more embodiments, the nitrourease includes SEQ ID NO: 5 or a fragment thereof effective to convert nitrourea to NH3, N2O, and CO2.

[0019] In one or more embodiments, the composition further includes a buffering agent, a desiccant, or structural material. In one or more embodiments, the structural material includes a polymer, diatomaceous earth, or silica.

[0020] In another aspect, this disclosure describes a method of nitroguanidine bioremediation. The method includes contacting nitroguanidine with a composition that includes a nitroguanidinase enzyme and an enzyme stabilizer together in an amount effective to reduce the amount of nitroguanidine.

[0021] In one or more embodiments, disolved nitroguanidine is present in solution at a concentration of up to 34 mM.

[0022] In one or more embodiments, at least some of the nitroguanidine is present as a solid. In one or more embodiments, the nitroguanidine is present in soil.

[0023] In one or more embodiments, the composition is contacted with the nitroguanidine prior to an event capable of washing the nitroguanidine out of an area of contamination.

[0024] In one or more embodiments, the method further includes washing at least a portion of the nitroguanidine from the soil to form a rinsate that includes nitroguanidine removed from the soil. In one or more embodiments, washing the soil occurs in situ. In one or more embodiments, washing the soil occurs ex situ.

[0025] In one or more embodiments, the method further includes adjusting the pH of the vicinity of nitroguanidine to a pH of from 7.0 to 12.0.

[0026] The above summary is not intended to describe each disclosed embodiment or every implementation of the present invention. The description that follows more particularly exemplifies illustrative embodiments. In several places throughout the application, guidance is provided through lists of examples, which examples can be used in various combinations. Ineach instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list.

[0027] BRIEF DESCRIPTION OF THE FIGURES FIG. 1. Biodegradation of guanidinium compounds. (A) Known route for the biodegradation of guanidine carried out by guanidine carboxylase, CgdAB and allophanate hydrolase in Pseudomonas stutzeri. (B) Hypothetical pathway for nitroguanidine based on observed nitrourea and nitrous oxide formed in vivo by a newly identified Pseudomonas strain, designated herein as Pseudomonas sp. strain NGU. Genes in the genome of this newly identified strain and their numbering are shown. Genes 1861-1864 have the same order as the guanidine operon in Pseudomonas stutzeri. Pseudomonas sp. strain NGU genes 1826 and 1827 are distant homologs of cgdAB and 1841 is distantly related to allophanate hydrolase.

[0028] FIG. 2. Bioremediation of nitroguanidine in water at concentration above solubility limit. The panel shows five images taken at two-minute intervals of a 40 mmol per liter suspension of nitroguanidine in 10 mM MOPS-NaOH pH 7.5 treated with 3.65 μg enzyme. The images were taken of the same sample consecutively at time 0, two minutes, four minutes, six minutes, and eight minutes after enzyme treatment using a stereomicroscope (M165C, Leica Microsystems GmbH, Wetzlar, Germany) fitted with a polarizing lens and an attached camera. Near complete clearance is achieved at eight minutes.

[0029] FIG. 3. Soil enzyme treatments to remove nitroguanidine (NQ). Two grams of sandy loam soil containing 1 mM nitroguanidine were treated. (A) Reaction catalyzed by treatment with nitroguanidinase (NGase). (B) Reaction catalyzed by treatment with nitroguanidinase (NGase) and nitrourease (NUase). (C) HPLC trace of nitroguanidine in soil treated with 0.57 mg / ml of nitroguanidinase for two hours. (D) HPLC trace of nitroguanidine in soil treated with 0.57 mg / ml of nitroguanidinase plus 0.01 mg / ml nitrourease for two hours. (E) Use of m-cresol purple pH indicator to follow the degradation of nitroguanidine in a water sample by the combination of nitroguanidinase and nitrourease as shown in panel (B), leading to a measurable pH change.

[0030] FIG. 4. Pathways for the biodegradation of guanidinium compounds showing the lack of connectedness of the newly evolved pathway for nitroguanidine (NQ). Two enzymes are involved in the complete detoxification and mineralization of nitroguanidine.FIG. 5. Sequence alignment of Pseudomonas stutzeri CgdA (SEQ ID NO: 1) and Pseudomonas sp. strain NGU nitroguanidinase subunit A (SEQ ID NO:2), showing 43.98% sequence identity despite having distinct functions.

[0031] FIG. 6. Sequence alignment of Pseudomonas stutzeri CgdB (SEQ ID NO: 3) and Pseudomonas sp. strain NGU nitroguanidinase subunit B (SEQ ID NO:4), showing 58.54% sequence identity despite having distinct functions.

[0032] FIG. 7. Crystal structure of nitroguanidinase. The left hexamer side view is of the holoenzyme. The middle panel shows the heterodimer pair. Only one subunit contains the active site in which a zinc atom if coordinated to three cysteine residues and a water. The substrate, nitroguanidine (NQ), is docked in the active site.

[0033] FIG. 8.1H-NMR of nitrosoguanidine as synthesized from nitroguanidine by reduction using powdered zinc.

[0034] FIG. 9.13C-NMR of nitrosoguanidine as synthesized from nitroguanidine by reduction using powdered zinc.

[0035] FIG. 10. Nitrosoguanidine bioremediation via nitroguanidinase. (Top) Photographic images of nitrosoguanidine bioremediation via nitroguanidinase showing the loss of the faint yellow color of nitrosoguanidine visually (left) and via a spectrophotomer At right, copious bubbles are seen immediately, indicating gas production. (Bottom) Chemical reactions involved in nitrosoguanidine bioremediation via nitroguanidinase.

[0036] FIG. 11. Schematic illustration of the process for purifying nitrourease.

[0037] FIG. 12. Nitroguanidine bioremediation in soil. (Top) Chemical reactions involved in bioremediation of nitroguanidine. (Bottom) Equipment used in exemplary analysis of nitroguanidine bioremediation.

[0038] FIG. 13. Generation of N2O in an exemplary test of nitroguanidine bioremediation of soil using nitroguanidinase and nitrourease.

[0039] FIG. 14. Assays detecting nitroguanidinase activity. (A) Current commercial assay measuring nitroguanidinase degradation of nitroguanidine to ammonia. (B) Exemplary alternative assay measuring nitroguanidinase conversion of 2-nitroethylene- 1,1 -diamine to nitroacetamide.

