GB2332432A - Biodegradation of Trinitrotoluene (TNT) - Google Patents

Abstract

The use of a strain of the bacterium Enterobacter cloacae, designated PB2, capable of producing the enzyme pentaerythritol tetranitrate reductase (PETN), for the degradation of the explosive, trinitrotoluene (TNT) is described. Alternatively, the enzyme itself may be used to degrade TNT. A sequence of the PETN gene is illustrated. In the degradation process nitrites are liberated but toluene and dinitrotoluenes are not produced. The methods are intended to be used for the bioremediation of water and soil.

Description

BIODEGRADATION OF EXPLOSIVES 2332432 This invention relates to the enzymic

detection and destruction of 2,4, 6trinitrotoluene (TNT) particularly in manufacturing waste streams and in the environment.

TNT has been manufactured in large quantities for use in munitions. Manufacture, storage, testing, use and disposal of such munitions have resulted in the contamination of large amounts of soil and water with TNT and related compounds. Further such contamination is likely to occur in the future. TNT is highly recalcitrant to biodegradation and as a result contamination has persisted in the environment (Rosenblatt el al, 1991, 'Organic explosives and related compounds, pp 195-234, 'Handbook of Environmental Chemistry', Springer-Verlag, Berlin). TNT is toxic to mammals, fish, algae and other organisms and is considered a priority pollutant by the United States Environmental Protection Agency (Keith and Telliard, 1979, 'Priority Pollutants L A perspective view', Environ.Sci. Technol. 13, pp 416-423). Other nitroaromatic compounds such as dinitrotoluenes, 2,4,6 trinitrophenol (picric acid) and pesticides/herbicides based on 2,4dinitrophenol may also be significant pollutants.

Soil contaminated with TNT may be treated by incineration, however, this is very expensive and can give rise to undesirable products. Studies have been made investigating the transformation of TNT by micro-organisms and plants, with a view towards developing bioremediation processes (Rieger and Knackmuss, 'Basic knowledge and perspectives on biodegradation of 2, 4, 6-trinitrotoluene and related nitroaromatic compounds in contaminated soil', pp 1- 18, 1995, Spain, J. (ed) 'Biodegradation of nitroaromatic compounds', Plenum Press N.Y.). Many organisms are capable of reducing TNT to nitroso-, hydroxylamino- and amino-derivatives. However, these compounds are still toxic and are often recalcitrant to further degradation. One organism, a strain of Pseudomonas sp. designated clone A, has been shown to grow with TNT as sole nitrogen source (Duque et al, 199'), 'Construction of a Pseudonionas hybrid strain that mineralises 2, 4, 6trinitrotoluene', J.Bacteriol. 175, pp 2278-2283). TNT was denitrated to produce dinitrotoluenes, mononitrotoluenes and toluene. It was proposed that the initial denitration step proceeds via the hydride- Meisenheimer complex of TNT (Haldour and Ramos, 1996, 'Identification of products resulting from the biological reduction of 2, 4, 6- trinitrotoluene, 2, 4-dinitrotoluene and 2, 6-dinitrotoluene by Pseudomonas sp.', Environ. S ci. Technol. 3 0, pp 2365-2370). This is a reduced derivative of TNT bearing a negative charge and can easily be produced by chemical reduction of TNT using boron hydrides (Kaplan and Seidle, 1970, 'Studies in boron hydrides. 4. Stable hydride Meisenheimer adducts', J.Org.Chem. 36, pp 937-939). Certain other bacteria have been shown to reduce TNT to the hydrideMeisenheimer complex, but not to grow with TNT as sole nitrogen source (Vorbeck et al, 1994, 'Identification of a hydride-Meisenheimer complex as a metabolite of 2, 4, 6trinitrotoluene by a Mycobacterium strain, J.Bacteriol. 176, pp 932-934).

It is therefore an object of the present invention to provide a process for the detection and biodegradation of TNT and for the bioremediation of environments contaminated with TNT which do not suffer from the above mentioned disadvantages.

International patent application No. PCT/GB96/01629, the contents of which is incorporated by reference herein, discloses a strain of Enlerobacier cloacae, designated strain PB2, which is capable of growth with pentaerythritol tetranitrate (PETN), a nitrate ester explosive, as sole source of nitrogen for growth. An NADPH-dependent PETN reductase was purified from this organism and was shown to liberate nitrogen as nitnte from PETN as well as from glycerol trinitrate (nitroglycerine) and other nitrate esters. The gene encoding PETN reductase, designated onr (for organic nitrate reductase) was cloned, sequenced and overexpressed in Escherichia coli. PETN reductase shows considerable promise for enzymic detection and bioremediation of nitrate esters. A culture of this organism was deposited under the terms of the Budapest Treaty on the International Recognition of the Deposit of Micro-organisms for the purposes of patent procedures at the UK National Collection of Industrial and Marine Bacteria, 23 St Machar Drive, Aberdeen AB2 I RY, Scotland on the 14th April 1995 under deposit number NCIMB 40718. The nucleotide sequence of the onr gene showing the coding region for PETN reductase and the amino acid sequence of PETN reductase are included herewith as SEQ ID NO: I and SEQ ID NO:2 respectively.

