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Home , Acid hydrolase, Amidase, Barbiturase

USOO8367389B2

(12) United States Patent (10) Patent No.: US 8,367,389 B2 Sadowsky et al. (45) Date of Patent: Feb. 5, 2013

(54) METHODS, COMPOSITIONS AND DEVICES EMBL printout (Morlle thermoacetica ATCC 39073) UTILIZING STRUCTURALLY STABLE Genbank: ABC20412.1 http://www.ncbi.nlm.nih.gov/protein? 83573860 downloaded Sep. 21, 2012.* CYANURIC ACID Cantu et al., “An HPLC Method with UV Detection, pH Control, and Reductive Ascorbic Acid for Cyanuric Acid Analysis in Water'. Anal. (75) Inventors: Michael J. Sadowsky, St. Paul, MN Chem., 72, 5820-5828 (2000). (US); Jennifer L. Seffernick, St. Paul, de Souza et al. "Atrazine Chlorohydrolase from Pseudomonas sp. MN (US); Lawrence P. Wackett, St. ADP: Gene Sequence, Purification and Protein Character ization”. J. Bacteriol., 178, 4894-4900, Supp. p. 695 (1996). Paul, MN (US) Devers et al., “Detection and Organization of Atrazine-degrading (73) Assignee: Regents of the University of Genetic Potential of Seventeen Bacterial Isolates Belonging to Diver gent Taxa Indicate a Recent Common Origin of Their Catabolic Minnesota, Saint Paul, MN (US) Functions”. FEMS Microbiol Lett., 273, 78-86 (2007). Fontaine et al., “A New Type of Glucose Fermentation by (*) Notice: Subject to any disclaimer, the term of this Clostridium Thermoaceticum”. J. Bacteriol. 43, 701-715 (1942). patent is extended or adjusted under 35 Hennig86, “A PC-DOS Program for Phylogenetic Analysis”. U.S.C. 154(b) by 292 days. Cladistics, 5, 163-166 (1989). Karns, J.S., “Gene Sequence and Properties of an S-triazine ring (21) Appl. No.: 12/879,903 cleavage enzyme from Pseudomonas sp. Strain NRRLB-12227”. Appl. Environ. Microbiol. 65, 3512-3517 (1999). (22) Filed: Sep. 10, 2010 Li et al., “Thermostable Cyanuric Acid Hydrolase from Moorella Thermoacetica ATCC 39073”. Applied and Environmental (65) Prior Publication Data Microbiology, vol. 75, No. 22,6986-6991 (2009). Marsilietal. “Microbial Biofilm Voltammetry: Direct Electrochemi US 2011 FO127208A1 Jun. 2, 2011 cal Characterization of Catalytic Electrode-Attached Biofilms'. Appl Environ Microbiol. 74,7329-7337 (2008). Puschner et al., “Assessment of Melamine and Cyanuric Acid in Related U.S. Application Data Cats', J Wet Diagn Invest 19, 616-624 (2007). (60) Provisional application No. 61/241,797, filed on Sep. Seffernicket al., “Hydroxyatrazine N-ethylaminohydrolase (AtzB): 11, 2009. an Superfamily enzyme catalyzing deamination and dechlorinations”. J. Bacteriol., 189, 6989-6997 (2007). Seffernicket al., "Atrazine Chlorohydrolase from Pseudomonas sp. (51) Int. Cl. Strain ADP is a metalloenzyme'. Biochemistry, 41, 14430-14437 CI2N 9/14 (2006.01) (2002). (52) U.S. Cl...... 435/195; 435/212 Shapir et al., “Purification, Range, and Metal Center of (58) Field of Classification Search ...... 435/195, AtzC: the N-isopropylammelide involved in Bacte 435/212 rial Atrazine ”, J. Bacteriol., 184. 5376-5384 (2002). See application file for complete search history. Shapir et al., “TrzN from Arthrobacter Aurescens TC1 is a Amidohydrolase”, J Bacteriol., 188,5859-5864 (2006). (56) References Cited Smith et al., “Cooperative Catabolic Pathways Within an Atrazine degrading Enrichment Culture Isolated from Soil'. FEMS Microbiol U.S. PATENT DOCUMENTS Ecol., 53,265-273 (2005). Thompson et al., “CLUSTALW. Improving the Sensitivity of Pro 4,071.409 A 1/1978 Messing et al. gressive Multiple Sequence Alignment Through Sequence Weight 4,090,919 A 5, 1978 Chibata et al. ing, Position-Specific Gap Penalties and Weight Matrix Choice”. 4,258,133 A 3, 1981 Mirabel et al. 4,532,040 A 7, 1985 Meeks et al. Nucleic Acids Res., 22, 4673-4680 (1994). 4,888,285 A 12/1989 Nishimura et al. Wall der Maarel et al., “Demethylation of 4,935,116 A 6, 1990 LeMire Dimethylsulfoniopropionate to 3-S-methylmercaptopropionate by 5,055, 183 A 10, 1991 Buchan marine sulfate-reducing bacteria'. Appl Environ Microbiol. 62, 5,177,013 A 1/1993 USui et al. 3927-3984 (1996). 5,310,469 A 5/1994 Cunningham et al. Yan-A-X. et al., “Recent Progress on Immobilization of on 5,478,467 A 12/1995 LeMire et al. Molecular Sieves for Reactions in Organic Solvents'. Applied Bio 5,855,777 A 1/1999 Bachand et al. chemistry and Biotechnology, vol. 101(2), 113-130(18) (2002). 5,980,761 A 11/1999 Boissie et al. 5.998, 183 A 12/1999 LeFevre et al. 6,257,242 B1 7/2001 Stavridis * cited by examiner 6,325,929 B1 12/2001 Bassett 6,673,582 B2 1/2004 McTavish Primary Examiner — Susan Hanley 6,905,733 B2 6, 2005 Russell et al. (74) Attorney, Agent, or Firm — Viksnins Harris & Padys 6,987,079 B2 1/2006 Wormsbecher PLLP 2003/0096.383 A1 5, 2003 Shimizu et al. FOREIGN PATENT DOCUMENTS (57) ABSTRACT WO WO 2007/107981 A2 9, 2007 The present invention relates to stable cyanuric acid hydro lase enzymes, compositions, and devices for use in the treat OTHER PUBLICATIONS ment of a liquid, Such as water. The present invention also Soong et al. J. Biol. Chem. (2002) 227(9): 7061-7068.* relates to methods of using these enzymes, compositions and Eaton et al. J. Bacteriol. (1991) 173(3): 1363-1366.* devices for the treatment of a liquid, Such as water. Fruchey et al. Appl. Environ. Microbiol. (2003): 69(6): 3653-3657.* Pierce et al. Environ. Microbiol. (Oct. 2008) 10(10): 2550-2573.* 17 Claims, 5 Drawing Sheets U.S. Patent Feb. 5, 2013 Sheet 1 of 5 US 8,367,389 B2

FG. I. U.S. Patent Feb. 5, 2013 Sheet 2 of 5 US 8,367,389 B2

FIG. 2. Protein tree of cyanuric acid and their close relatives.

Arthrobacter AD25 Pseudomonas sp. strain ADP (AtzO) Bradyrhizobium japonicum USDA 100 Moorella thermoacetica ATCC 39073 Chelatobacter heintzi Pseudomonas sp. strain 12227 (Trzo) Rhodococcus erythropolis JCM 3132 (barbiturase) U.S. Patent Feb. 5, 2013 Sheet 3 of 5 US 8,367,389 B2

FIG. 3. A. Substrates ul-RO 2-, R = H, CH o B. Not Substrates

Barbituric acid derivatives instO 9. 39 2

Pyrimidines H SH N1 n ass

H 1. H H /N2, H NO

S-Triazines U.S. Patent Feb. 5, 2013 Sheet 4 of 5 US 8,367,389 B2

FIG. 4.