[0040] FIG. 15. Exemplary alternative assay measuring nitrourease conversion of nitroacetamide to nitroacetic acid.DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS This disclosure describes strategies for bioremediation of nitroguanidine from soils and water samples. Nitroguanidine is a major artillery munition component that poses a cancer threat from soil mineral reduction to nitrosoguanidine. This disclosure identifies enzymes that transform nitroguanidine to fertilizer (ammonia) and non-toxic gases (CO2 and N2O). Water and soil remediation were demonstrated using enzymes.

[0041] Evidence for nitroguanidine biodegradation by a non-reductive route

[0042] Spilled or unexploded munitions can provide nitroguanidine to soils and water sources, where the nitroguanidine can be converted to a carcinogen. While described herein in the context of munitions as a source of nitroguanidine in the environment, the strategies and methods described herein can involve bioremediation of nitroguanidine from any source. Exemplary nonmunition sources of nitroguanidine include, but are not limited to, construction sites, propellant compositions, and as a precursor in the manufacture of certain pesticides. A strain of Pseudomonas extremaustr alius, NQ5, degrades nitroguanidine over two days, suggesting the presence of enzymes that degrade nitroguanidine have evolved under selective pressure for releasing assimilable nitrogen from nitroguanidine (Kim et al., 2024, Science of The Total Environment, 912, p. 169184). Further, nitrourea is an intermediate in the biodegradation pathway, but no urease is known to participate in degrading nitroguanidine (Perreault et al., 2012, Environmental Science & Technology, 46f ), pp. 6035-6040).

[0043] Here, a nitroguanidine-degrading bacterium was isolated and denoted as Pseudomonas sp. strain NGU (shown by the shorthand as Pseudomonas strain NG in FIG. IB). Previously described isolates of NQ5, NQ4, and NQ7 grew with doubling times of 10 hours, 28 hours, and 41 hours, respectively, when grown on nitroguanidine as the sole nitrogen source (Kim et al., 2024, Science of The Total Environment, 912, p. 169184). In the present study, strain NGU doubled in 1.2 hours on nitroguanidine. Nitrourea was observed to form transiently at low levels and nitrous oxide was evolved by strain NGU grown with nitroguanidine (FIG. 1). The genome of strain NGU was sequenced by short and long read methods to clone the genome and assemble an accompanying plasmid element. Comparative genomics of strains NGU with strains NQ4, NQ5, and NQ7 revealed that only the NGU and NQ5 strains, that grew the fastest, containedmultiple gene regions potentially related to guanidine metabolism (FIG. IB). Guanidine is carboxylated by the CgdAB heterodimer, which makes carboxyguanidine that is isosteric with nitroguanidine (FIG. 1A).

[0044] The carb oxy guanidine deiminase (CgdAB) protein from Pseudomonas syringae catalyzes a hydrolytic deamination reaction with carboxy guanidine, but also can react with nitroguanidine to release one equivalent of ammonia with a KM of 17.2 mM and kcat of 2.2 s’1. Pseudomonas sp. strain NGU contained a second gene pair that was distantly related in sequence similarity to cgdAB. The genes were codon optimized for expression in A. coli with 6*His-tag and purified. The new protein, denoted here as nitroguanidinase, showed 11 -fold higher activity with nitroguanidine than the carboxyguanidine deiminase, or CgdAB, from strain NGU.

[0045] The Pseudomonas sp. strain NGU genes 1826 and 1827, encoding the nitroguanidinase and nitrourease enzymes, respectively, were not the typical genes previously reported for guanidine metabolism, consistent with these genes being independent from typical guanidine metabolic pathways. Early reports of microbial degradation of nitroguanidine focused on either reduction or displacement of the nitro group (Rios-Valenciana et al., 2022. Environmental Pollution, 309, p.l 19788; Heitholt et al., 1990. Bulletin of Environmental Contamination and Toxicology; (USA), 44(5)). Nitro group transformation has been considered an undesirable route due to formation of carcinogenic nitrosoguanidine. The nitroguanidinase gene region lacked evidence of nitro group metabolism and guanidine carboxylase, and it contained multiple transposons, suggesting a recent evolutionary capture by the genome of strain NGU.

[0046] Handling the metabolic intermediate and explosive compound nitrourea

[0047] In addition to ammonia, the co-product of the CgdAB-catalyzed reaction was nitrourea, which will decompose non-enzymatically in neutral aqueous solutions with a ti / 2 of about 24 hours. However, in whole cell studies only very low levels of nitrourea were observed and it rapidly disappeared after nitroguanidine was exhausted. This strongly suggested enzymatic transformation was occurring in vivo. Given that nitrourea is structurally analogous to allophanate (FIG. 1), purified allophanate hydrolase from Pseudomonas sp. ADP (Shapir et al., 2005. Journal of Bacteriology, 187(\ 1), pp, 3731-3738) was tested but no activity with nitrourea was detected.In light of these negative results, protein extracts from Pseudomonas sp. NGU were prepared, fractionated, and assayed using the strong absorbance of nitrourea at 259 nm as a means of following its disappearance. Extracts were subjected to a series of ammonium sulfate fractionation, ion exchange, size exclusion chromatography, and hydroxyapatite fractionation steps until one protein band was obtained, which showed an approximate subunit molecular weight of 46 kDa. Mass spectrometry of proteolyzed fragments identified a sequence match with a genome-predicted protein of about 46 kDa that was annotated as an amidase. The match was to the predicted protein sequence encoded by gene 1841 (FIG. IB). A synthetic gene encoding the predicted nitrourease protein was expressed in E. coli and purified. The purified protein encoded by gene 1841 catalyzed deamination of nitrourea with a specific activity of 32 pmol per min per mg. By way of comparison, the specific activity with allophanate was 0.9 pmol per min per mg.

[0048] In the genome, the nitrourease gene was adjacent to two hypothetical genes and all were flanked by transposase DNA (FIG. IB). Comparing the nitrourease to well characterized proteins showed the highest sequence identity of 44% to the structurally determined allophanate hydrolase from Pseudomonas sp. ADP, which had been previously shown not to be reactive with nitrourea (Shapir et al,, 2005, Journal of Bacteriology, 187(\ 1), pp. 3731-3738). The nitrourease sequence from strain NGU showed much higher identity (73%) with an enzyme from the P. extremaustralus nitroguanidine degrader (Kim et al., 2023, Microbiology Resource Announcements, 12(9), pp. e00467-23). Both had subunit molecular weights of approximately 48 kDa, significantly shorter than the characterized allophanate hydrolases that have subunit molecular weights of approximately 60 kDa. In total, these data suggest a newly evolved function as a nitrourease involved in nitroguanidine biodegradation.