It has now been unexpectedly found that E. cloacae PB2 is capable of growth also with the nitro- substituted aromatic TNT as sole nitrogen source. TNT is consumed during growth. Dinitrotoluenes are not produced and cannot be used as sole nitrogen sources for growth, indicating, that the degradation pathway followed by E. cloacae PB2 is different from that reported for Pseudomonas sp. clone A (Duque el al, supra).

Thus according to a first aspect of the present invention there is provided an Enterobacter cloacae bacterial strain referred to as "PB2" and deposited as NCIMB 40718, and mutants or variants thereof, for use in the biodegradation of TNT.

Cells of E.cloacae PB2 or similar organisms could be grown according to well known techniques and applied to contaminated water, soil etc. either in situ or in specialised bioreactors. Further, compositions could be produced containing E. cloacae P132 to be added to particular environments for the biodegradation of TNT. Therefore another aspect of the present invention involves the use of E. cloacae PB2 in the preparation of a composition used for the biodegradation of TNT in an environment.

In a second aspect of the invention there is provided a method for the biodegradation of TNT in an environment comprising the steps of inoculating the environment with a sample of bacterial isolate E. cloacae PB2 and allowing the isolate to degrade the TNT in the environment. The environment could be a waste stream or could be a ground or water sample contaminated with TNT.

As a further aspect of the present invention there is provided PETN reductase having the amino acid sequence shown in SEQ ID NO:2 or a derivative thereof for use in the biodegradation of TNT. By derivative is meant herein a version of the amino acid sequence SEQ ID NO:2 containing insertions, deletions and/or substitutions of the amino acid sequence such that the functionality of the enzyme is retained.

This could be used for the bioremediation of a contaminated environment such as a waste stream, or a soil or water sample and could be carried out in situ or in a bloreactor. This method of bioremediation has the aforementioned advantages i.e. that toluene and nitrotoluenes are not produced. Two further aspects of the present invention are the use of PETN reductase in preparation of a composition used for the biodegradation of TNT in an environment and a method of bioremediation of TNT in an environment comprising the steps of adding to the environment a quantity of PETN reductase enzyme of claim 4 and maintaining the mixture under conditions appropriate for degradation of the contaminant by PETN reductase enzyme.

According to a yet further aspect of the present invention there is provided a method for the biodegradation of TNT in an environment comprising the steps of introducing to the environment a quantity of recombinant organisms expressing the onr gene having the nucleotide sequence of SEQ ID NO: I or a derivative thereof and maintaining the environment under conditions appropriate for degradation of the contaminant by the recombinant organism. Such organisms could include bacteria, fungi or plants and could be grown in contaminated environments such as waste streams or soil or water samples either in situ or in bioreactors.

By derivative of the gene is meant herein homologues of the gene having a coding sequence which is at least 70% identical to the onr gene, involving any and all single or multiple nucleotide additions, deletions and/or substitutions thereto.

It has been shown that PETN reductase, and Escherichia coli overexpressing this enzyme, are able to reduce TNT to the hydride- Meisenheimer complex, which is further reduced to unstable, negatively- charged soluble compounds. Nitrite is liberated from TNT, demonstrating removal of nitro groups. The reaction products have not been identified, but similar products are formed on chemical reduction of the hydride- Melsenheimer complex of TNT using sodium borohydride. These reaction products appear to be soluble and nonaromatic and to contain less nitrogen than TNT. They are therefore likely to be much less toxic and more amenable to further biodegradation than is TNT. By contrast, other enzymes active against TNT typically reduce the aromatic nitro groups to nitroso, hydroxylamino and amine groups (Rosenblatt et al, supra). The resulting nitrogencontaining aromatic compounds are still highly undesirable in the environment. E. cloacae PB2, PETN reductase, and recombinant organisms expressing PETN reductase, therefore show great promise for the bioremediation of soil or water contaminated with TNT.