120

60 40

2

Temprature(C) U.S. Patent Feb. 5, 2013 Sheet 5 of 5 US 8,367,389 B2

F.G. 5

O 8&la. CO (A) o ()NH map O NH ano- O -( NH2 N-g N - N -4 O O O Uraci Barbitric acid Ureidorraionic acid

Cyarturic acid armidohydrolase O No. NHC2His N (B) ci -K –4.N and was a o S- N annuinea- han-K N 3ry O Atrazine Cyanuric acid Bire Oketo form) US 8,367,389 B2 1. 2 METHODS, COMPOSITIONS AND DEVICES certain embodiments, the CAH has a K value for cyanuric UTILIZING STRUCTURALLY STABLE acid of 25-150 uM. In certain embodiments, the CAH has a CYANURIC ACID HYDROLASE K value for cyanuric acid of 100-130 uM. In certain embodi ments, the CAH has a k value for cyanuric acid of 4.8-76 REFERENCE TO RELATED APPLICATIONS 5 s'. In certain embodiments, the enzyme is thermostable. In certain embodiments, the CAH is thermostable such that the This application claims priority under 35 U.S.C. 119(e) enzyme retains at least 30% (e.g., at least 40%, at least 50%, from U.S. Provisional Application Ser. No. 61/241,797 filed at least 95%, or any other value between 30% and 100%) Sep. 11, 2009, which is incorporated herein by reference. enzymatic activity at a temperature above 25°C., and has 10 activity up to 70° C. In certain embodiments, the enzyme is SEQUENCE LISTING stable when stored at between room temperature and -80°C. (e.g., between 20° C. and -80° C., or any other value in The instant application contains a Sequence Listing which between) for at least 8 weeks. In certain embodiments, the has been submitted in ASCII format via EFS-Web and is enzyme is stable for at least 1 year. In certain embodiments, hereby incorporated by reference in its entirety. Said ASCII 15 the enzyme retains enzymatic activity at a pH of from 5.5 to copy, created on Jan. 19, 2011, is named 09531291.txt and is 10.5. In certain embodiments, the enzyme is from Moorella 13,619 bytes in size. thermoacetica, such as from Moorella thermoacetica ATCC 39073. BACKGROUND OF THE INVENTION In certain embodiments, the CAH enzyme contains at least 19 Lys residues. In certain embodiments, the CAH enzyme S-Triazine compounds have diverse applications as herbi contains at least 40 Arg and/or Lys residues. In certain cides, resins, and disinfectants. The S-triazine herbicides, embodiments, the CAH enzyme contains at least 50 Asp Such as atrazine, help promote high-yields and Sustainability and/or Glu residues. in agricultural crops. In certain embodiments, the CAH enzyme comprises Melamine, or triamino-s-triazine, is a high Volume indus 25 sequence TEGNG(C/G/L)(V/M/A)ND(Y/F)(T/ trial chemical. Melamine-based polymers have outstanding S)R (SEQID NO:15), such as sequence TEGNGCVNDFTR thermosetting properties, are ideal for their use in kitchen (SEQID NO:16). In certain embodiments, the CAH enzyme utensils and plates, and as high-pressure laminates such as comprises amino acid sequence (M/F/L/I)(V/I/M)(M/F/W) Formica, and as whiteboards. Di- and tri-chloroinated isocya SGG (D/E/G/P)G(V/I/L/G/A)(L/I/M/A)(S/TIA/C)PHX(T/I/ nuric acids find widespread application as disinfectants, algi 30 L/S)(V/I/L)(F/I/V) (SEQID NO:17), whereinX is any amino cides, and bactericides. The chlorinated isocyanuric acids are acid, such as sequence FIMSGGEGVMTPHTVF (SEQ ID used in water treatment, in the textile industry as bleaching NO:18). In certain embodiments, the enzyme comprises compounds, and in preventing and curing diseases in hus amino acid sequence TEGNG(C/G/L)(V/M/A)ND(Y/F)(T/ bandry and fisheries. A major use of these compounds is for S)R (SEQID NO:15) and amino acid sequence (M/F/L/I)(V/ Swimming pool chlorination. They have outstanding perfor 35 I/M)(M/F/W)SGG (D/E/G/P)G(V/I/L/G/A)(L/I/M/A)(S/T/ mance for maintaining an elevated, stable, chlorine content A/C)PHX(T/I/L/S)(V/I/L)(F/I/V) (SEQID NO:17), wherein by dissolving slowly in water, allowing a continuous metered X is any amino acid. In certain embodiments, the enzyme dosing of chlorine. comprises amino acid sequence TEGNGCVNDFTR (SEQ Degradation of these and other S-triazine compounds ID NO:16) and amino acid sequence FIMSGGEGVMTPH results in the production of cyanuric acid (FIG. 1). Cyanuric 40 TVF (SEQID NO:18). acid has come under increased scrutiny because of its poten In certain embodiments, the enzyme is 350-380 amino tial involvement in co-mediating toxicity resulting from the acids in length. In certain embodiments, the enzyme has a pl ingestion of melamine (Puschner, B., et al., 2007.J Wet Diagn of about 5-6. Invest 19:616–24). Recently, melamine has been found in In certain embodiments, the CAH is SEQID NO:3: adulterated pet food and baby formula. Melamine and its 45 metabolite cyanuric acid co-crystallize at low concentrations and are implicated in acute renal failure in cats that have MOKVEVFRIPTASPDDISGLATLIDSGKINPAEIVAILGKTEGNGCVND consumed adulterated food products (Puschner, B., et al., 2007.J Wet Diagn Invest 19:616–24). Cyanuric acid degrada FTRGFATOSLAMYLAEKLGISREEVVKKVAFIMSGGTEGVMTPHITVFW tion is also of interest from the perspective of environmental 50 remediation. The use of di- or tri-chloroisocyanuric acid in RKDVOEPAKPGKRLAVGVAFTRDFLPEELGRMEOWNEVARAVKEAMKDA pool water results in spontaneous chemical dechlorination QIDDPRDVHFVOIKCPLLTAERIEDAKRRGKDVVVNDTYKSMAYSRGAS that disinfects the water, but also produces as a by- large amounts of cyanuric acid. High levels of cyanuric acid ALGWALALGEISADKISNEAICHDWNLYSSWASTSAGWELLNDEIIWWG perturb the equilibrium of dissolution of chloro-cyanuric 55 NSTNSASDLWIGHSWMKDAIDADAWRAALKDAGLKFDCCPPAEELAKIW acids preventing dechlorination by additional chlorinated iso cyanuric acid. If this happens, disinfection is not achieved. As NWLAKAEAASSGTWRGRRNTMLDDSDINHTRSARAWWNAWIASWWGDPM a result, Swimming pools must be emptied and refilled, using water and causing discharge issues. It would be desirable to WYVSGGAEHOGPDGGGPIAVIARV. remediate pool water in situ, maintaining disinfection ability, 60 In certain embodiments, the CAH has an SDS-PAGE pro conserving water, saving money and extending pool water tein band corresponding to about 40 kDa. In certain embodi SC. ments, the CAH has a specific activity of 12-18 umol/min/mg with cyanuric acid as Substrate and a specific activity of less SUMMARY OF THE INVENTION than 2 Limol/min/mg with as Substrate. In 65 certain embodiments, the CAH has a specific activity of 15.7 The present invention provides an isolated or purified umol/min/mg with cyanuric acid as Substrate, but does not structurally stable cyanuric acid hydrolase (CAH) enzyme. In show detectable activity with barbituric acid. In certain US 8,367,389 B2 3 4 embodiments, the CAH enzyme shows less that 10% detect is plastic, nylon, activated carbon, cellulose, agarose, chitin, able activity with barbituric acid as compared to the enzyme’s chitosan, collagen and/or polystyrene. In certain embodi activity with cyanuric acid. ments, the matrix is an inorganic matrix and the inorganic In certain embodiments, the CAH enzyme contains at least matrix is glass, Zeolite, silica, alumina, titania, Zirconia, cal 19 Lys residues. In certain embodiments, the CAH enzyme cium alginate and/or celite. In certain embodiments, the contains at least 40 Arg and/or Lys residues. In certain device further comprises a permeable layer. In certain embodiments, the CAH enzyme contains at least 50 Asp embodiments, the enzyme is imbedded in or on the permeable and/or Glu residues. layer. In certain embodiments, the device further comprises at The present invention provides an isolated or purified least one casing, wherein water flowing through the at least nucleic acid molecule comprising SEQID NO: 4: 10 one casing contacts the enzyme. The present invention provides methods of remediating a liquid comprising contacting the liquid from a circulating CTACACCCTGGCAATAACAGCAATTGGGCCACCGCCATCAGGCCCTTGA reservoir with the enzyme, composition, or the device described herein above to reduce the concentration of