[0049] Bioremediation of nitroguanidine in water

[0050] There is current interest in degrading nitroguanidine in soils and in water suspensions. Water suspensions of crystalline nitroguanidine at a nominal concentration of 40 mM, which is higher than its water solubility limit of 33 mM, were treated with 3.56 pg of purified nitroguanidinase. The suspension cleared completely in eight minutes (FIG. 2). This not only illustrates the high activity of the enzyme but also indicates rapid nitroguanidine mass transfer from the solid to liquid phase. These are both advantageous for bioremediation purposes.The incubation of nitroguanidine with a mass of strain NGU cells more than a thousandfold greater than the enzyme used in FIG. 2 and for 1440 minutes resulted in negligible degradation of nitroguanidine. The bacterium had been grown using nitroguanidine as the sole source of nitrogen and so nitroguanidinase must have been expressed. The most likely explanation is that the ABC transporters upstream of the nitroguanidinase gene play a role in active transport of nitroguanidine and that energy-dependent transport of nitroguanidine into the cells is not occurring with resting cells. This highlights the greater potential of using cell-free enzyme compositions for remediation of nitroguanidine.

[0051] Bioremediation of nitroguanidine in soil

[0052] Remediation of surface soil containing high levels of nitroguanidine may be favorable in the case of spills or unexploded ordinance where time, or lack of precipitation, precludes leaching of the contaminant and confines nitroguanidine to a limited area. Although some microorganisms release enzymes into soil, there are limited studies of enzyme amendment to soil and there is a general perception that enzyme treatment of pollutants is too expensive and / or of limited effectiveness. However, enzymes are increasingly being used in large-scale processes including the commercial degradation and recycling of the polymer polyethylene terephthalate. Moreover, enzyme treatments can be effective decontaminating environments that contain highly toxic agents (e.g., nerve agents) and to reduce the infectivity of prion agents that spread in soils and cause chronic wasting disease.

[0053] Enzymatic remediation of nitroguanidine is facilitated by the toxicant not being sequestered by soil, facile transfer from the solid to the dissolved phase, and the enzyme remaining active for a reasonable time. Nitroguanidine is not hydrophobic and is uncharged between pH 1 to 12, consistent with its reported propensity to remain unbound in soils, even those with high organic content. The stability of the enzyme in soil and feasibility of in-soil biodegradation was studied in two different soil types. One was a loamy sand with a low organic matter content. The other soil was a silty clay loam high in organic matter. Organic matter often leads to tighter binding of anthropogenic chemicals, which can impede enzymatic degradation.

[0054] Two different methods were developed to measure and corroborate enzyme activities in the soils (FIG. 3). One method takes advantage of the non-binding properties of nitroguanidine to soils, allowing facile water extraction coupled to HPLC analysis of the extract. The secondmethod involved determining pH change as the combined activities of nitroguanidinase and nitrourease releases 2 mol ammonia per mole of nitroguanidine, raising the pH as monitored using the pH indicator m-cresol purple.

[0055] First, the enzyme could be added to a surface soil, for example after a spill, and significantly degrade nitroguanidine and thus reduce the likelihood and / or extent to which nitroguanidine leaches into a much larger area. Secondly, nitroguanidine degradation was rapid, showing availability of nitroguanidine, consistent with a low extent of binding. The rapid and complete degradation shows the feasibility of enzyme-based bioremediation. Third, adding 20 mg 3-morphilinopropane sulfonic acid (MOPS) adjusted to pH 8.0 significantly enhanced enzymatic nitroguanidine degradation. The pls of the proteins range from 4.93 to 5.98 and enzymes are largely most stable and show greater activities several pH units away from their isoelectric point. This suggests that it may be desirable for a commercially formulated enzyme bioremediation composition to contain additives that stabilize and enhance enzyme activity. Lastly, nitroguanidinase alone transformed nitroguanidine to nitrourea. While a one-enzyme remediation may be sufficient in practice, the current study showed that a two-enzyme treatment rapidly converts nitroguanidine to fertilizer (NH3) and safe gases carbon dioxide and nitrous oxide (N2O).

[0056] Rapid Enzyme Evolution to Handle Anthropogenic Chemicals

[0057] Previous approaches to nitroguanidine biodegradation focused on reactions at the nitro group and its displacement. Indeed, the Eawag-bbd Pathway Prediction System (Indest et al., 2017. J Ind Microbiol Biotechnol. 44(7):987-995) depicted nitroguanidine biodegradation via denitration and deamination to make urea. The nitro group was metabolized via a six-electron reduction process. This approach includes nitrosoguanidine as an intermediate, which is mutagenic and carcinogenic and, therefore, presents safety concerns. This disclosure provides a reaction pathway that proceeds through two hydrolytic enzymatic amino group removals and, therefore, avoids producing nitrosoguanidine.

[0058] The highly active nitroguanidinase and nitrourease enzymes described here show evidence of having recent evolutionary origins (FIG. 4). In a study in 2017 on biodegradation of IMX munition components, 2,4-dinitroanisole and 3-nitro-l,2,4-triazol-5-one were completely biodegraded but nitroguanidine was largely untransformed in 30 days (Indest et al., 2017. J IndMicrobiol Biotechnol. 44(7):987-995). The nitroguanidinase gene employed in the bioremediation approach described herein is distantly related to an ancestral carboxyguanidine deimininase (CgdAB) and may have come from that lineage. The gene was not in an organized operon and showed evidence of acquisition by a transposition into the genome of the nitroguanidine degrading strain NGU (FIG. 1). The nitrourease gene is flanked by transposable elements and it provides the means to efficiently detoxify nitrourea, which can be highly inhibitory to some bacteria. Nitrourease is related to allophanate hydrolases but its substrate specificity has almost completely crossed over to nitrourea. The evolved nitrourease shows a very high apparent beat of 26 s’1, and the reaction with allophanate occurs at < 1 s’1. The native allophanate hydrolases tested here showed no activity with nitrourea.