Since the initial products of reduction of TNT by PETN reductase are brightly coloured, PETN reductase may also be useful in enzyme-based assays for the presence of TNT, According to a further aspect therefore, this invention concerns a method of detecting TNT in a sample comprising the steps of adding a quantity of PETN reductase, or a derivative thereof, to the sample in the presence of NADPH and detecting the occurrence of a reaction. Such detection mieffit be throuch detection of the oxidation of the cofactor 0 W NADPH, for example by spectrophotometric, fluorometric or luminometric methods, or through detection of the coloured products produced by enzymic transformation of the substrate, for example by visual or spectrophotometric detection of the colour produced. Organisms overexpressing PETN reductase or the onr gene or derivatives thereof could also be used.

In a yet further aspect of the present invention there is provided a biosensor for the detection of TNT in a sample which comprises means for contacting the sample with PETN reductase enzyme or a derivative thereof in the presence of NADPH and means for detecting the occurrence of a reaction, catalysed by the PETN reductase enzyme, of TNT when TNT is present in the sample.

The invention will now be described by way of reference only with reference to the accompanying drawings of which; Figure I shows the growth curves for growth of E. cloacae PB2 with TNT as the sole nitrogen source, Figure 2 shows the degradation of TNT during growth of E. cloacae PB2, Figure 3 shows UV-visible absorbance spectra of ion-pair EPLC peaks following reduction of TNT Figure 4 shows the development of colour and release of nitrite during reduction of TNT by PETN reductase.

Example I - Growth of Enterobacter Cloacae PB2 with TNT as a sole nitroszen source Growth of Enterobacier cloacae PB2 with TNT as sole nitrogen source was assessed in a minimal medium with the following composition: 19.5 m.M KH2PO4; _3 10. 5 MM Na2HP04; 4 mIA trace elements (0. 5 M HCl; 25 mM MgO; 20 mM CaC03.- 20 m.M FeS04; 5 mM ZnS04; 5 mM MnS04; I MM CUS04; I MM COS04; I mM H31304). The carbon source was 22 MM D-glucose. As an inoculum, E. cloacae PB2 was grown for 5 days at.)O'C in the above medium with the addition of 15 mM NaN02 as nitrogen source. To 50 ml of medium containing no nitrogen, 0.5 mM TNT or 1.0 mM TNT as nitrogen source, 0.5 ml inoculurn was added. The cultures were incubated at 30'C with rotary agitation at 150 rpm. Each day, samples of I ml were removed and growth was estimated by measuring light-scattering at 600 nm. Cells were then removed from the samples by centrifugation and the supernatants were stored at -20'C for HPLC analysis.

C The concentration of TNT and presence of metabolites were determined by HPLC analysis using a Techsphere 50DS reverse phase column (HPLC Technology, Macclesfield, U.K.). The mobile phase consisted of 60% v/v methanol, 40% v/v water, and was delivered at a flowrate of 1.0 ml/min. Compounds were detected at 260 rim. This solvent system resolved TNT, 2,6dinitrotoluene, 2,4-dinitrotoluene, 2-nitrotoluene and 4-nitrotoluene. For ion-pair HPLC, the same column was used, with a mobile phase consisting of 45% v/v acetonitrile, 55% v/v 20 mM tetrabutylammonium phosphate buffer, pH 7. Peaks were detected at 260 nm and 500 nm and UVvisible spectra of peaks were measured using a Waters 994 Programmable Photodiode Array detector.

The growth curves obtained are shown as Figure 1. Growth, estimated by turbidity, was observed only in the presence of TNT and was proportional to the amount of TNT present in the growth medium. In similar experiments where TNT was replaced by 2,4dinitrotoluene, 2,6-dinitrotoluene, 2nitrotoluene or 4-nitrotoluene, growth did not occur.

HPLC analysis showed that TNT was removed from the medium during growth. Figure 2 shows the degradation of TNT with growth of E. cloacae P132 with initial amounts of TNT of 0.5 mM and 1.0 mM. Peaks corresponding to dinitrotoluene and mononitrotoluene were not detected. Two peaks were detected which may represent metabolites of TNT. One of these was similar in elution position and UV-visible spectrum to products resulting from the action of cloned E.cloacae nitroreductase (Bryant el al, 1991, 'Cloning, nucleotide sequence and expression of the nitroreductase gene from Enlerobacter cloacae', J.Biol.Chem. 266, pp 4126-4130) on TNT. This peak may represent a stable nitroreductase product such as aminodinitrotoluene. Such products are commonly observed when bacteria are incubated with TNT (Rosenblatt et al, 199 1, supra). The second peak observed migrated at the solvent front in standard HPLC, but was retarded by the column in ion-pair HPLC in the presence of the tetrabutylammonium counter-Ion, suggesting that this peak represents a negatively charged molecule. This experiment is not sufficient to determine whether or not these peaks represent products derived from TNT.

