WO1999032636A1

WO1999032636A1 - Biodegradation of explosives

Info

Publication number: WO1999032636A1
Application number: PCT/GB1998/003646
Authority: WO
Inventors: Stephen Nicklin; Neil Charles Bruce; Christopher Edward French
Original assignee: The Secretary Of State For Defence
Priority date: 1997-12-19
Filing date: 1998-12-07
Publication date: 1999-07-01
Also published as: GB9726813D0; AU1494499A; GB2332432A; ZA9811409B

Abstract

A bacterial strain Enterobacter cloacae PB2, deposited as NCIMB 40718, is provided which is capable of producing an enzymic activity which degrades 2, 4, 6-trinitrotoluene (TNT) as is a method for the biodegradation of TNT in an environment using E.cloacae PB2. The enzyme, called PETN reductase as it also catalyses the removal of nitrite from pentaerythritol tetranitrate (PETN), may be used for the biodegradation of TNT and in the preparation of compositions for the biodegradation of TNT and the invention also relates to methods of bioremediation using PETN reductase. Toluene and nitrotoluenes are not produced in the degradation. The invention also relates to methods of bioremediation of TNT in an environment using recombinant organisms expressing the onr gene encoding PETN reductase. Methods of detection of TNT in a sample and a biosensor using PETN reductase are also provided.

Description

BIODEGRADATION OF E.XPLOSINES

Tliis invention relates to the enzymic detection and destruction of 2,4,6- trinitrotoluene (TΝT) particularly in manufacturing waste streams and in the environment.

TΝT 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 TΝT and related compounds. Further such contamination is likely to occur in the future. TΝT is .highly recalcitrant to biodegradation and as a result contamination has persisted in the environment (Rosenblatt et al, 1991, Organic explosives and related compounds', pp 195-234, 'Handbook of Environmental Chemistiy', Springer-Nerlag, Berlin). TΝT 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 I. 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 2,4- dinitrophenol may also be significant pollutants.

Soil contaminated with TΝT may be treated by incineration, however, this is veiy expensive and can give rise to undesirable products. Studies have been made investigating the transformation of TΝT 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 Ν.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, 1993, 'Construction of a. Pseudomonas hybrid strain that mineralises 2, 4, 6- trinitrotoluene', 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 (Haϊdour 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.Sci.Technol. 30, pp 2365-2370). TWs is a reduced derivative of TNT be.aring a negative charge and can easily be produced by chemic 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 hydride- Meisenheimer complej , 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, 6- trinitrotoluene by aMycobacterium 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 wWch do not suffer from the above mentioned disadvantages.

International patent application No. PCT/GB96/01629, the contents of which is incoiporated by reference herein, discloses a strain of Enter obacter cloacae, designated strain PB2, wWch 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 tWs organism and was shown to liberate nitrogen as nitrite 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 en.zymic 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 1RY, 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 NOJ 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 et al, supra).

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

Cells of Kcloacae 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 PB2 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 another 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 Kcloacae PB2 and allowing the isolate to degrade the TNT in the environment. A method for degradation of PETN and TNT in the same environment by the isolate is also provided. 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 bioreactor. 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. A kit for biodegradation of an environment a store of PETN reductase and a means of contacting the environment with PETN reductase in the presence of NADPH and maintaining the environment under conditions appropriate for the degradation of TNT by PETN reductase is also provided.

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: 1 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 phnts and could be grown in contaminated environments such as waste streams or soil or water samples either in situ or in bioreactors. A method of biodegradation of both PETN and TNT in the same environment is so provided.

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-Meisenheimer complex of TNT using sodium borohydride. These reaction products appear to be soluble and non- aromatic 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 nitrogen- containing 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 might be through detection of the oxidation of the cofactor NADP.H, for example by spectrophotometric, fluorometric or luminometric methods, or through detection of the coloured products produced by enzymic transfoπnation 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 1 shows the growth curves for growth of Kcloacae PB2 with TNT as the sole nitrogen source,

Figure 2 shows the degradation of TNT during growth of E.cloacae PB2,

Figure 3 shows UN-visible absorbance spectra of ion-pair HPLC peaks following reduction of TΝT

Figure 4 shows the development of colour and release of nitrite during reduction of TΝT by PETΝ reductase. Example 1 - Growth of Enterobacter Cloacae PB2 with TNT as a sole nitrogen source

Growth o.f Enterobacter cloacae PB2 with TNT as sole nitrogen source was assessed in a minima medium with the following composition: 19.5 mM aKH₂P0₄; 30.5 MM Na₂.HPO₄; 4 ml/1 trace elements (0.5 M HC1; 25 mM MgO; 20 mM CaCO₃; 20 mM FeSO₄; 5 mM ZnSO₄; 5 mM MnSO₄; 1 MM CuS0₄; 1 MM CoSO₄; 1 mM H₃BO₄). The carbon source was 22.MM D-glucose. As an inoculum, E. cloacae PB2 was grown for 5 days at 30°C in the above medium with the addition of 15 mM NaNO₂ as nitrogen source. To 50 ml of medium containing no nitrogen, 0.5 mM TNT or 1.0 M TNT as nitrogen source, 0.5 ml inoculum was added. The cultures were incubated at 30°C with rotary agitation at 150 rpm. Each day, samples of 1 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.