[0059] The nitrourease is also distantly related to known members of the amidase signature family, which is a fertile ground for the evolution of pesticide-degrading enzymes. Most of the enzymes in that family share less than 50% sequence identity to the nitrourease described here. Nitroguanidine was first synthesized more than one century ago, but its adoption by global militaries for the replacement of TNT has occurred largely within the last 15 years.

[0060] The Pseudomonas sp. strain NGU contains nitrile hydratase and guanylurea hydrolase (guuH) genes and, consequently, grew rapidly with cyanoguanidine (dicyandiamide), a fertilizer additive used over the last century. Cyanoguanidine is degraded by a Rhodococcus species and jack bean urease, both stopping metabolism at cyanourea. The pathway here proceeds through a nitrile hydratase, guanylurea hydrolase, and guanidine and that leads to complete degradation to ammonia and carbon dioxide.

[0061] Pseudomonas strain NGU did not contain mfmAB genes encoding metforminase and did not degrade metformin. The nickel-enzyme metforminase displaces dimethylamine from the pharmaceutical metformin. Metformin is heavily prescribed for type II diabetes and people pass most of the dose, so environmental microbes have been exposed widely. The use of nitroguanidine is not as widespread as metformin and its heaviest use has largely occurred in the last two decades. A search of the Global Microbial Gene Catalog, UniProt, and NCBI databases revealed only two sequences that could be nitroureases, suggesting this gene and pathway may be very rare and in an early stage of evolution.

[0062] NitroguanidinaseThis disclosure therefore describes compositions and methods effective for nitroguanidine bioremediation. The compositions and methods may be useful for removing nitroguanidine from an area of contamination such as, but not limited to, a surface, soil, water, wastewater, and the like. Generally, the composition includes a nitroguanidinase enzyme and an enzyme stabilizer. In one or more embodiments, the nitroguanidinase enzyme includes the amino acid sequence of SEQ ID NO:2, a fragment thereof effective to convert nitroguanidine to nitrourea, or a polypeptide comprising a degree of structurally similarity to SEQ ID NO:2 effective to convert nitroguanidine to nitrourea. In one or more embodiments, the nitroguanidinase enzyme includes the amino acid sequence of SEQ ID NO:4, a fragment thereof effective to convert nitroguanidine to nitrourea, or a polypeptide comprising a degree of structurally similarity to SEQ ID NO:4 effective to convert nitroguanidine to nitrourea. In one or more embodiments, the nitroguanidinase enzyme is a heterodimer having a first subunit and a second subunit, in which the first subunit includes any embodiment of polypeptide described above with reference to SEQ ID NO:2 and the second subunit includes any embodiment of polypeptide described above with respect to SEQ ID NO:4.

[0063] As used herein, a polypeptide is “structurally similar” to a reference polypeptide if the amino acid sequence of the polypeptide possesses a specified amount of identity compared to the reference polypeptide. Structural similarity of two polypeptides can be determined by aligning the residues of the two polypeptides (for example, a candidate polypeptide and the polypeptide of, for example, SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:5) to optimize the number of identical amino acids along the lengths of their sequences; gaps in either or both sequences are permitted in making the alignment in order to optimize the number of identical amino acids, although the amino acids in each sequence must nonetheless remain in their proper order. A candidate polypeptide is the polypeptide being compared to the reference polypeptide (e.g., SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:5). A candidate polypeptide can be isolated, for example, from an animal, plant, or microbe, or can be produced using recombinant techniques, or chemically or enzymatically synthesized.

[0064] A pair-wise comparison analysis of amino acid sequences can be carried out using the BESTFIT algorithm in the GCG package (version 10.2, Madison WI). Alternatively, polypeptides may be compared using the Blastp program of the BLAST 2 search algorithm, as described by Tatiana et al., (FEMS Microbiol Lett, 174, 247-250 (1999)), and available on theNational Center for Biotechnology Information (NCBI) website. The default values for all BLAST 2 search parameters may be used, including matrix = BLOSUM62; open gap penalty = 11, extension gap penalty = 1, gap x dropoff = 50, expect = 10, wordsize = 3, and filter on.

[0065] In the comparison of two amino acid sequences, structural similarity may be referred to by percent “identity” or may be referred to by percent “similarity.” “Identity” refers to the presence of identical amino acids. “Similarity” refers to the presence of not only identical amino acids but also the presence of conservative substitutions. A conservative substitution for an amino acid in a polypeptide may be selected from other members of the class to which the amino acid belongs. For example, it is well-known in the art of protein biochemistry that an amino acid belonging to a grouping of amino acids having a particular size or characteristic (such as charge, hydrophobicity and hydrophilicity) can be substituted for another amino acid without altering the activity of a protein, particularly in regions of the protein that are not directly associated with biological activity. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and tyrosine. Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, or glutamine. The positively charged (basic) amino acids include arginine, lysine, or histidine. The negatively charged (acidic) amino acids include aspartic acid or glutamic acid. Conservative substitutions include, for example, Lys for Arg or vice versa to maintain a positive charge; Glu for Asp or vice versa to maintain a negative charge; Ser for Thr or vice versa so that a free -OH is maintained; or Gin for Asn or vice versa to maintain a free -NH2. Likewise, biologically active analogs of a polypeptide containing deletions or additions of one or more contiguous or noncontiguous amino acids that do not eliminate a functional activity of the polypeptide are also contemplated.

[0066] An enzyme polypeptide as described herein can include a polypeptide with at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence similarity to the reference amino acid sequence.

[0067] An enzyme polypeptide as described herein can include a polypeptide with at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the reference amino acid sequence.

[0068] An enzyme polypeptide as described herein also can be designed to provide additional sequences, such as, for example, the addition of added C-terminal or N-terminal amino acids that can, for example, facilitate purification by trapping on columns or use of antibodies. Such tags include, for example, histidine-rich tags that allow purification of polypeptides on nickel columns. Such gene modification techniques and suitable additional sequences are well known in the molecular biology arts.

[0069] The crystal structure of a nitroguanidinase heterodimer of SEQ ID NO:2 and SEQ ID NO:4. was successfully crystallized and produced a dataset of 2.7 A (FIG. 7). The structure was solved and model built based on separate ALPHAFOLD models (European Molecular Biology Laboratory, Heidelberg, Germany) of the two subunits. The asymmetric unit contains six chains, organized as a heterodimer with three-fold symmetry around a central axis, forming an a3b3 heterohexamer with a 10 A pore on the axis of rotation. The three A subunits make up the core of the complex surrounding the central pore. The three B subunits fan out from this central structure, interacting with two neighboring A subunits and not with each other. The mass of this heterohexamer (164 kDa) is in reasonable agreement the size calculated from size exclusion chromatography (132 kDa), so the asymmetric unit is likely the biological construct.