cyanu TGCTCTGCACCACCGGAAACGTAGACCATAGGATCTCCTACCACGCTGG 15 ric acid in the liquid. In certain embodiments, the enzyme is CAATAACAGCATTTACTACTGCTCGCGCCGAGCGGGTATGATTGATATC present in pellet form. In certain embodiments, the liquid is contacted with the device described hereinabove by passing AGAGTCATCAAGCATCGTGTTACGCCTACCCCTTACTGTACCAGAAGAT the water over or through the device. In certain embodiments, the circulating liquid reservoir is a pool, a Swimming pool, a GCGGCCTCAGCCTTGGCCAGTACATTAACGATCTTAGCAAGCTCTTCTG spa, a hot tub, a whirlpool bath, a fountain or a waterslide. In CTGGCGGGCAACAATCAAATTTTAAACCGGCATCTTTAAGGGCAGCACG certain embodiments, the concentration of cyanuric acid in the liquid Subsequent to treatment is less than 100 ppm. In TACTGCATCAGCGTCAATGGCATCCTTCATAACAGAGTGGCCTATAACC certain embodiments, the concentration of cyanuric acid in AAATCACTGGCACTATTGGTAGAGTTTCCTACTACGATAATTTCGTCAT the liquid subsequent to treatment ranges from 70 ppm to 30 25 ppm. In certain embodiments, the enzyme is present at a TAAGAAGTTCAACCCCCGCTGACGTCGAAGCCACACTAGAGTAGAGATT concentration of at least 2.5 mg per one cubic meter of the liquid. In certain embodiments, the liquid flows through the CCAGTCATGACAAATTGCTTCGTTGCTAATCTTATCCGCAGATATCTCG device. In certain embodiments, the liquid treatment com CCCAGTGCGAGGGCCACTCCGAGAGCTGAGGCGCCACGTGAGTAAGCCA prises reducing a concentration of cyanuric acid in the liquid. 30 In certain embodiments, the liquid treatment is effected dur TTGATTTATAAGTGTCATTTACCACAACATCTTTCCCGCGTCGCTTGGC ing a time period of 20 hours or less. In certain embodiments, ATCCTCAATTCTTTCAGCAGTCAAAAGCGGGCACTTTATCTGAACAAAG the passing of the liquid through the device is effected at a flow rate of at least 10 cubic meters per hour. In certain TGAACGTCGCGGGGATCATCTATTTGGGCGTCTTTCATAGCCTCTTTTA embodiments, the liquid is water. 35 CAGCTCGAGCCACTTCGTTTACCTGTTCCATCCGGCCCAATTCTTCCGG BRIEF DESCRIPTION OF DRAWINGS CAGAAAGTCCCGCGTAAAAGCTACGCCTACTGCCAAGCGCTTTCCTGGC FIG. 1. Atrazine, ametryn, trichloroisocyanuric acid and TTAGCTGGTTCCTGGACATCTTTTCGGACAAAGACAGTAATGTGCGGCG melamine are all metabolized via cyanuric acid that is trans 40 TCATAACACCCTCAGTACCGCCTGACATTATAAACGCAACTTTTTTTAC formed to biuret by the action of cyanuric acid hydrolases. FIG. 2. Tree showing relatedness of cyanuric acid hydro AACTTCTTCGCGGCTTATTCCCAATTTTTCTGCTAGATACATTGCTAGA lases known or identified in different bacteria. The degree of relatedness shown is based on amino acid sequence identity. GATTGGGTAGCAAAACCGCGAGTAAAATCGTTAACACAACCATTACCTT FIG. 3. Substrate specificity of the cyanuric acid hydrolase CCGTCTTGCCCAGAATAGCTACAATTTCAGCCGGATTAATCTTCCCTGA 45 from M. thermoacetica ATCC 39073. (A) Compounds that are substrates. (B) Compounds that are not substrates. GTCAATCAAAGTAGCCAACCCGCTGATATCATCAGGTGAGGCTGTTGGG FIG. 4. Stability of enzyme activity vs. temperature at pH ATACGAAAGACTTCAACTTTTTGCAT. 8.0 for purified cyanuric acid hydrolases from M. thermoace tica ATCC 39073 (O) compared with Trz|D () and Atz) The present invention provides a composition for remedia 50 (A). Enzymes were maintained at the temperature indicated tion of a liquid comprising the CAH as described above, along for 30 minutes prior to assay under Standard conditions. with polyethylene glycol (PEG) and/or KC1. In certain FIG. 5. Comparative reaction and metabolic pathways for embodiments, the PEG is PEG4000. In certain embodiments, barbiturase (A) and cyanuric hydrolase (B) (Soong, C. L., J. the PEG is present at a concentration of 50-500 mM. In Ogawa, E. Sakuradani, and S. Shimizu. 2002. Barbiturase, a certain embodiments, the composition comprises 50-500 mM 55 novel zinc-containing amidohydrolase involved in oxidative KC1. In certain embodiments, the liquid is water. pyrimidine metabolism. J. Biol. Chem. 277:7051-7058). The present invention provides a device for remediation of a liquid comprising a matrix and one or more structurally DETAILED DESCRIPTION OF THE INVENTION stable cyanuric acid hydrolases or a composition for reme diation of a liquid as described above. In certain embodi 60 Hypochlorous acid (HOCl) is a common source of free ments, the liquid is water. In certain embodiments, the device chlorine. Chemical compounds that release free chlorine are further comprises a casing or housing for the matrix. In cer the most commonly used sanitizers in Swimming pools. tain embodiments, the matrix is water-insoluble. In certain Hypochlorous acid, however, is highly unstable, and readily embodiments, the water-insoluble matrix is granular and/or decomposes into inactive breakdown products, such as porous. In certain embodiments, the water-insoluble matrix is 65 hydrochloric acid, water and oxygen, via UV radiation-driven an organic matrix or an inorganic matrix. In certain embodi photochemical reactions upon exposure to direct Sun light, ments, the matrix is an organic matrix and the organic matrix and/or upon exposure to moderate and high temperatures. On US 8,367,389 B2 5 6 a typical summer day, up to 90% of the total active chlorine invention. The parameters of cyanuric acid hydro species are lost within two to three hours. In order to control lase on cyanuric acid, namely K values of 25-125 M as these effects and preserve the effectiveness of the chlorine, presented herein, signify that these enzymes can be used the chlorine-stabilizing agent cyanuric acid (also called S-tri effectively to reduce the concentration of cyanuric acid in the azinetrione or isocyanuric acid) is often added to the water. liquid, Such as water, so as to achieve a concentration lower Cyanuric acid, as well as cyanurate salts and various deriva than the chlorine-lock concentration of 100 ppm (correspond tives thereofare compounds which protect the chlorine from ing to 0.77 mM). Even at the highest allowable concentration the negative effects of UV and heat, and therefore practically of cyanuric acid in Such water, 0.62 mM, the enzyme is highly reduce the amount of chlorine which needs to be added to the effective and can produce the desired hydrolysis. The cyanu water in order to maintain safe conditions of disinfection. The 10 ric acid hydrolase of the present invention has k values for protection action of these compounds is achieved by the abil cyanuric acid of 4.8-76 s'. ity of free chlorine (Cl) to reversibly bind to the The present inventors discovered that the number of Lys atoms in the cyanuric acid ring. With a correct dosing, cya residues is considerably higher in Moorella as compared to nuric acid can reduce the chlorine consumption. However, other enzymes with similar structure and function. Atz) had incorrect balance of cyanuric acid can create an over-protec 15 7, Trz) had 12, but Moorella CAH had 21. Many of these are tive effect and hence substantially decrease the effectiveness conservative changes, replacing the lower numbers of Arg's, of chlorine as a disinfectant. but some are not. The inventors also looked at acidic and basic Excessive amounts of cyanuric acid drive the equilibrium residues as a potential metric for salt bridges. towards the uptake of free chlorine. Hence, excessive Arg+Lys=32 Atz)/36Trz D/41 Moorella (due mainly to the amounts of cyanuric acid cause the chlorine to become pro Lys as mentioned above) gressively over-stabilized, reducing the availability of free Asp--Glu-43 atZd/50 trzd/50 Moorella chlorine and interfering with its disinfection function. The The global amino acid composition of these various pro phenomenon, known as "chlorine-lock, takes place when teins was also examined. The CAH from Bradyrhizobium the concentration of cyanuric acid reaches over 100 ppm USDA 110 was also compared as a control for random devia (0.77 mM). Chlorine-lock expresses itself similarly to inad 25 tions. The relative stability for these proteins is as follows: equately low chlorine level, in clouding of the pools water Bradyrhizobium CAH (loses activity in weeks)