Example 2 - Dep gradation of TNT by PETN reductase PETN reductase was purified from recombinant E. coli bearing the plasn-d pONR I by affinity chromatography (French el al, 1996, 'Sequence and Properties of pentaerythritol tetranitrate reductase from Enterobacter cloacae PB2', J.Bacteriol. 178, pp 6623-6627). Reaction mixtures were set up containing 7 tg/rnl PETN reductase, 0.2 mM NADPH and 0.05 mM TNT, 2,4dinitrotoluene, 2,6-dinitrotoluene, 2-nitrotoluene, 4-nitrotoluene or no substrate, in 50 mM potassium phosphate buffer, pH 7, at 30'C. Oxidation of NADPH was followed based on the loss of absorbance at 340 run. The background rate of NADPH oxidation in the absence of substrate was 0. 10 gmol NADPH. min-'. mg protein-'. Thisrate was not significantly enhanced in the presence of 0.05 mM 2,4-dinitrotoluene, 2,5dinitrotoluene, 2nitrotoluene or 4-nitrotoluene. However, in the presence of 0.05 n-1M TNT, the observed rate of NADPH oxidation increased to 0.50 jimol NADPH.min-l. mg protein-', suggesting that TNT is able to oxidize the reduced form of the enzyme, presumably becoming reduced in the process. Errors in these rate measurements were estimated as less than 1%. It was further observed that reaction mixtures containing PETN reductase, NADPH and TNT developed an orange colouration suggesting the formation of a coloured product from TNT. No such colouration developed in the absence of enzyme, of TNT or of NADPH.

Known reduction products of TNT include products of nitro-group reduction, such as aminodinitrotoluene, which are uncharged and essentially colourless (Rosenblatt et al, 1991, supra), and products of aromatic ring reduction, such as the hydnde-Meisenheimer complex, which is negatively charged and brightly coloured (Kaplan and Seidle, 1970, supra). However, the UV-visible absorbance spectrum of the orange product observed when TNT was reduced by PETN reductase did not match the spectrum of the hydrideMeisenheimer complex of TNT reported in the literature.

Example 3 - Nature of the products of TNT reduction.

To investigate the nature of the coloured product or products produced by the action of PETN reductase on TNT, a reaction mixture was set up containing 0.02 mg/ml PETN reductase, 0.4 m.M NADPH and 0.5 mM TNT in 50 mM potassium phosphate buffer, pH 7. Samples of 100 mI were taken at intervals and diluted with 1.9 nil HPLC mobile phase (45% v/v acetonitrile, 55% v/v 20 mM tetrabutylammonium phosphate buffer, pH 7). These samples were analysed by ion-pair F1PLC as described in Example 1. Peaks having ultraviolet absorbance were detected at 260 rim and peaks having visible absorbance were detected at 500 run. The UV-visible spectra of detected peaks were measured using a Waters 994 programmable photodiode array detector. A simflar experiment was performed using, in place of PETN reductase, recombinant Enterobacter cloacae nitroreductase (Bryant et al, 1991, supra), a relatively well characterized enzyme which reduces the aromatic nitro groups of TNT to amino groups via nitroso and hydroxylamino intermediates.

During the reduction of TNT by PETN reductase, a UV peak at a retention time of 77, corresponding to TNT, decreased. Another UV peak at a retention time of 5.4, appeared and increased in size. An identical peak was observed when PETN reductase was replaced by nitroreductase. This peak is presumed to represent a nitroreductase product such as hydroxylaniinodinitrotoluene or aminodinitrotoluene. This suggests that PETN reductase has nitroreductase activity. In addition, with PETN reductase, six peaks with both UV and visible absorbance were detected, with retention times of 3.0' (peak A), 18' (peak B), 4.2' (peak C), 4.8' (peak D), 8.6' (peak E), and 11. 6' (peak F). These peaks were not observed with nitroreductase. Peak A was confounded with the peaks of NADPH and NADP+, so that the shape of the spectrum below 400 rim could not be determined; however, the spectrum above 400 rim was identical to the spectrum of peak B in this region. The UV-visible spectra of peaks C and D appeared to be identical to one another, 0 as did the spectra of peaks E and F, as shown in Figure 3. It is therefore unclear whether these peaks represent six distinct compounds, or three compounds, each of which migrates as two peaks due to some peculiarity of the ion-pair HPLC system, such as the formation of ion- pairs with difFerent numbers of tetrabutylammonium ions.

If reaction mixtures were left for several hours, the observed peaks decreased in size, with no peaks appearing to replace them. Visible colour in the reaction mixtures also faded. This suggests that the coloured products are unstable and degrade to give nonaromatic (non UV- absorbing) products.