[0070] All subunits have a similar fold of about 8-10 antiparallel β-sheets organized into two layers in a jelly roll-like fold. Each subunit also has three or more α-helices that largely sit at the interface between the subunit interface and may contribute to the heterodimer quaternary structure. The average pruned Root-Mean-Squared-Deviation (RMSD) between the A subunit and theB subunit are 0.155 A and 0.185 A, respectively. Despite the fairly low sequence identity between the two subunits (31%) the intersubunit RMSD is 0.915 A. One difference between the two subunits is a zinc atom chelated by three cysteine residues (C109, C128, C194) in the B subunit of which only C194 is conserved in subunit A. The sulfurs, along with a water ligand, form a tetrahedral geometry around the Zn atom, which is consistent with inductively coupled plasma optical emission spectroscopy (ICP-OES) results that showed a stoichiometry of 1 Zn per heterodimer. Sitting 2.6 A from the water is a Oδ of an aspartate residue (D49), well positionedto deprotonate the bound water. This putative active site sits buried in the B subunit near the interface with one of the neighboring A subunits.

[0071] Nitroguanidine was docked into the putative active site in chain D using SwissDock Vina (Bugnon, et al., 2024, Nucleic Acids Res. 2024, 52 (Wl), W324-W332). The carbon of nitroguanidine is positioned very close to the water ligand. Two asparagine residues (N151 and N153) along with a lysine residue of subunit A (K89) are well positioned to hydrogen bond with the guanidinium moiety.

[0072] Nitroguanidinase is reactive with nitrosoguanidine

[0073] Nitrosoguanidine was synthesized by zinc reduction of nitroguanidine. The structure and purity of nitroguanidine was confirmed by1H-NMR (FIG. 8) and13C-NMR (FIG. 9). The nitroguanidinase was highly reactive with nitrosoguanidine and completely degraded it to ammonia and gases (FIG. 10). Nitrosoguanidine is faintly yellow and has an absorbance at 400 nm. The image labeled “faint yellow” in FIG. 10 is a photo of a solution of the chemical in a cuvette. A spectrophotometric scan of the cuvette shows the absorbance at 400 nm. Upon adding nitroguanidinase enzyme, the faint color decreased, the absorbance band at 400 nm decreased, and copious bubbles were seen arising in the cuvette. Based on known chemistry, the gases are carbon dioxide and nitrogen. Ammonia was demonstrated by enzyme assay. These results show that any mixture of nitroguanidine and nitrosoguanidine will be jointly degraded by nitroguanidinase.

[0074] Over time, nitroguanidine present in soil or water will get converted to nitrosoguanidine, which is highly mutagenic, likely a carcinogen, and therefore more dangerous in the environment than nitroguanidine. Consequently, the ability of nitroguanidinase to degrade both nitroguanidine and nitrosoguanidine means that bioremediation of nitroguanidine using nitroguanidinase also effectively clears nitrosoguanidine.

[0075] Buffers and Enzyme Stabilizers

[0076] Enzyme activity may be enhanced by buffers that control and / or maintain a pH above 7. Exemplary buffers include, but are not limited to, tris(hydroxymethyl)aminomethane (Tris), N-(Hydroxyethyl)piperazine-N'-2-hydroxypropanesulfonic acid (HEPPSO), triethanolamine (TEA), N-(tri(hydroxymethyl)methyl)glycine (Tricine), glycylglycine, N-cyclohexyl-2-aminoethanesulfonic acid (CHES), borate, ethanolamine, triethanolamine, sodium carbonate, N-[Tris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid (TAPS), and / or (2-Amino-2-methyl-1,3-propanediol) (AMPD).

[0077] Stabilizing agents include proteins or other compounds that inhibit denaturation.

[0078] Conventional enzyme stabilizers are known in the art. Exemplary suitable enzyme stabilizers include those conventionally used in detergents, foods, feeds, leather, paper, textiles, pharmaceuticals, and the like. Additional suitable stabilizing agents include, but are not limited to, beta-cyclodextrins, polyols, carboxylic acids, magnesium chloride, calcium chloride, boric acid, proteins (e.g., bovine serum albumin, whey protein, casein, etc.) glycerol, D-mannitol, trehalose dihydrate, L-arginine, Pol oxamer 188, magnesium chloride hexahydrate, sorbitol, sucrose, polyethylenimine, diatamceous earth, a poly amine, and / or a polyethylene glycol.

[0079] Nitrourease

[0080] In the bacterium that grows on nitroguanidine, Pseudomonas sp. strain NGU. the metabolic pathway degrades nitroguanidine to two ammonia molecules, carbon dioxide, and nitrous oxide. The first step of the metabolic pathway is catalyzed by nitroguanidinase that makes ammonia and nitrourea. The second enzyme, nitrourease, converts nitrourea to ammonia, carbon dioxide, and nitrous oxide (FIG. 4). The nitrourease enzyme was purified as illustrated in FIG. 11.

[0081] Thus, in one or more embodiments, the composition can further include a nitrourease. In one or more embodiments, the nitrourease includes the amino acid sequence of SEQ ID NO:5, a fragment thereof effective to convert nitrourea to NH3, N2O, and CO2, or a polypeptide comprising a degree of structural similarity to SEQ ID NO: 5 effective to convert nitrourea to NH3, N2O, and CO2.

[0082] Bioremediation of cyanoguanidine and metformin

[0083] In one or more embodiments, the composition can further include a nitrile hydratase, a metforminase, or both. A composition that includes a nitrile hydratase can be used in bioremediation methods that degrade cyanoguanidine. A composition that includes a metforminase can be used in bioremediation methods that degrade metformin. (FIG. 4).Compositions

[0084] The compositions described herein may be formulated with a carrier. As used herein, “carrier” includes any solvent, dispersion medium, vehicle, coating, diluent, isotonic agent, buffer, carrier solution, suspension, colloid, foam, or the like. The use of such media and / or agents for enzymatically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active enzymes in the composition, its use in the enzymatic compositions is contemplated.