Among the additives were 10% ethylene glycol, 0.2 mM and 0.05 molar metal to subunit stoichiometries, respectively. DTT, 25% glycerol, and 25% PEG4000. The enzymatic activ These results indicated that no catalytically-significant met ity was monitored every 2 weeks for a total of 8 weeks. The als were present in the enzyme preparation having a ki/K, enzyme without additives showed no loss of activity after 8 of 1.0 10 M's', a significant catalytic rate. Similar results weeks at both storage temperatures. This suggested that the 25 were obtained with Atz) (Fruchey, I., et al., 2003. Appl enzyme was stable at low temperatures. Though the enzy Environ Microbiol. 69:3653-7). matic activity was unchanged in the frozen sample after 8 Discussion weeks, the specific activity of the sample stored at 4° C. The cyanuric acid hydrolase enzymes Atz) from increased to 142% and 13.6% of initial specific activity com Pseudomonas sp. strain ADP (Fruchey, I., et al., 2003. Appl pared to the sample without additives and 0.2 mM DTT, 30 Environ Microbiol. 69:3653-7) and Trz) from Pseudomonas respectively. The addition of 25% PEG 4000 had a stimula sp. strain NRRLB-12227 (Smith, D., S. Alvey, and D. E. tory affect upon activity prior to storage, increasing activity Crowley. 2005. FEMS Microbiol Ecol. 53:265-73) have been by 270%. However, activity for the PEG-stimulated enzyme purified and characterized. These enzymes were identified in Subsequently decreased to around 1.4-1.6 times initial activ organisms isolated for their ability to degrade atrazine and ity without additives over the 8 week time course at both temperatures. All other additives, including 10% ethylene 35 melamine, respectively. Both organisms mineralize the com glycol and 25% glycerol, had little or no positive effect on pounds via metabolic pathways that proceed through cyanu Sustaining activity. ric acid as an intermediate. Cyanuric acid also forms abioti Effect of metal ions and chelators. The affects of divalent cally via the spontaneous decomposition of chlorinated metals upon native enzyme activity were monitored (Table 2). isocyanuric acids (Cantu, R., et al., 2000. Anal. Chem. 40 72:5820-8). The breakdown is an intended process as it serves TABLE 2 to disinfect pools by slowly generating hypochlorite. After a time, however, cyanuric acid accumulates, rendering further Effect of metal ions on the activity of purified cyanuric chlorination ineffective. Various methods to remove cyanuric acid hydrolase from M. thermoacetica 45 acid chemically have been tried, but none have proven com Metal Concentration (mM) Relative activity mercially successful to date. To deal with this issue, enzy CoCl2 O.2 925 matic treatment of cyanuric acid in pool water has been con MnSO O.2 96.3 sidered. One impediment to this application has been the ZnSO O.2 514 relative instability of the cyanuric acid hydrolases known to CuCl2 O.2 833 50 date. FeCl O.2 1054 NiCl, O.2 793 The known cyanuric acid hydrolases, Atz) and Trzl), have CaCl2 1.O 1217 been observed to lose activity during freezing, a typical MgCl2 2.0 119 + 2 method used to maintain enzyme stability and deter microbial Values are the mean+ standard error of three replicates. and proteolytic breakdown of proteins. The goal of this study 55 was to identify a more stable cyanuric acid hydrolase enzyme Zn(II) had the greatest inhibitory affect, with slight for use in bioremediation applications. In this context, the decreases in activity observed in the presence of Cu(II) and inventors identified an Atz)/TrzD homolog from M. ther Ni(II). At higher concentrations, Ca(II) and Mg(II) caused moacetica ATCC 39073. This is the first cyanuric acid hydro slight increases to 121% and 119% of native activity, respec lase purified from an organism not previously shown to tively. Chelators, including EDTA, o-phenanthroline, and 60 degrade S-triazine compounds. Genome context gives little 8-hydroxyquinoline-5-sulfonic acid, even with 24h incuba indication of a native function for this gene. Upstream of tions, failed to alteractivity. These data Suggested that either Moth 2120 are genes that encode for a CdaR transcriptional a metal could not be removed or that metals were not neces regulator, two hypothetical proteins, an FdrA protein impli sary for catalytic activity. cated in regulating diverse cellular processes through FtsH. Quantitation of bound metal. The metal content of the 65 and another hypothetical protein. Downstream there is a enzyme was analyzed by ICP. The only metals detected above hypothetical protein, uracil-Xanthine permease, carbamate background were Zinc and nickel which were present at 0.08 kinase, and methyltransferase genes. US 8,367 389 B2 29 30 The Atz) gene homolog from M. thermoacetica was Because of the lack of sequence and evolutionary links to cloned, the recombinant enzyme was purified. Characteristics other well-characterized enzymes, this family must of the enzyme were studied with respect to catalysis and be considered independently. Consistent with this, a metal stability. Table 3 compares the properties of this new enzyme was not necessary for catalysis by the M. thermoaceticum with those of Atz) and Trz). cyanuric acid hydrolase. Previously, active Atz) was found TABLE 3 Comparison of Cyanuric Acid Hydrolases