When samples were re-analysed in the same mobile phase but with the tetrabutylammonium counter-Ion omitted, all visible absorbance, presumably corresponding -g- to peaks A, B, C, D, E and F, eluted at the solvent front. The TNT and presumed nitroreductase product peaks were unafFected. This suggests that the visible peaks A to F represent negatively charged molecules.

The UV-visible spectra of peaks E and F were distinctive and were identical to the spectrum of the hydride-Meisenheimer complex of TNT reported in the literature (Kaplan and Seidle, 1970; Vorbeck et al, 1984, supra). As shown in Figure 3 the spectra of peaks A, B, C and D were distinctly different from this spectrum, lacking significant absorbance above 550 rim.

Comparative Example - Comparison with chemical reduction of TNT.

To determine whether peaks E and F represented the hydride-Meisenheimer complex of TNT, the authentic hydride-Meisenheimer complex was prepared by chemical reduction of TNT using sodium borohydride (Kaplan and Seidle, 1970; Haldour and Ramos, 1996, supra). To 1 ml of 10 mM TNT in acetonitrile was added 2.8 ing solid sodium borohydride (NaBH4). The reaction mixture instantly developed a deep brownishpurple colour and the UV-visible spectrum, measured in 50% v/v acetonitrile, 50% v/v water, was identical to that reported for the hydride-Meisenheimer complex of TNT. However, it was noticed that after standing at room temperature for several hours, orange colouration and a red precipitate developed in the reaction mixture. If water was added to the reaction mixture at an early stage, so that excess borohydride was consumed throullh reaction with water, the purple colouration was stable over days and no orange colour developed. This suggests that the orange colouration represents a slow further reduction of the hydride-Meisenheimer complex.

Chemical reaction mixtures were analysed by ion-pair HPLC as described above. Initially, TNT disappeared and peaks identical to peaks E (large) and F (small) appeared. As the reaction proceeded and orange colouration developed, peaks apparently identical in retention time and UV-visible spectrum to peaks A, B, C and D appeared. A large ultraviolet peak lacking visible absorbance also appeared at the solvent front. No peak corresponding to the nitroreductase product peak appeared.

These results suggest that PETN reductase reduces TNT via two competing reactions. In one reaction, the nitro groups are reduced in a similar fashion to that seen with nitroreductase. This is not surprising since the aromatic nitro group is a facile electron acceptor and is readily reduced by a variety of enzymes (Bryant et al, 199 1). In the other reaction, which is predominant in the case of PETN reductase but does not occur with nitroreductase, the aromatic ring of TNT is reduced to give the hydfide-Meisenheimer complex as with chemical reduction of TNT by sodium borohydride. This is further reduced to give negatively charged orange products. With PETN reductase, the reduction of the hydfide- Meisenheimer complex is rapid so that the complex is seen only transiently, however, in the case of reduction with sodium borohydride, the reduction of the complex is much slower than the initial reduction of TNT so that initially the hydfide-Melsenheimer complex is the only product.

Example 4 - Reduction of the hydride-Meisenheimer complex of TNT by PETN reductase.

To confirm that the orange products are produced through further reduction of the hydride-Meisenheimer complex, hydride-Meisenheimer complex was prepared chemically as described above and the chemical reduction was quenched with aqueous buffer. An enzymic reaction mixture was set up containing 0.4 mM NADPH, 0.04 mg/ml PETN reductase, and the amount of chemical reduction product corresponding to 2 mM TNT, in 50 niNI potassium phosphate buffer, pH 7. The brown-purple colour of the chemical reduction product, presumed to be the hydride-Meisenheimer complex of TNT, was rapidly replaced by an orange colour identical to that seen in enzymic reduction of TNT by PETN reductase. The UV-visible absorbance spectrum of the reaction mixture was identical to that seen during enzymic reduction of TNT. When nitroreductase replaced PETN reductase, the orance colouration and the distinctive UV-visible spectrum associated with 0 the orange products were not seen.

Example 5 - Liberation of nitrite from TNT by PETN reductase It was further noted that, during enzymic reduction of TNT by PETN reductase, nitrite was liberated. A reaction mixture was set up containing 0.04 mg/ml PETN reductase, 2.0 MM NADPH, and 0.5 mM TNT, in 50 mM potassium phosphate buffer, pH 7. Visible absorbance at 440 nm, corresponding to the visible absorbance peak in aqueous buffers of the orange products, was monitored. Samples were periodically removed and assayed for nitrite as follows: 6 pil was added to 594 gi water. To this diluted sample were added 200 ptl of 10 mg/ml sulphanilamide in 0.68 M 11C1, and 40 pLI of 10 mg/ml N-(lnaphthyl)ethylenediamine in water. Visible absorbance at 540 nm was measured. Sodium nitrite was used as a standard. Results are shown as Figure 4. Over -33 h of incubation, 0.066 mM nitrite was released from 0.5 mM TNT, or 0. 13 mol nitrite/mol TNT. In reaction mixtures which had been left standing for several days, up to 1. 0 mol nitrite/mol TNT was detected. It is not clear from these experiments whether or not this represents a limiting value.