[0085] An enzymatic composition can include one or more supplementary materials. Suitable supplementary materials include materials that are not enzymatically, biologically, or otherwise undesirable, i.e., the material does not cause an unsuitable degree of undesirable chemical or biological effects or interact in a deleterious manner with any of the other components of the enzymatic composition. Exemplary supplementary materials include, but are not limited to, a buffering agent, a desiccant, or structural material. Suitable structural materials include, but are not limited to, a polymer, diatomaceous earth, silica, nanoparticles, metal-organic frameworks, cellulose, starch, alginate, agarose, chitosan, or zeolites. In one or more embodiments, the polymer can be, or can include, polyethylenimine, polyethylene glycol, a polysaccharide, or a block co-polymer. Exemplary desiccants include, but are not limited to, silica gel adsorbent, clays, or calcium sulfate.

[0086] Thus, the enzymatic composition may be provided in any suitable form including but not limited to dry granules, a solution, a suspension, an emulsion, a spray, an aerosol, a foam, a gel, or any form of mixture. A formulation may be prepared by methods well known in the art. Methods of preparing the enzymatic composition can include the step of bringing the enzyme or enzymes of the composition into association with one or more additional ingredients.

[0087] Soil experiments

[0088] To determine whether nitroguanidinase can successfully clear nitroguanidine from soil, a 2-mL solution of various concentrations of nitroguanidine was added to 2 g of soil. First, the solution was incubated in soil without any additives overnight on a rotisserie. HPLC analysis showed no significant loss of nitroguanidine due to adsorption to soil, suggesting that the nitroguanidinase enzyme would have access to the substrate. Next, 2 mg of purified nitroguanidinase was added to the soil. Significant degradation of nitroguanidine occurred withthe concurrent appearance of nitrourea in the low-organic soil after two hours of incubation at room temperature. The nitrourea peak was not formed when 40 pg of nitrourease was included, due to further degradation of nitrourea by nitrourease (FIG. 12, top). When 50 mM MOPS at pH 8.0 was added, nitroguanidine entirely disappeared after two hours of incubation, suggesting that the buffer stimulated the biodegradation.

[0089] Next, soil testing was performed using continuous gas monitoring. The enzymatic degradation of one molecule of nitroguanidine produces one molecule of nitrous oxide (FIG. 12, top). This 1:1 correspondence allows one to monitor nitroguanidine degradation continuously. The level of nitroguanidine and nitrourease enzymes were 40 pg each.

[0090] In the soil experiment, 100 ppm N2O was generated in 15 minutes (FIG. 13). This translated to a specific activity of 1.1 pM of nitroguanidine degraded per minute per milligram of nitroguanidinase enzyme. The specific activity of the linear phase was 1.5 pM of nitroguanidine degraded per minute per milligram of nitroguanidinase enzyme overall.

[0091] Improved Assays for determining Nitroguanidinase and Nitrourease enzyme activities Existing commercial nitroguanidinase assays based on detecting ammonia are cumbersome and expensive (FIG 14A). This disclosure describes alternative assays using surrogate substrates that are faster, more direct, and cheaper. In one embodiment, the assay involves monitoring the nitroguanidinase conversion of 2-nitroethylene- 1,1 -diamine to nitroacetamide (FIG. 14B). The enzymatic conversion can be monitored by observing a shift in absorbance over time in wells. The substrate in the new assay absorbs above 300 nm where the nitroguanidinase enzyme and the reaction product have no absorbance. Thus 2-nitroethylene- 1,1-diamine can be used directly to monitor the activity of the enzyme by measuring its decrease over time.

[0092] This disclosure also describes an alternative assay for the nitrourease enzyme that is faster, more direct, and cheaper. This assay uses nitroacetamide, a structural analog of nitrourea. This also has the added benefit that nitrourea is not widely available commercinally whereas nitroacetamide is. Morever, nitroacetamide is more stable and easy to handle. The assay measures the direct descrease in absorbance of the substrate as it is degraded by the enzyme nitrourease.Bioremediation methods

[0093] In another aspect, this disclosure describes a method of contaminant bioremediation in which the contaminant is nitroguanidine, cyanoguanidine, or metformin. Generally, the method includes contacting the contaminant with an enzyme composition as described herein formulated and in an amount effective to reduce the amount of the contaminant. Thus, the method may include applying the composition to an area of contamination. In one or more embodiments, the area of contamination may be an environmental area such as, but not limited to, soil, water, or wastewater. In one or more embodiments, the area of contamination may be a surface such as, but not limited to, pavement, floor, wall, ceiling, clothing, container (e.g., shipping container), machinery, clothing, protective gear, and the like. In one or more embodiments, the area of contamination may be a spill, manufacturing waste, area of military operation (e.g., testing ranges, unexploded munitions, etc.), and the like.

[0094] In one or more embodiments, the contaminant may be present as a solid or in solution. In one or more embodiments in which the contaminant is present in soil, it may be advantageous that the contaminant is not bound to soil particles, thereby making the contaminant available to the enzyme composition. In embodiments in which the contaminant is nitroguanidine present in solution, the nitroguanidine may be present at a concentration of up to 34 mM. In one or more embodiments in which the contaminant is at least partially in solution, undissolved contaminant may be pulled into solution, by equilibrium, as dissolved material is degraded to end products.

[0095] In one or more embodiments, the composition is applied or otherwise contacted with the contaminant prior to an event capable of washing the contaminant out of an area of contamination. In some cases, such an event may be, for example, a period of rain that may disperse the contaminant out of the area of contamination and / or dilute the contaminant within the area of contamination. In one or more embodiments, contaminated water may be suctioned or flushed from soil and sequestered (e.g., contained within a tank, trough, or other manner of containment) and then treated after having been sequestered. In one or more embodiments, the method can include a step of “soil washing,” either in situ or ex situ, to remove the contaminant from soil by, for example, flushing contaminated soil with a fluid (e.g., water) and then subjecting the rinsate to treatment with the enzyme composition. For example, the method may include flushing water through a soil with a horizontal flow to remove the contaminant from the soil and collect the rinsate that now contains the contaminant. For example, a company mighttreat its wastes in a lined trough located outside of a plant such that water could flow from one end and move horizontally through the trough, go through a filter and be collected outside the trough. That rinsate could then be treated with enzyme to degrade one or more contaminants in the waste.