Properties Moorelia Trz) Atz) Mol. wt. (calc.) (kDa) 38.9 39.4 38.2 pH optimum 8.0 8.0-8.5 (8) 8.2(7) Temp, optimum (C.) 70 45-50(8) 30(6) Thermostability (C.) 20-65 20-45 20-40 k(s) 10.6 250(8) 6.8 + 0.7(7) Ka (M) 110 50(8) 57 + 10(7) k/K (s'M') 1.0 10 5 106 1.210 Known substrates Cyanuric acid, N Cyanuric acid (8) Cyanuric acid, N methylisocyanuric methylisocyanuric acid acid (7) Metal content None n.d. None(7) Metal affects on Cu(II), Zn(I), Ni(II) Mg(II), Mn(II) no affect No stimulatory activity inhibitory at 0.2 mM: 1 mM: Co(II), Cu(II), affects with Zn(II), Ca(II), Mg(II) slight Fe(II) slight inhibitory Cu(II), Fe(II), Co(II), increase at 1.0 and 1 mM, Zn(II) 1 mM or Ni(II)(7) 2.0 mM, respectively greatly inhibitory (8) Influence of chelators No affect EDTA, o No affect EDTA(8) No affect EDTA and on activity phenanthroline, or 8 o-phenanthroline hydroxyquinoline-5- Sulfonic acid Temperature where activity above 50% after a 30 min incubation. n.d. = not determined 30 The cyanuric acid hydrolase from M. thermoacetica ATCC not to contain significant levels of metals (Fruchey, I., N. et 39073 was shown to have cyanuric acid hydrolase activity, al., 2003. Appl Environ Microbiol. 69:3653-7). with K, and k values for cyanuric acid of 110 uM and 10.6 In conclusion, the cyanuric acid hydrolase from M. ther s', respectively. The enzyme displayed a high degree of 35 moacetica ATCC 39073 is not a metalloenzyme. Further Substrate discrimination, only catalyzing reactions with cya more, it is an outstanding thermophilic enzyme that is stable nuric acid and its close structural analog N-methylisocyanu at high or low temperatures. ric acid, but with no other analogous structure. This mirrors the restricted reactivity found with Atz D (Fruchey, I., et al., EXAMPLE 2 2003. Appl Environ Microbiol. 69:3653-7) and indicates that, 40 although the sequences of the cyanuric acid hydrolases are Defining the Cyanuric Acid Hydrolase/Barbiturase only 57-64% identical, the proteins share a similar substrate Protein Family range. The key difference between the M. thermoacetica cya nuric acid hydrolase and Atz)/TrzD is in the greater stability As sequence databases grown and alignment methods of the former enzyme. The new enzyme had its highest cata 45 become more Sophisticated, many protein Superfamilies have lytic activity at 70° C. (the highest temperature that our assays swelled to include tens or hundreds of thousands of members. were able to maintain), was stable when stored for 30 min at Dozens of those members typically have available X-ray 50° C., and was able to be stored frozen for long periods of structures. This provides structure and function information time. In combination, these properties make this a Superior that newly discovered members of the Superfamily can be enzyme for cyanuric acid remediation. 50 anchored to and thus provide immediate clues as its structure. A notable exception to this paradigm is the Small cluster of Barbiturase, a cyanuric acid homolog, has been proposed related sequences that belong to proteins known to be, or to be a member of the amidohydrolase Superfamily (Soong, annotated as, cyanuric or barbituric acid hydrolases. These C. L., et al., 2002. J Biol Chem. 277:7051-8). In general, related proteins catalyze the opening of structurally analo amidohydrolase Superfamily members contain one or two 55 gous nitrogen heterocyclic rings (FIG. 1). The barbituric acid metal atoms per subunit, with Zinc being the most commonly hydrolases, also known as barbiturases, react with a pyrimi identified metal. The enzymes AtZA, AtzB, AtzC and TrzN. dine ring and carry out recycling of cytosine and uracil. The which are in the pathway of atrazine degradation, are all cyanuric acid hydrolases have been largely identified in bac metalloenzymes and members of this superfamily (Seffer teria that catabolize anthropogenic S-triazine ring compounds nick, J. L., et al., 2007. J Bacteriol. 189:6989-97: Seffernick, 60 Such as the herbicide atrazine, or the monomer melamine. The J. L., et al., 2002. Biochemistry 41: 14430-7: Shapir, N., et al., barbiturases and cyanuric acid hydrolases characterized to 2002. J. Bacteriol. 184:5376-84; Shapir, N., et al., 2006. J date do not show cross-reactivity with each other substrates: Bacteriol. 188:5859-64). However, sequence analysis of the they are each quite specific. Atz)/TrzD/barbiturase family of proteins revealed that they In pairwise comparisons, the barbiturases and cyanuric are not members of this common Superfamily. Instead, these 65 acid hydrolases generally show 40-60% relatedness to each enzymes are very distant from any other proteins of known other. There are annotated proteins that show approximately function, and cluster as an isolated island in sequence space. 50% sequence relatedness to each of a known cyanuric acid US 8,367,389 B2 31 32 hydrolase and a known barbiturase. Thus, it can be difficult to TABLE 4-continued discern from sequence alone if a protein is a cyanuric acid hydrolase or a barbiturase. It also is possible that some of the Kinetic Properties of Cyanuric Acid Hydrolases annotated proteins catalyze a different reaction altogether. kcar K. kca?Kn In light of these issues, the current study sought to further Organism (s) (IM) (s-M-1) define the cyanuric acid/barbiturase protein family. The total Pseudomonas sp. ADP-AtzD 6.8 O.7 57 - 10 1.2 10 membership in this family to-date was defined. Bioinformat (literature) ics initially delineated each protein as a cyanuric acid hydro Pseudomonas sp. ADP-AtzD 73 6 237 3.2 106 lase, barbiturase, or an unknown function protein. Several (current study) 10 Moorelia thermoacetica 10.6 110 1.0 10 proteins from diverse were purified to homogeneity and most ATCC 39073 (literature) were shown to have cyanuric acid hydrolase activity and were Bradyrhizobium japonicum USDA 9.3 - 0.7 SO 10 1.9 10 not active with barbituric acid. One protein was a barbiturase. 110 Another protein was observed to be unreactive with any sub Rhizobium leguminosarum 5 - 1 130 60 3.8 10 strate tested. A previous study had suggested that barbiturase bv. viciae 3841 was a member of the amidohydrolase Superfamily and thus 15 Methyliobacterium sp. 4-46 17 - 2 69 16 2.5 10s was a metalloenzyme. The present study finds no sequence Note: relatedness to the amidohydrolase Superfamily and no evi All enzymes in the current study were stored at 4°C. and never frozen. Stability was dence for the participation of metals in catalysis by any mem monitored to ensure no deactivation occurred throughout the study, ber of the cyanuric acid/barbiturase family of proteins. Although the foregoing specification and examples fully Materials and Methods disclose and enable the present invention, they are not Spectrophotometric assay for cyanuric acid degradation. intended to limit the scope of the invention, which is defined The rate of cyanuric acid hydrolysis catalyzed by pure or by the claims appended hereto. partially purified enzyme preparation was determined by All publications, patents and patent applications are incor monitoring the disappearance of cyanuric at 220 nm over porated herein by reference. While in the foregoing specifi time. The spectrophotometer was blanked with 1 ml of 25 25 cation this invention has been described in relation to certain mM Tris-HCl, pH 8.0. Enzyme was added to create an absor bance of 0.05 to 0.4, and reblanked. To this solution, 1 ul of embodiments thereof, and many details have been set forth 0.15 M cyanuric acid was added. The observed extinction for purposes of illustration, it will be apparent to those skilled coefficient for cyanuric acid in this range is 5.654 A220 in the art that the invention is susceptible to additional nm/mM. When 25 mM Tris-HCl, pH 8.25 was used, the 30 embodiments and that certain of the details described herein extinction coefficient for cyanuric acid was 5.848 A220 may be varied considerably without departing from the basic nm/mM. The absorbance of cyanuric acid was monitored principles of the invention. over time and used to calculate the activity of the enzyme. The use of the terms 'a' and “an and “the' and similar Preparation of soluble crude extract. E. coli was grown referents in the context of describing the invention are to be overnight at 30°C. with shaking at 225 rpm in 1 LLB broth 35 construed to cover both the singular and the plural, unless containing 50 ug/ml amplicillin. Isopropyl-1-thio-B-D-galac otherwise indicated herein or clearly contradicted by context. toside (IPTG) was added to a final concentration of 1 mM The terms “comprising.” “having,” “including,” and “con when the culture reached OD600 nm 0.5. Cells were har taining are to be construed as open-ended terms (i.e., mean vested by centrifugation at 5000xg for 15 minutes at 4°C., ing “including, but not limited to') unless otherwise noted. washed with 500 ml sterile PBS, and resuspended in 40 ml 25 40 Recitation of ranges of values herein are merely intended to mM MOPS, pH 7.0. The cells were lysed by one passage serve as a shorthand method of referring individually to each through a cold French pressure cell at 18,000 lb/in2. The separate value falling within the range, unless otherwise indi extract was clarified initially by centrifugation at 13,000xg cated herein, and each separate value is incorporated into the for 15 minutes at 4° C., followed by centrifugation at specification as if it were individually recited herein. All 25,000xg for 90 minutes at 4°C. This supernatant was des 45 methods described herein can be performed in any suitable ignated the crude, cell-free soluble enzyme fraction and was order unless otherwise indicated herein or otherwise clearly used for further enzyme purification. contradicted by context. The use of any and all examples, or Results exemplary language (e.g., “Such as”) provided herein, is The subunit sizes of the different cyanuric acid/barbiturase intended merely to better illuminate the invention and does homologs were typically 350-380 amino acids with a molecu 50 not pose a limitation on the scope of the invention unless lar weight of approximately 40,000. The polypeptides are otherwise claimed. No language in the specification should be generally acidic with p values in the range of 5-6. construed as indicating any non-claimed element as essential The k and K values for the cyanuric acid hydrolases are to the practice of the invention. within an order of magnitude of each other. K ranges from Embodiments of this invention are described herein, 5-73 s and the K, from 19-58 uM. These givek/K, in the 55 including the best mode known to the inventors for carrying range of 10 to 10°. These values suggest that cyanuric acid out the invention. Variations of those embodiments may hydrolysis is the true physiological function of these become apparent to those of ordinary skill in the art upon enzymes. reading the foregoing description. The inventors expect skilled artisans to employ Such variations as appropriate, and TABLE 4 60 the inventors intend for the invention to be practiced other Kinetic Properties of Cyanuric Acid Hydrolases wise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the kcar K. kca?Kn Subject matter recited in the claims appended hereto as per Organism (s) (IM) (s-M-1) mitted by applicable law. Moreover, any combination of the Trz D (literature) 250 50 5 106 65 above-described elements in all possible variations thereof is Trz D (current study) 14.2 O.4 587 2.5 10 encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context. US 8,367,389 B2 33