Conversion of TNT to the hydride-Meisenheimer complex does not result in liberation of nitrite. In our enzymic reaction mixtures, the hydrideMeisenheimer complex appeared to be a minor and transient product. Possibly nitrite was released either during further reduction of the hydride-Meisenheimer complex to the orange products, or during breakdown of the orange products to unidentified colourless, non-UV-absorbing products.

SEQUENCE LISTING (1) GENERAL INFORMATION:

(i) APPLICANT: (A) NAME: The Secretary of State for Defence (B) STREET: Defence Evaluation and Research Agency (C) CITY: Farnborough (D) STATE: Hampshire (E) COUNTRY: England (F) POSTAL CODE (ZIP): GU 14 OLX (ii) TITLE OF INVENTION: Biodegradation of Explosives (iii) NUMBER OF SEQUENCES: 2 (iv) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER-. IBM PC compatible (C) OPERATING SYSTEM: PC-DOSIMS-DOS (D) SOFTWARE: Patentln Release W1.0, Version #1.30 (EPO) (2) INFORMATION FOR SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1531 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE: (A) NAMEIKEY: CDS (B) LOCATION: 2 8L.137 5 (ix) FEATURE: (A) NAA4E/KEY: matpeptide (B) LOCATION: 284---1375 (ix) FEATURE (A) NAMEIKEY.. terminator (B) LOCATION. 1396..1419 (ix) FEATURE: (A) NAMEIKEY: RBS (B) LOCATION: 271..27 5 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1 i