[0096] In one or more embodiments, the method can include providing the contaminant with a local environment having a pH of pH 7-12, preferably pH 8-9, which is higher than most soils and many environmental water sources. Thus, the method can include adding a safe material to the area in which the enzyme composition is applied to raise the localized pH in the vicinity of the enzyme to achieve a pH of, for example, 8-9. Suitable materials for increasing the pH of the local environment to which the enzyme composition is applied include, but are not limited to, ammonium bicarbonate, an amine, or an organic buffers with high pKa.

[0097] In the preceding description and following claims, the term “and / or” means one or all of the listed elements or a combination of any two or more of the listed elements; the terms “comprises,” “comprising,” and variations thereof are to be construed as open ended — i.e., additional elements or steps are optional and may or may not be present; unless otherwise specified, “a,” “an,” “the,” and “at least one” are used interchangeably and mean one or more than one; and the recitations of numerical ranges by endpoints include all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).

[0098] As used herein, “have,” “has,” “having,” “include,” “includes,” “including,” “comprise,” “comprises,” “comprising” or the like are used in their open-ended inclusive sense, and generally mean “include, but not limited to,” “includes, but not limited to,” or “including, but not limited to.” Further, wherever embodiments are described herein with the language “have,” “has,” “having,” “include,” “includes,” “including,” “comprise,” “comprises,” “comprising” and the like, otherwise analogous embodiments described in terms of “consisting of’ and / or “consisting essentially of’ are also provided. The term “consisting of’ means including, and limited to, that which follows the phrase “consisting of.” That is, “consisting of’ indicates that the listed elements are required or mandatory, and that no other elements may be present. The term “consisting essentially of’ indicates that any elements listed after the phrase are included, and that other elements than those listed may be included provided that those elements do notinterfere with or contribute to the activity or action specified in the disclosure for the listed elements.

[0099] As used herein, the word “exemplary” means to serve as an illustrative example and should not be construed as preferred or advantageous over other embodiments.

[0100] As used herein, the terms “preferred” and “preferably” refer to embodiments of the invention that may afford certain benefits under certain circumstances. However, other embodiments may also be preferred under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful and is not intended to exclude other embodiments from the scope of the invention.

[0101] In the preceding description, particular embodiments may be described in isolation for clarity. Reference throughout this specification to “one embodiment,” “an embodiment,” “certain embodiments,” “one or more embodiments,” or “some embodiments,” etc., means that a particular feature, configuration, composition, or characteristic described in connection with the embodiment is included in at least one embodiment of the disclosure. Thus, the appearances of such phrases in various places throughout this specification are not necessarily referring to the same embodiment of the disclosure. Furthermore, the particular features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more embodiments. Thus, features described in the context of one embodiment may be combined with features described in the context of a different embodiment except where the features are necessarily mutually exclusive.

[0102] In several places throughout the above description, guidance is provided through lists of examples, which examples can be used in various combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list.

[0103] For any method disclosed herein that includes discrete steps, the steps may be performed in any feasible order. And, as appropriate, any combination of two or more steps may be performed simultaneously.

[0104] EXAMPLESThe present invention is illustrated by the following examples. It is to be understood that the particular examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention as set forth herein.

[0105] HPLC Analysis

[0106] Soil experiments were conducted with a sandy loam soil from Becker, Minnesota. 2 g of soil was mixed with 2 mL of solution consisting of 1 mM nitroguanidine, 570 pg / mL nitroguanidinase, 10 pg / mL nitrourease, and 50 mM MOPS-NaOH pH 8.0. This was incubated at room temperature on a rotisserie. After two hours, the samples were centrifuged at 5.8 krcf for 5 minutes. 900 pL of the supernatant was removed and mixed with 100 pL of 1 M HC1. This sample was centrifuged for five minutes at 17 krcf. The supernatant was filtered through a 0.22 μm PTFE filter.

[0107] 10 uL of sample was injected onto a 5 μm 4.6×250 mm reverse phase HPLC column (ECLIPSE plus C18, Agilent Technologies, Inc., Santa Clara, CA) and eluted isocratically with 75% acetonitrile, 1.25 mM potassium phosphate pH 8.0. Elution was monitored at 260 nm.

[0108] In other experiments (FIG. 12), 1.2 g nitroguanidine was added to 30 g of soil with 5 ml 50 mM sodium borate, pH 9.0 to simulate a spill of nitroguanidine into a surface soil. The soil and nitroguanidine mixture was added to a 130 cm3glass vessel with a gas-tight cap and a port on the cap. The gas contents of the container containing the soil mixture was connected to a trace gas analyzer via gas-impermeable tubing (LI-7820, Li-COR Biosciences, Lincoln, NE). The inner volume of the tubing was 17 cm3and the gas volume inside the gas analyzer was 6 cm3. The experiment was run under atmospheric pressure. The ideal gas law was used with the known volumes to calculate the amount of nitrous oxide gas generated during the enzymatic degradation of nitroguanidine. The moles of nitrous oxide gas produced was 1:1 with respect to the nitroguanidine degraded.

[0109] To start the enzymatic degradation, 0.04 mg each of purified nitroguanidinase and nitrourease was added to the soil and the vessel was sealed to allow all of the gas produced to be measured by the LiCOR nitrous oxide gas analyzer. Significant degradation of nitroguanidine occurred as indicated by the 1:1 appearance of nitrous oxide after two hours of incubation at room temperature. The rate of degradation in this experiment was calculated to be 1.1 pmol nitroguandine degraded by mg of each enzyme per minute.The complete disclosure of all patents, patent applications, and publications, and electronically available material (including, for instance, nucleotide sequence submissions in, e.g., GenBank and RefSeq, and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB, and translations from annotated coding regions in GenBank and RefSeq) cited herein are incorporated by reference in their entirety. In the event that any inconsistency exists between the disclosure of the present application and the disclosure(s) of any document incorporated herein by reference, the disclosure of the present application shall govern. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The invention is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the invention defined by the claims.

[0110] Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless otherwise indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0111] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. All numerical values, however, inherently contain a range necessarily resulting from the standard deviation found in their respective testing measurements.

[0112] All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.

