Biodegradation of explosives

Details Biodegradation of explosives Biodegradation of explosives

1998-12-23

2001-08-14

Ware, Deborah K. (Department: 1651)

Chemistry: molecular biology and microbiology

Micro-organism, per se ; compositions thereof; proces of...

Bacteria or actinomycetales; media therefor

C435S029000, C435S071100, C435S262500, C435S822000

Reexamination Certificate

active

06274368

ABSTRACT:

This invention relates to the field of explosives detection and biodegradation and in particular to novel bacterial isolate and a novel enzymic activity, i.e. a single enzyme or group of enzymes, derived therefrom. This invention further relates to the aerobic biodegradation of hexahydro-1,3,5-trinitro-1,3,5-triazine (hereinafter referred to by the commonly used abbreviation RDX) and to methods and apparatus for the detection of RDX using the RDX degrading enzymic activity.
The novel enzymic activity has been shown to liberate nitrite from RDX, a heterocyclic nitramine.
Nitramines, although apparently extremely rare in nature, are produced in significant quanties by the chemical industry and comprise, for example, an important class of energetic materials having applications as explosives and propellants, RDX is currently the most important mlitary explosive in the United States. The manufacture, handling and disposal of RDX can all lead to the contamination of the environment with RDX. There are concerns regarding the environmental fate of nitramines due to their relative recalcitrance and therefore there exists a need for a means of removing such contaminants from the environment without producing other undesirable pollutants. There is also an urgent requirement for a better method of detecting RDX as the currently proposed analytical systems rely mostly on bulky and sophisticated pieces of equipment such as High Performance Liquid Chromatography and/or require specially trained laboratory technicians for their application.
It is an aim of this invention to provide an enzyme which is capable of catalysing the biodegradation of RDX and which may be employed in a bioremediation system for the environmental contamination of the RDX pollutant. It is a further aim of this project to provide an enzyme which is useful for RDX detection systems.
Thus according to a first aspect of the invention there is provided a
Rhodococcus rhodochrous
11Y bacterial strain preferred to as “11Y” and deposited as NCIMB 40820, and mutants and variants thereof, capable of producing enzymic activity which degrades hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) and in a second aspect of the invention there is provided an RDX degrading enzymic activity characterised in that it catalyses the release of nitrite from hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) and is obtained from cells of
Rhodacoccus rhodochrous
11Y or mutants or variants thereof.
This enzymic activity shows lesser activity against the similar heterocyclic nitramine octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) and less activity again against the nitrate ester pentaerythritol tetranitrate (PETN). The activity was found to be membrane-associated and could be solubilised from the membrane with 5% triton. There is no requirement for a co-factor such as NADPH, NADH, PES or FAD for enzymic activity and the enzymic activity exhibits stablilty to the presence of relatively high concentrations of denaturants and the activity is increased in the presence of urea. The enzymic activity is also relatively stable to heat denaturation. Further, a large proportion of the enzymic activity remains soluble when the pH is reduced to 3.5with glacial acetic acid.
The bacterial strain from which the enzymic activity of the current invention is obtained was isolated from nutur and is a strain of
Rhodococcus rhodochrous
herein designated 11Y. A sample of the novel isolate has been deposited under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the purpose of patent procedure at the UK National Collection of Industrial and Marine Bacteria, 23 St. Machar Drive, Aberdeen, AB2 1RY, Scotland on Aug. 7, 1996 under the deposit number NCIMB 40820.
Gram stain
+ve
Spores
−ve
Motility
−ve
Growth
37° C.
+ve
41° C.
+ve
45° C.
−ve
Catalase
+ve
Oxidase
−ve
Fermentative
No change
in Glucose OF
A cell wall and fatty acid analysis provided the following information.
Mycolic acids are present.
The cell wall diamino acid is mesoDAP.
The fatty acid profile shows that the major acids present are straight chain saturated and unsaturated acids together with a small amount of a 10-methyl branched acid i.e. tuberculostearic acid.
The enzymic activity can be produced by culturing
R. rhodochroits
on ROX or NH
4
Cl as a nitrogen source. To obtain the enzymic activity the cells can be disrupted in any conventional way. Conveniently a cell free extract is made. The extract may then be fractionated by ultracentrifugation and the pellet taken to provide active membrane fraction.
Alternatively the supernatant from ultracentrifugation provides active soluble protein. The soluble protein may be partially purified using anion exchange chromatography and eluted with a linear salt gradient from 0-2 M sodium chloride. The protein elutes as a single peak at approximately 350 mM sodium chloride.
The enzymic activity obtained as cell extract requires the presence of dithiothreitol (DTT) for RDX degrading enzymic activity.
Instead of the precise starting organism deposited, a mutant thereof, eg derived by gamma-ray irradiation or the use of a chemical mutant, induction by culture on another medium etc. or a transconjugant thereof with another bacterium or an artificially produced variant can be used. The ability of any such organism to give the enzymic activity can be readily determined by the skilled person.
The ability of the novel enzymic activity to catalyse the removal of nitrite from RDX allows the enzymic activity to be used in the detection of RDX. According to a further aspect of the invention therefore, there is provided a method of detecting the presence of RDX in a sample which comprises exposing the sample to the RDX-degrading enzymic activity and detecting any nitrite liberated from said sample. Conveniently such detection would be by means of a colorimetric method as is well known is the art.
The removal of nitrite from RDX may create an unstable intermediate which then spontaneously degrades to give a range of smaller molecules including formaldehyde and ammonia. In a further aspect of the present invention therfore there is provided a method of detecting the presence of RDX in a sample which comprises exposing the sample to the RDX degrading enzymic activity and detecting any formaldehyde produced. Such detection could again be by means of a colorimetric method as known in the art.
In a further aspect, the present invention also provides a biosensor for the detection of RDX in a sample which comprises means for contacting the sample with the RDX degrading enzymic activity and means for detecting occurrence of a reaction, catalysed by the enzymic activity, of RDX when RDX is present in the sample. Alternatively in a further aspect there is provided a biosensor for the detection of RDX in a sample which comprises means of innoculating the sample with a culture of the bacterial isolate
Rhodococcus rhodochrous
11Y and maintaining the sample under conditions appropriate for degradation of the contaminent by the isolate and means of detecting the occurrence of a reaction, catalysed by the isolate, of RDX when RDX is present in the sample. The means for detecting the occurrence of a reaction may conveniently comprise a colorimetric transducer. Such sensors can be used as the basis for highly portable detectors for analysing the extent of contamination of the RDX pollutant within the environment.
A further aspect of this present invention is the provision of a method for the bioremdial treatment of an environment contaminated with RDX, which method comprises the steps of adding to the contaminated environment a quantity of cell extract containing the enzymic activity and maintaining the mixture under conditions appropriate for degradation of RDX by the enzymic activity such that the RDX present in the material is consumed.
The material consumed may be, for example, a waste stream of material containing RDX originating from the destruction of an explosives charge containing RDX or

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