CCATGGATAA AGGAGCCAGC GG=GATTG CCCTGTTGGC TCAGGCG=G GAGAGWGGC 60 GCAATGAAAA AACWTC= TTC=GWG ATCCGCTCAC GCAGGCACAG GTG=TATT 120 CCCTCTGGTT AGGCGCCAAC CTGCAAGCAA AAATG=CG CAGCGWGTG CCG=GAAA i80 GWCWTGGC GCATGTGAAA AAWGTATTA. CCGCG=GG WTGTAGCW GCGTTTTTAT 240 TTACCCTTTT ACTAGTCGAC TGGTCTACTC AGGAGCCGTT ATG TCC GCT GAA AAG Met Ser Ala Glu Lys -1 1 CTG TTT ACC CCA CTG AAA GTG GGT GCC GTT ACT GCC CCA AAC CGC GTG Leu Phe Thr Pro Leu Lys Val Gly Ala Val Thr Ala Pro Asn Arg Val 10 15 20 TTT ATG GCC CCA CTT ACC CGT CTG CGC AGC ATC GAG CCG GGC GAT ATC Phe Met Ala Pro Leu Thr Arg Leu Arg Ser Ile Glu Pro Gly Asp Ile 30 35 CCA ACG CCA TTG ATG GGT GAG TAT TAC CGC CAG CGC GCC AGC GCG GGC Pro Thr Pro Leu Met Gly Glu Tyr Tyr Arg Gin Arg Ala Ser Ala Gly 45 50 CTG ATT ATC TCC GAA GCC ACG CAG ATT TCT GCT CAG GCA AAA GGC TAC Leu Ile Ile Ser Glu Ala Thr Gin Ile Ser Ala Gin Ala Lys Gly Tyr 60 65 GCC GGT GCA CCG GGT CTG CAC AGC CCG GAA CAG ATC GCC GCG TGG AAA Ala Gly Ala Pro Gly Leu His Ser Pro Glu Gin Ile Ala Ala Trp Lys 75 80 AAA ATC ACC GCA GGC GTG CAT GCT GAA GAT GGC CGT ATT GCG GTT CAG Lys Ile Thr Ala Gly Val His Ala Glu Asp Gly Arg Ile Ala Val Gin 90 95 100 CTG TGG CAC ACC GGT CGT ATC TCA CAC AGC AGC ATC CAG CCT GGC GGT Leu Trp His Thr Gly Arg Ile Ser His Ser Ser Ile Gin Pro Gly Gly 110 115 CAG GCG CCG GTT TCT GCC TCT GCC CTG AAC GCC AAT ACC CGC ACT TCC Gin Ala Pro Val Ser Ala Ser Ala Leu Asn Ala Asn Thr Arg Thr Ser 125 130 CTG CGC GAT GAA AAC GGT AAT GCG ATC CGC GTC GAC ACC ACC ACG CCA Leu Arg Asp Glu Asn Gly Asn Ala Ile Arg Val Asp Thr Thr Thr Pro 140 145 CGC GCG CTG GAG CTG GAC GAG ATC CCG GGT ATC GTG AAT GAT TTC CGT Arg Ala Leu Glu Leu Asp Glu Ile Pro Gly Ile Val Asn Asp Phe Arg 155 160 CAG GCC GTC GCC AAC GCC CGG GAA GCG GGC TTC GAC CTG GTT GAG CTT Gin Ala Val Ala AsnAla Arg Glu Ala Gly Phe Asp Leu Val Glu Leu 170 175 180 CAC TCT GCG CAC GGT TAC CTG CTG CAT CAG TTC CTG TCC CCG TCT TCC His Ser Ala His Gly Tyr Leu Leu His Gin Phe Leu Ser Pro Ser Ser 190 195 AAC CAG CGT ACC GAC CAG TAC GGC GGC AGC GTT GAA AAC CGC GCG CGT Asn Gin Arg Thr Asp Gin Tyr Gly Gly Ser Val Glu Asn Arg Ala Arg 205 210 CTG GTG CTT GAA GTG GTG GAT GCT GTC TGT AAT GAG TGG AGC GCA GAC Leu Val Leu Glu Val Val Asp Ala Val Cys Asn Glu Trp Ser Ala Asp 215 220 225 CGC ATT GGT ATT CGT GTC TCC CCG ATC GGT ACT TTC CAG AAC GTC GAC Arg Ile Gly Ile Arg Val Ser Pro Ile Gly Thr Phe Gin Asn Val Asp295 343 391 439 487 535 563 679 727 775 823 871 919 967 1015 230 235 240 AAC GGT CCG AAC GAA GAA GCA GAC GCG CTG TAT CTG ATT GAA GAG CTG Asn Gly Pro Asn Glu Glu Ala Asp Ala Leu Tyr Leu Ile Glu Glu Leu 245 250 255 260 GCG AAA CGC GGT ATC GCC TAT CTG CAC ATG TCC GAG ACG GAC TTG GCA Ala Lys Arg Gly Ile Ala Tyr Leu His Met Ser Glu Thr Asp Leu Ala 265 270 275 GGC GGC AAG CCT TAC AGT GAA GCC TTC CGT CAG AAA GTG CGC GAG CGC Gly Gly Lys Pro Tyr Ser Glu Ala Phe Arg Gln Lys Val Arg Glu Arg 280 285 290 TTC CAC GGC GTG ATT ATC GGG GCG GGT GCG TAT ACG GCA GAA AAA GCC Phe His Gly Val Ile Ile Gly Ala Gly Ala Tyr Thr Ala Glu Lys Ala 295 300 305 GAG GAT TTG ATC GGT AAA GGC CTG Glu Asp Leu Ile Gly Lys Gly Leu 310 315 ATC GAC GCC GTG GCC TTT GGC CGT Ile Asp Ala Val Ala Phe Gly Arg 320 GAC TAC ATT GCT AAC CCG GAT CTG GTT GCC CGT TTG CAG AAA AAA GCC Asp Tyr Ile Ala Asn Pro Asp Leu Val Ala Arg Leu Gln Lys Lys Ala 325 330 335 340 GAA CTG AAC CCG CAG CGT CCT GAA AGC TTC TAT GGC GGC GGC GCG GAA Glu Leu Asn Pro Gln Arg Pro Glu Ser Phe Tyr Gly Gly Gly Ala Glu 345 350 355 GGT TAT ACC GAC TAC CCT TCA CTG TAATCCCGCT TTGTACATTG ATAGCGGCGA Gly Tyr Thr Asp Tyr Pro Ser Leu 360 1063 1111 1159 1207 1255 1303 1351 CCTTTCGCCG CTATACTAAA ACATCG=C TGTTCAAAAA GATAATWAT TWACTGG= 1465 AATGAGGAAA TTATGW= ACTTCACACC ATWTG=G TTGGWACCT GCAA==C 1525 ATWAT (2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 365 arnino acids (B) TYPE: arnino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Met Ser Ala Glu Lys Leu Phe Thr Pro Leu Lys Val Gly Ala Val Thr - 1 1 5 10 15 Ala Pro Asn Arg Val Phe Met Ala Pro Leu Thr Arg Leu Arg Ser Ile 25 30 Glu Pro Gly Asp Ile Pro Thr Pro Leu Met Gly Glu Tyr Tyr Arg Gln 40 45 Arg Ala Ser Ala Gly Leu Ile Ile Ser Glu Ala Thr Gln Ile Ser Ala 1531 -is60 Gln Ala Lys Gly Tyr Ala Gly Ala Pro G1Y Leu His Ser Pro Glu Gln 70 75 Ile Ala Ala Trp Lys Lys Ile Thr Ala Gly Val His Ala Glu Asp Gly 85 90 95 Arg Ile Ala Val Gln Leu Trp His Thr Gly Arg Ile Ser His Ser Ser 105 110 Ile Gln Pro Gly Gly Gln Ala Pro Val Ser Ala Ser Ala Leu Asn Ala 120 125 Asn Thr Arg Thr Ser Leu Arg Asp Glu Asn Gly Asn Ala Ile Arg Val 135 140 Asp Thr Thr Thr Pro Arg Ala Leu Glu Leu Asp Glu Ile Pro Gly Ile 150 155 Val Asn Asp Phe Arg Gln Ala Val Ala Asn Ala Arg Glu Ala Gly Phe 165 170 175 Asp Leu Val Glu Leu His Ser Ala His GlY Tyr Leu Leu His Gln Phe 185 190 Leu Ser Pro Ser Ser Asn Gln Arg Thr Asp Gln Tyr Gly Gly Ser Val 200 205 Glu Asn Arg Ala Arg Leu Val Leu Glu Val Val Asp Ala Val Cys Asn 210 215 220 Glu Trp Ser Ala Asp Arg Ile Gly Ile Arg Val Ser Pro Ile G1Y Thr 225 230 235 Phe Gln Asn Val Asp Asn Gly Pro Asn Glu Glu Ala Asp Ala Leu Tyr 240 245 250 255 Leu Ile Glu Glu Leu Ala Lys Arg Gly Ile Ala Tyr Leu His Met Ser 260 265 270 Glu Thr Asp Leu Ala Gly Gly Lys Pro Tyr Ser Glu Ala Phe Arg Gln 275 280 285 Lys Val Arg Glu Arg Phe His Gly Val Ile Ile Gly Ala Gly Ala Tyr 290 295 300 Thr Ala Glu Lys Ala Glu Asp Leu Ile Gly Lys Gly Leu Ile Asp Ala 305 310 315 Val Ala Phe Gly Arg Asp Tyr Ile Ala Asn Pro Asp Leu Val Ala Arg 320 325 330 335 Leu Gln Lys Lys Ala Glu Leu Asn Pro Gln Arg Pro Glu Ser Phe Tyr 340 345 350 Giv Giy Gly Ala Glu Gly Tyr Thr Asp Tyr Pro Ser Leu 355 360