[0113] Sequence Listing Free TextSEQ ID NO: 1 - Pseudomonas stutzeri CgdA (urea amidolyase protein UAAP1) MTDSTTLYPV FAEELLPGGG HRSFILKRGE LLRLTDLNGN ANVSLTLLNA HEKTERLNLP DSLKCQHTAK LSNGHCLYSD MGRVLAAIVT DTCGWSDSIG GVLNAQEVAE KYGQGRYQEL RNGFFRNGVD NLLVELGKWG LGLSDLLMTL NLFSRVDVSE TGTLHFAPNN SKAGDYIELY APMDTLVVLT ALQHPMDPNP DYVPQPLTLS WMKADASVAE HCRTSRPENE RGFINTDRLF A

[0114] SEQ ID NO:2 - Subunit A of Pseudomonas sp. NGU gene 1826 protein (nitroguanidinase subunit A)

[0115] MSNPNINKVM TFDDLPPLAR ASVPEHSTAY SPDLTLWKEE LPGGAHWSGL IRRGNTLRIS TLGPKANVSA LFYNFDDRVE RYNMPDTLKA QSTAYLRKGL VCYSDMGRIL CSVADSSVAW HDTIGGITDA VITEQKYGVK RYQEHHNAMH RNAKDGFLTQ LGKWGLGKRD LVPNVNLFSR VSVDDDGNMV LAPDNSTTGD FVDLRFEMNT IVVLSTCPHR LDPSPEYSPA DIQIECWRSG TAGKDDVCRL SMPENGRGFI NTERYYL

[0116] SEQ ID NO: 3 - Pseudomonas stutzeri CgdB (urea amidolyase protein UAAP2) MNSHNHHCCD ATIAAGEPFL AEVKAGQTVR ILDLEGNQAV DTLFFSLANS RERYDVQRTL RRQNSVYLTT GSVLFSNLGR PMLTIIDDTC GRHDTLGGAC AQESNTVRYA LEKRYMHSCR DNYLRACLHD GRLTKADIGP NINFFMNVPV TADGGLTFED GISAPGKYVE LRAEMDVIVL ISNCPQLNNP CNGYNPTPAQ LLIRD

[0117] SEQ ID NO:4 - Subunit A of Pseudomonas sp. NGU gene 1827 protein (nitroguanidinase subunit B

[0118] MNVRIVESQL DPADAIFEEV QPPGTPWMHE VKKGQIIRIV DLGGNQAVDT LFYSAKNPEE RYSLSHTMLA QENIYLGVGT QIMSSEDRPM LTIIADTCGR HDTVGGGACS SESNTVRYAL EKHCMHNCRD NFVMGITNHH SGLEKRDVVS NINFFMNVPM TPQGRLTFED GISGPGKYVE MRAEMDVYIL VSNCPQLNNP ANGYNPTPIQ YFVWETPEQG IEV

[0119] SEQ ID NO: 5 - Pseudomonas putida gene 1841 protein (nitrourease)

[0120] MQNTIEPNIK KLVQKAIKDE LHPLTIVEEV IHRLERQKDN PIFISKVSPE ALRQRAMELE DLPSDEKAKM PLYGVPFAVK DNIDVAGMPT TAGCPEYAYT PKENAFWER LLLAGAILVG KCNLDQFATG LTGMRSVYGS PVNPYSDDHV TGGSSSGSAV AVSSGIVQFS LGTDTAGSGR IPAGFNNIVG YRPSSGLLSN SGVVPCAKSL DSVSVFGRCV DDVRRVLSVA CGFDAADPYS ADIKTSEGNF TSPVFGILDP QIDFFLGDEQ ARSAYIRGIE HLKSLGYRLE VIDYDSFKEI SDMTYFGPSM SERTAVLGNF ISNNPDATYT PVVRDAIANA GDFSAVDAFT MLHRLKFLER KIELEVWSKV DMLCVPTAPT IHTWEVVKQD PGYGSTSLGA YTNFAPFLGI PAISVPNGFR ADSLPTGIMF VGKRANDMRV LEAAEAFSF

Claims

What is claimed is:

1. A composition comprising:a nitroguanidinase enzyme; andan enzyme stabilizer.

2. The composition of claim 1, further comprising a nitrourease.

3. The composition of claim 1 or claim 2, further comprising a nitrile hydratase, a metforminase, or both.

4. The composition of any preceding claim, formulated as a solid.

5. The composition of any preceding claim, formulated as a liquid.

6. The composition of any preceding claim, formulated as a foam.

7. The composition of any preceding claim, wherein the nitroguanidinase enzyme comprises:a first subunit comprising:the amino acid sequence of SEQ ID NO:2,a fragment thereof effective to convert nitroguanidine to nitrourea, or a polypeptide comprising a degree of structurally similarity to SEQ ID NO:2 effective to convert nitroguanidine to nitrourea;a second subunit comprising:the amino acid sequence of SEQ ID NO:4,a fragment thereof effective to convert nitroguanidine to nitrourea, or a polypeptide comprising a degree of structurally similarity to SEQ ID NO:4 effective to convert nitroguanidine to nitrourea; ora heterodimer of the first subunit and the second subunit.

8. The composition of any one of claims 2-7, wherein the nitrourease comprises SEQ ID NO:5 or a fragment thereof effective to convert nitrourea to NH3, N2O, and CO2.

9. The composition of any preceding claim, further comprising a buffering agent, a desiccant, or structural material.

10. The composition of claim 9, wherein the structural material comprises a polymer, diatomaceous earth, or silica.

11. The composition of claim 10, wherein the polymer comprises polyethylenimine.

12. A method of nitroguanidine bioremediation comprising contacting nitroguanidine with the composition of any preceding claim in an amount effective to reduce the amount of nitroguanidine.

13. The method of claim 12, wherein dissolved nitroguanidine is present in solution at a concentration of up to 34 mM.

14. The method of claim 12, wherein at least a portion of the nitroguanidine is present as a solid.

15. The method of any one of claims 12-14, wherein the nitroguanidine is present in soil.

16. The method of claim 15, wherein the composition is contacted with the nitroguanidine prior to an event capable of washing the nitroguanidine out of an area of contamination.

17. The method of claim 15, further comprising washing at least a portion of the nitroguanidine from the soil to form a rinsate comprising nitroguanidine removed from the soil.

18. The method of claim 17, wherein washing the soil occurs in situ.

19. The method of claim 17, wherein washing the soil occurs ex situ.

20. The method of any one of claims 12-19, further comprising adjusting the pH of the vicinity of nitroguanidine to a pH of from 7.0 to 12.0.

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