SEQUENCE LISTING

<16O is NUMBER OF SEO ID NOS : 18

<210s, SEQ ID NO 1 &211s LENGTH: 31 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic prlmer

<4 OOs, SEQUENCE: 1 gcqaatticca tatgcaaaaa gttgaagttct t

<210s, SEQ ID NO 2 &211s LENGTH: 29 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic prlmer

<4 OOs, SEQUENCE: 2 gccaagctitc tacaccctgg caataa.ca.g

<210s, SEQ ID NO 3 &211s LENGTH: 367 212. TYPE: PRT <213> ORGANISM: Moorella thermoacetica

<4 OOs, SEQUENCE: 3 Met Glin Llys Val Glu Val Phe Arg Ile Pro Thr Ala Ser Pro Asp Asp 1. 5 1O 15 Ile Ser Gly Lieu Ala Thr Lieu. Ile Asp Ser Gly Lys Ile ASn Pro Ala 2O 25 3O Glu Ile Val Ala Ile Lieu. Gly Lys Thr Glu Gly Asn Gly Cys Val Asn 35 4 O 45 Asp Phe Thr Arg Gly Phe Ala Thr Glin Ser Leu Ala Met Tyr Lieu Ala SO 55 6 O Glu Lys Lieu. Gly Ile Ser Arg Glu Glu Val Val Llys Llys Val Ala Phe 65 70 7s 8O Ile Met Ser Gly Gly Thr Glu Gly Val Met Thr Pro His Ile Thr Val

Phe Val Arg Lys Asp Val Glin Glu Pro Ala Lys Pro Gly Lys Arg Lieu. 1OO 105 11 O Ala Val Gly Val Ala Phe Thr Arg Asp Phe Lieu Pro Glu Glu Lieu. Gly 115 12 O 125 Arg Met Glu Glin Val Asn. Glu Val Ala Arg Ala Wall Lys Glu Ala Met 13 O 135 14 O Lys Asp Ala Glin Ile Asp Asp Pro Arg Asp Wal His Phe Val Glin Ile 145 150 155 160 Lys Cys Pro Lieu. Lieu. Thir Ala Glu Arg Ile Glu Asp Ala Lys Arg Arg 1.65 17O 17s Gly Lys Asp Val Val Val Asn Asp Thr Tyr Lys Ser Met Ala Tyr Ser 18O 185 19 O Arg Gly Ala Ser Ala Lieu. Gly Val Ala Lieu Ala Lieu. Gly Glu Ile Ser 195 2OO 2O5 Ala Asp Llys Ile Ser Asn. Glu Ala Ile Cys His Asp Trp Asn Lieu. Tyr 21 O 215 22O US 8,367,389 B2 35 36 - Continued Ser Ser Val Ala Ser Thr Ser Ala Gly Val Glu Lieu. Lieu. Asn Asp Glu 225 23 O 235 24 O Ile Ile Val Val Gly Asn. Ser Thir Asn. Ser Ala Ser Asp Lieu Val Ile 245 250 255 Gly His Ser Val Met Lys Asp Ala Ile Asp Ala Asp Ala Val Arg Ala 26 O 265 27 O Ala Lieu Lys Asp Ala Gly Lieu Lys Phe Asp Cys Cys Pro Pro Ala Glu 27s 28O 285 Glu Lieu Ala Lys Ile Val Asn Val Lieu Ala Lys Ala Glu Ala Ala Ser 29 O 295 3 OO Ser Gly Thr Val Arg Gly Arg Arg Asn. Thir Met Lieu. Asp Asp Ser Asp 3. OS 310 315 32O Ile Asn His Thr Arg Ser Ala Arg Ala Val Val Asn Ala Val Ile Ala 3.25 330 335 Ser Val Val Gly Asp Pro Met Val Tyr Val Ser Gly Gly Ala Glu. His 34 O 345 35. O Glin Gly Pro Asp Gly Gly Gly Pro Ile Ala Val Ile Ala Arg Val 355 360 365

<210s, SEQ ID NO 4 &211s LENGTH: 1104 &212s. TYPE: DNA <213> ORGANISM: Moorella thermoacetica