Claims (1)

1. Claims

   I An Enterobacter cloacae bacterial strain referred to as "PB2" and deposited as NCDAB 40718, and mutants and variants thereof, for use in the biodegradation of TNT.

   The use of bacterial strain E. cloacae PB2 of claim I in the preparation of a composition used for the biodegradation of TNT in an environment.

   3. A method for the biodegradation of TNT in an environment comprising the steps of inoculating the environment with a sample of bacterial isolate E. cloacae PB2 of claim I and allowing the isolate to degrade the TNT in the environment.

   A PETN reductase enzyme having the amino acid sequence shown in SEQ ID NO: 1 1 or a derivative thereof for use in the biodegradation of TNT.

   The use of PETN reductase enzyme of claim 4 in the preparation of a composition used for the biodegradation of TNT in an environment.

   A method for the biodegradation of TNT in an environment comprising the steps of adding to the environment a quantity of PETN reductase enzyme of claim 4 and maintaining the mixture under conditions appropriate for degradation of the contaminant by PETN reductase enzyme.

   A method for the biodegradation of TNT in an environment comprising the steps of introducing to the environment a quantity of recombinant organisms expressing the onr gene having the nucleotide sequence of SEQ ID NO I or a derivative thereof and maintaining the environment under conditions appropriate for degradation of the contaminant by the recombinant organism.

   8. A method according to any of claims 3, 6 or 7 wherein the environment is a waste stream containing TNT or a ground or water sample.

   9. A method for detecting TNT in a sample comprising the steps of adding a quantity of PETN reductase of claim 4 to the sample in the presence of NADPH and detecting the occurrence of a reaction.

   10. A method according to claim 9 wherein oxidation of NADPH is detected.

   A method according to claim 9 wherein coloured reaction products are detected.

   A biosensor for the detection of TNT in a sample which comprises means for contacting the sample with PETN reductase enzyme of claim 4 in the presence of IZ NADPH and means for detecting the occurrence of a reaction, catalysed by the PETN reductase enzyme, of TNT when TNT is present in the sample.