The concentration of TNT and presence of metabolites were determined by .HPLC analysis using a Techsphere 5ODS 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 nm. This solvent system resolved TNT, 2,6-dinitrotoluene, 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 UN- visible 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 TΝT and was proportional to the amount of TΝT present in the growth medium. In similar experiments where TΝT was replaced by 2,4- dinitrotoluene, 2,6-dinitrotoluene, 2-nitrotoluene or 4-nitrotoluene, growth did not occur.

HPLC analysis showed that TΝT was removed from the medium during growth. Figure 2 shows the degradation of TΝT with growth of E.cloacae PB2 with initial amounts of TΝT of 0.5 mM and 1.0 mM. Peaks coiresponding to dinitrotoluene and mononitrotoluene were not detected. Two peaks were detected which may represent metabolites of TΝT. One of these was similar in elution position and UN-visible spectrum to products resulting from the action of cloned E.cloacae nitroreductase (Bryant et al, 1991, 'Cloning, nucleotide sequence and expression of the nitroreductase gene from Enterobacter 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, 1991, 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 - Degradation of TNT by PETN reductase

PETN reductase was purified from recombinant E. coli bearing the plasmid pONRl by affinity chromatography (French et 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 μg/ml PETN reductase, 0.2 mM NiADPH and 0.05 mM TNT, 2,4-dinitrotoluene, 2,6-dinitrotoluene, 2-nitrotoluene, 4-nitrotoluene or no substrate, in 50 mM potassium phosphate buffer, pH 7, at 30°C. Oxidation of N.ADPH was followed based on the loss of absorbance at 340 nm. The background rate of NADPH oxidation in the absence of substrate was 0J0 μmol NaADPH.min^'1 .mg protein^"1. This rate was not significantly enhanced in the presence of 0.05 mM 2,4-dinitrotoluene, 2,5- dinitrotoluene, 2-nitrotoluene or 4-nitrotoluene. However, in the presence of 0.05 mM TNT, the observed rate of N^ DPH oxidation increased to 0.50 μmol N^ DPH.min^.mg protein^'1, 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 obseived that reaction mixtures containing PETN reductase, N.ADPH 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 hydride-Meisenheimer complex, which is negatively charged and brightly coloured (Kaplan and Seidle, 1970, supra). However, the UN-visible absorbance spectrum of the orange product observed when TΝT was reduced by PETΝ reductase did not match the spectrum of the hydride- Meisenheimer complex of TΝT 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 mM NADPH and 0.5 mM TNT in 50 mM potassium phosphate buffer, pH 7. Samples of 100 ml were taken at intervals and diluted with 1.9 ml HPLC mobile phase (45% v/v acetonitrile, 55% v/v 20 mM tetrabutylammonium phosphate buffer, pH 7). These samples were analysed by ion-pair HPLC as described in Example 1. Peaks having ultraviolet absorbance were detected at 260 nm and peaks having visible absorbance were detected at 500 nm. The UN-visible spectra of detected peaks were measured using a Waters 994 programmable photodiode array detector. A similar experiment was performed using, in place of PETΝ reductase, recombinant Enterobacter cloacae nitroreductase (Bryant et al, 1991, supra), a relatively well characterized enzyme which reduces the aromatic nitro groups of TΝT to amino groups via nitroso and hydroxylamino intermediates.

During the reduction of TΝT by PETΝ reductase, a UN peak at a retention time of 7.7', corresponding to TΝT, decreased. Another UN peak at a retention time of 5.4' appeared and increased in size. An identical peak was observed when PETΝ reductase was replaced by nitroreductase. This peak is presumed to represent a nitroreductase product such as hydroxylaminodinitrotoluene or aminodinitrotoluene. This suggests that PETΝ reductase has nitroreductase activity. In addition, with PETΝ reductase, six peaks with both UN and visible absorbance were detected, with retention times of 3.0' (peak A), 3.8' (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 N.ADPH and NADP+, so that the shape of the spectrum below 400 nm could not be determined; however, the spectrum above 400 nm was identical to the spectrum of peak B in tWs region. The UN-visible spectra of peaks C and D appeared to be identical to one another, 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. TWs suggests that the coloured products are unstable and degrade to give non- aromatic (non UN-absorbing) products.