<4 OOs, SEQUENCE: 4 ctacac cctd gcaataacag caattgggcc accoccatca gg.ccc.ttgat gctctgcacc 6 O accoggaaacg tag accatag gat ct cotac cacgctggca ataac agcat titact actgc 12 O tcgc.gc.cgag cqgg tatgat tatat caga gtcatcaagc atcgtgttac gcc tacic cct 18O tact.gtacca gaagatgcgg cct cagoctt ggc.cagtaca ttaacgatct tagcaa.gctic 24 O ttctgctggc gggcaacaat caaattittaa accggcatct tta agggcag cacgtactgc 3OO atcagogt ca atggcatcct t catalacaga gtggcctata accaaat cac togg cactatt 360 ggtagagttt cotactacga taattitcgt.c attaagaagt toaac coccg citgacgt.cga 42O agccacacta gagtagagat tccagt catg acaaattgct tcgttgctaa tottatcc.gc 48O agat at Ct c cccagtgcga gggcc actico gagagctgag gcgc.cacgtgagtaa.gc.cat 54 O tgatttataa gtgtcattta ccaca acatc titt cocqcqt cqcttggcat cottcaattct 6OO ttcagcagtic aaaag.cgggc actittatctgaacaaagtga acgt.cgcggg gat Catctat 660 ttggg.cgt.ct tt catago ct ctitttacago togagcc act tcgtttacct gttccatcc.g 72 O gcc caattct tcc.ggcagaa agt ccc.gc.gt aaaagctacg cct actgcca agcgctitt.cc 78O tggcttagct ggttcCtgga Catcttitt.cg gacaaagaca gtaatgtgcg gcgt.cataac 84 O accct cagta cc.gc.ctgaca ttataaacgc aacttitttitt acaacttctt cqcggctitat 9 OO t cccaattitt totgctagat acattgctag agattgggta gcaaaac cqc gagtaaaatc 96.O gtta acacaa ccattacct t c cqtc.ttgcc cagaatagot acaattt cag ccggattaat 1 O2O Cttic cctgag ticaatcaaag tagccaac cc gctgatat catcaggtgagg Ctgttgggat 108 O acgaaagact tcaactttitt gcat 1104

<210s, SEQ ID NO 5 &211s LENGTH: 14 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic US 8,367,389 B2 37 38 - Continued peptide FEATURE: NAME/KEY: MOD RES LOCATION: (1) ... (1) OTHER INFORMATION: Gly or Ala FEATURE: NAME/KEY: MOD RES LOCATION: (8) ... (8) OTHER INFORMATION: Cys or Gly FEATURE: NAME/KEY: MOD RES LOCATION: (12) ... (12) OTHER INFORMATION: Tyr or Phe FEATURE: NAME/KEY: MOD RES LOCATION: (13) . . (13) OTHER INFORMATION: Thir or Ser

SEQUENCE: 5

Xaa Lys Thr Glu Gly Asn Gly Xaa Wall Asn Asp Xaa Xaa Arg 1. 5

SEQ ID NO 6 LENGTH: 13 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide FEATURE: NAME/KEY: MOD RES LOCATION: (1) ... (1) OTHER INFORMATION: Wall or Ile FEATURE: NAME/KEY: MOD RES LOCATION: (9) ... (9) OTHER INFORMATION: Val, Ala, or Gly FEATURE: NAME/KEY: MOD RES LOCATION: (10) . . (10) OTHER INFORMATION: Leu or Met FEATURE: NAME/KEY: MOD RES LOCATION: (11) . . (11) OTHER INFORMATION: Ser, Ala, or Thr

SEQUENCE: 6 Xaa Met Ser Gly Gly Chir Glu Gly Xaa Xala Xaa Pro His 1. 5

SEO ID NO 7 LENGTH: 5 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic

NAME/KEY: MOD RES LOCATION: (2) ... (2) OTHER INFORMATION: Glu, His, Asp, or Ala FEATURE: NAME/KEY: MOD RES LOCATION: (3) ... (3) OTHER INFORMATION: Any amino acid FEATURE: NAME/KEY: MOD RES LOCATION: (5) . . (5) <223> OTHER INFORMATION: Arg or Thr SEQUENCE: 7 Glu Xaa Xala Gly Xaa 1. 5 US 8,367,389 B2 39 40 - Continued SEQ ID NO 8 LENGTH: 13 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide FEATURE: NAME/KEY: MOD RES LOCATION: (1) ... (1) OTHER INFORMATION: Asp or Glin FEATURE: NAME/KEY: MOD RES LOCATION: (2) ... (2) OTHER INFORMATION: Leu, Wall, or Ala FEATURE: NAME/KEY: MOD RES LOCATION: (4) ... (4) OTHER INFORMATION: Phe, Tyr, or Luell FEATURE: NAME/KEY: MOD RES LOCATION: (7) . . (7) OTHER INFORMATION: Wall or Ile

<4 OOs, SEQUENCE: 8

Xaa Xala H is Xaa Val Glin Xaa Lys Cys Pro Lieu. Lieu. Thr 1. 5

<210s, SEQ ID NO 9 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: &223s OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: Gly, Ala, or Ser 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (4) ... (4) <223> OTHER INFORMATION: Tyr, Phe, or Luell 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (5) . . (5) 223 OTHER INFORMATION: Seir or ASn 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (7) . . (7) <223> OTHER INFORMATION: Gly or Arg 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (9) ... (9) <223 is OTHER INFORMATION: Ser or Ala

<4 OOs, SEQUENCE: 9 Ser Met Xaa Xala Xaa Arg Xaa Ala Xaa Ala Lieu. Gly 1. 5

SEQ ID NO 10 LENGTH: 22 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide FEATURE: NAME/KEY: MOD RES LOCATION: (1) ... (1) OTHER INFORMATION: Ser, Ala, or Gly FEATURE: NAME/KEY: MOD RES LOCATION: (2) ... (2) OTHER INFORMATION: Ala, Thir, or Cys FEATURE: US 8,367,389 B2 41 - Continued <221s NAME/KEY: MOD RES <222s. LOCATION: (4) ... (4) <223> OTHER INFORMATION: Ser, Ala, or Gly 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (6) . . (6) <223> OTHER INFORMATION: Ile, Val, Gly, or Ser 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (9) . . (10) <223> OTHER INFORMATION: Any amino acid 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (11) . . (11) <223> OTHER INFORMATION: Asn., Asp, Cys, or His 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (12) ... (12) 223 OTHER INFORMATION: Wall or Gul 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (13) . . (16) <223> OTHER INFORMATION: Any amino acid 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (18) ... (18) 223 OTHER INFORMATION: Met or ASn 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (19) . . (19) <223 is OTHER INFORMATION: Ser or Ala 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (2O) . . (21) <223> OTHER INFORMATION: Any amino acid 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (22) ... (22) <223> OTHER INFORMATION: Ser, Ala, or Trp <4 OOs, SEQUENCE: 10 Xaa Xala Ser Xaa Gly Xaa Glu Lieu. Xaa Xala Xaa Xaa Xaa Xala Xala Xala 1. 5 1O 15 Gly Xaa Xala Xala Xaa Xaa 2O

<210s, SEQ ID NO 11 &211s LENGTH: 9 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (2) ... (2) <223> OTHER INFORMATION: Any amino acid 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (3) ... (3) 223 OTHER INFORMATION: Wall or Gul 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (5) (5) <223> OTHER INFORMATION: Any amino acid 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (7) . . (7) <223> OTHER INFORMATION: Ala or Gly 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (8) ... (8) 223 OTHER INFORMATION: Ile or Met

<4 OOs, SEQUENCE: 11 His Xaa Xala Met Xaa Asp Xaa Xala Asp US 8,367,389 B2 43 44 - Continued

<210s, SEQ ID NO 12 &211s LENGTH: 3 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 12 Lys Ala Glu 1.

<210s, SEQ ID NO 13 &211s LENGTH: 21 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: Arg or Asp 22 Os. FEATURE <221 > NAMEAKEY OD RES <222s. LOCATION: (2) ... (2) <223> OTHER INFORMATION: Gly or Asn 22 Os. FEATURE: <221 > NAMEAKEY OD RES <222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: Any amino acid 22 Os. FEATURE: <221 > NAMEAKEY OD RES <222s. LOCATION: (5) . . (5) 223s OTHER INFORMATION: His or Asn 22 Os. FEATURE: <221 > NAMEAKEY OD RES <222s. LOCATION: (6) . . (6) 223 OTHER INFORMATION: Thir or Ile 22 Os. FEATURE: <221 > NAMEAKEY OD RES <222s. LOCATION: (8) ... (8) <223> OTHER INFORMATION: Leu, His or Asn 22 Os. FEATURE: <221 > NAMEAKEY OD RES <222s. LOCATION: (9) ... (9) <223> OTHER INFORMATION: Ser, Thr, Asp, or Glu 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (11) . . (11) 223 OTHER INFORMATION: Seir or Thr 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (13) . . (13) 223 OTHER INFORMATION: Ile or Wall 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (14 (14) <223> OTHER INFORMATION: Asn. Ser, or Ala 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (15) . . (15) <223> OTHER INFORMATION: Any amino acid 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (18) ... (18) 223s OTHER INFORMATION: His or Ser 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (21) ... (21) <223> OTHER INFORMATION: Ala or Gly <4 OOs, SEQUENCE: 13 Xaa Xala Xala Arg Xaa Xaa Met Xaa Xala Asp Xaa Asp Xaa Xala Xala Thr US 8,367,389 B2 45 46 - Continued

1O 15 Arg Xaa Ala Arg Xaa

SEQ I D NO 14 LENGT H: 17 TYPE : PRT ORGAN ISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide FEATURE: NAME/KEY: MOD RES LOCATION: (1) ... (1) OTHER INFORMATION: Val, Ile, or Luell FEATURE: NAME/KEY: MOD RES LOCATION: (2) ... (2) OTHER INFORMATION: Phe or Tyr FEATURE: NAME/KEY: MOD RES LOCATION: (7) . . (7) OTHER INFORMATION: Ser, Ala, or Gly FEATURE: NAME/KEY: MOD RES LOCATION: (13) . . (13) OTHER INFORMATION: Ala, Asp, or Pro <4 OOs, SEQUENCE: 14 Xaa Xaa Val Ser Gly Gly Xaa Glu. His Glin Gly Pro Xaa Gly Gly Gly 1. 5 15

Pro

SEO ID NO 15 LENGTH: 12 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide FEATURE: NAME/KEY: MOD RES LOCATION: (6) . . (6) OTHER INFORMATION: Cys, Gly, or Luell FEATURE: NAME/KEY: MOD RES LOCATION: (7) . . (7) OTHER INFORMATION: Val Met, or Ala FEATURE: NAME/KEY: MOD RES LOCATION: (10) . . (10) OTHER INFORMATION: Tyr or Phe FEATURE: NAME/KEY: MOD RES LOCATION: (11) . . (11) OTHER INFORMATION: Thir or Ser

<4 OOs, SEQUENCE: 15 Thr Glu Gly Asn Gly Xaa Xaa Asn Asp Xaa Xaa Arg 1. 5

SEQ ID NO 16 LENGTH: 12 TYPE : PRT ORGAN ISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

SEQUENCE: 16 Thr Glu Gly Asn Gly Cys Val Asn Asp Phe Thr Arg 1. 5 US 8,367,389 B2 47 48 - Continued

SEO ID NO 17 LENGTH: 17 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide FEATURE: NAME/KEY: MOD RES LOCATION: (1) ... (1) OTHER INFORMATION: Met, Phe, Lieul, or Ile FEATURE: NAME/KEY: MOD RES LOCATION: (2) ... (2) OTHER INFORMATION: Wall, Ile, or yet FEATURE: NAME/KEY: MOD RES LOCATION: (3) ... (3) OTHER INFORMATION: Met, Phe, or Trp FEATURE: NAME/KEY: MOD RES LOCATION: (7) . . (7) OTHER INFORMATION: Asp, Glu, Gly, or Pro FEATURE: NAME/KEY: MOD RES LOCATION: (9) ... (9) OTHER INFORMATION: Wall, Ile, Lieul, Gly, or Ala FEATURE: NAME/KEY: MOD RES LOCATION: (10) . . . OTHER INFORMATION: Ile, Met, or Ala FEATURE: NAME/KEY: MOD RES LOCATION: (11) . . . OTHER INFORMATION: Thir, Ala, or Cys FEATURE: NAME/KEY: MOD RES LOCATION: (14) . . . OTHER INFORMATION: acid FEATURE: NAME/KEY: MOD RES LOCATION: (15) . . ( OTHER INFORMATION: Lieul, or Ser FEATURE: NAME/KEY: MOD RES LOCATION: (16) ... ( OTHER INFORMATION: or Luell FEATURE: NAME/KEY: MOD RES LOCATION: (17) . . ( OTHER INFORMATION: or Wall

<4 OOs, SEQUENCE: 17 Xaa Xala Xala Ser Gly Gly Xaa Gly Xaa Xala Xaa Pro His Xaa Xala Xala 1. 5 15

Xaa

SEQ ID NO 18 LENGTH: 16 TYPE : PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 18 Phe Ile Met Ser Gly Gly Glu Gly Val Met Thr Pro His Thr Val Phe 1. 5 15 US 8,367,389 B2 49 50 What is claimed is: 10. A composition for remediation of a liquid comprising 1. An isolated or purified structurally stable cyanuric acid the CAH of claim 1 and polyethylene glycol (PEG) and/or hydrolase (CAH) enzyme having at least 95% sequence iden KC1. tity with the amino acid sequence SEQID No. 3. 11. The composition of claim 10, wherein the PEG is 2. The CAH of claim 1 having a K value for cyanuric acid PEG4OOO. of 25-150 uM. 12. The composition of claim 10, wherein the PEG is 3. The CAH of claim 1 having ak value for cyanuric acid present at a concentration of 5-50% by weight. of 4.8-76 s. 13. The composition of claim 10, wherein KCl is present at 4. The CAH of claim 1, wherein the enzyme is thermo a concentration of 50-500 mM. stable. 5. The CAH of claim 1, wherein the enzyme retains at least 10 14. The composition of claim 10, wherein the liquid is 30% enzymatic activity at a temperature above 25°C. Water. 6. The CAH of claim 1, wherein the enzyme has a pi of 15. A device for remediation of a liquid comprising a about 5-6. matrix and the stable cyanuric acid hydrolase of claim 1. 7. The CAH of claim 1, wherein the enzyme is from 16. The device of claim 15, wherein the device further Moorella thermoacetica. 15 comprises a casing or housing for the matrix, wherein water 8. The CAH of claim 7, wherein the enzyme is from flows through the at least one casing and contacts the enzyme. Moorella thermoacetica ATCC 39073. 17. The device of claim 15, further comprising a permeable 9. The CAH of claim 1, wherein the enzyme has a specific layer. activity of 12-18 umol/min/mg with cyanuric acid as Sub strate and less than 2 umol/min/mg with barbituric acid as 20 substrate.