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

The UN- visible spectra of peaks E and F were distinctive and were identical to the spectrum of the hydride-Meisenheimer complex of TΝT reported in the literature (Kaplan and Seidle, 1970; Norbeck 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 nm.

Comparative Example - Comparison with chemical reduction of TΝT.

To determine whether peaks E and F represented the hydride-Meisenheimer complex of TΝT, the authentic hydride-Meisenheimer complex was prepared by chemical reduction of TΝT using sodium borohydride (Kaplan and Seidle, 1970; Haϊdour and Ramos, 1996, supra). To 1 ml of 10 mM TΝT in acetonitrile was added 2.8 mg solid sodium borohydride (NaBFL). The reaction mixture instantly developed a deep brownish- purple colour and the UN- visible spectrum, measured in 50% v/v acetonitrile, 50% v/v water, was identical to that reported for the hydride-Meisenheimer complex of TΝT. 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 through 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, TΝT 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 UN-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 suφrising since the aromatic nitro group is a facile electron acceptor and is readily reduced by a variety of enzymes (Bryant et al, 1991). 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 hydride-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 hydride-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 hydride-Meisenheimer complex is the only product.

Example 4 - Reduction of the hydride-Meisenheimer complex of TNT bv 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 mM potassium phosphate buffer, pH 7. The brown-puφle 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 UN- visible absorbance spectrum of the reaction mixture was identical to that seen during enzymic reduction of TΝT. When nitroreductase replaced PETΝ reductase, the orange colouration and the distinctive UN-visible spectrum associated with the orange products were not seen.

Example 5 - Liberation of nitrite from TΝT by PETΝ reductase

It was further noted that, during enzymic reduction of TΝT by PETΝ reductase, nitrite was liberated. A reaction mixture was set up containing 0.04 mg/ml PETΝ reductase, 2.0 MM ΝADPH, 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 μl was added to 594 μl water. To this diluted sample were added 200 μl of 10 mg/ml sulphanilamide in 0.68 M HC1, and 40 μl of 10 mg/ml N-(l- naphthyl)ethylenediamine in water. Visible absorbance at 540 nm was measured. Sodium nitrite was used as a standard. Results are shown as Figure 4. Over 3 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 hydride-Meisenheimer 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.

Example 6 - Growth of Transgenic Plants

In order to investigate the ability of transgenic plants to grow with TNT as a nitrogen source transgenic tobacco plants were produced. Tobacco was chosen due to its ease of genetic manipulation.

The onr gene was modified by PCR to introduce a plant consensus start sequence AACAATGG which resulted in the alteration of the first .amino acid from serine to alanine. To check the activity of the modified gene it was expressed in E. coli as described above and it was found that the activity was unaffected.

The modified gene was introduced into tobacco (Nicotiana tabacum cv xanthi) leaf discs by Agrobacterium-med ted transformation using the binary vector method of Cleave (A.P. Cleave, 1992, Plant Mol. Biol. 20, 1203-1207). Plants from 24 independent transformation events were regenerated. Genomic DNA was prepared from leaf tissue using a Phytopure kit (Scotlab). In all 24 lines the transgene was detected by PCR using the same primers as originally used for modification of the gene.

The primary plants were allowed to self fertilize and seeds were collected. These seeds were surface sterilized and germinated in Murashige and Skoog complete medium (ICN) amended with varying concentrations of explosives. These were contrasted with the growth of seeds from wild unmodified plants. TNT at 0.05 mM was found to seriously inhibit germination and growth of wild plants whereas the seeds of the transgenic plants grew comparably to seeds sown in media without explosive.

Claims

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

2. The use of bacterial strain E. cloacae PB2 of claim 1 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 1 and allowing the isolate to degrade the TNT in the environment.

4. A method according to claim 3 wherein TNT and PETN in the environment are degraded by the isolate.

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

6. The use of PETN reductase enzyme of claim 5 in the preparation of a composition used for the biodegradation of TNT in an environment.

7. 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 5 and maintaining the mixture under conditions appropriate for degradation of the contaminant by PETN reductase enzyme.

8. A kit for biodegradation of an environment contaminated with TNT comprising a store of PETN reductase of claim 5 and a means of contacting the environment with PETN reductase in the presence of N.ADPH and maintaining the environment under conditions appropriate for the degradation of TNT by PETN reductase.