Detection and biodegradation of explosives

Details Detection and biodegradation of explosives Detection and biodegradation of explosives

1998-02-02

1999-07-27

Wax, Robert A.

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

435 41, 435191, 4352523, 4352625, 4353201, 435822, 536 232, C12Q 100, C12N 906, B09B 300, C07H 2104

Patent

active

059288590

DESCRIPTION:

BRIEF SUMMARY
This invention relates to the field of explosives detection and biodegradation and in particular to a novel enzyme, to the amino acid sequence for the enzyme, to the gene which encodes the enzyme and to methods of producing the enzyme recombinantly. The invention further relates to a method of using the enzyme to catalyze the aerobic biodegradation of pentaerythritol tetranitrate (hereinafter referred to by the commonly used abbreviation PETN) and to a method and apparatus for the detection of PETN using the enzyme.
The novel enzyme of this invention has also been demonstrated to liberate nitrite from glycerol trinitrate (GTN) and from ethylene glycol dinitrate (EGDN) which are further examples of nitrate ester species. The enzyme is referred to as a PETN reductase.
Nitrate esters, though apparently extremely rare in nature, are produced in significant quantities by the chemical industry and comprise, for example, an important class of energetic materials having applicability as explosives and propellants. PETN itself has a diverse range of applications including as an explosive in blasting caps and detonators and in pharmaceutical compositions as the active ingredient of long-acting, slow onset coronary vasodilators for the prevention of angina attacks. The manufacture, handling and disposal of PETN can all lead to the contamination of the environment with PETN. There are concerns regarding the environmental fate of nitrate esters due to their relative recalcitrance and there therefore 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 PETN as the currently proposed analytical systems rely mostly on use of bulky and sophisticated pieces of equipment such as gas chromatograms or mass spectrometers 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 catalyzing the biodegradation of PETN and which may be employed in a bioremediation system for the environmental decontamination of the PETN pollutant. It is a further aim to provide an enzyme which is useful for PETN detection systems.
According to a first aspect of the present invention therefore, there is provided a PETN reductase enzyme which catalyses the removal of nitrite from PETN characterised in having the amino acid sequence shown in FIG. 4 or a derivative thereof By derivative herein is meant a version of the enzyme sequence of FIG. 4 containing insertions, deletions and/or substitutions of the amino acid sequence such that the functionality of the enzyme is retained.
Based on the evidence of the amino acid sequence it is possible to state that the enzyme is a member of the family of .alpha./.beta.-barrel flavoprotein oxidoreductases.
This PETN reductase enzyme has the characteristics that, in the presence of nicotinamide adenine dinucleotide phosphate (NADPH hereinafter), it:
1) catalyses the removal of nitrite from PETN; and
2) has reductase activity specifically at the nitrate ester linkage of PETN.
The enzyme further has a pH optimum of 6.5 and has a M.sub.r of about 40,000 Daltons as determined by gel filtration. The subunit M.sub.r, as estimated by SDS-PAGE (polyacrylamide gel electrophoresis), was also 40 000. These results suggest that PETN reductase is a monomer of M.sub.r approximately 40,000.
The PETN reductase enzyme of this invention may be further identified by additional characteristics such as its pH optimum, catalytic activity, thermal stability or molecular weight. Details of such further characteristics are given in Example 3 below but it must be stressed that these characteristics are variable to a degree depending upon the conditions under which the microorganism producing the enzyme is grown and upon the degree of purification of the crude product. Variations of this kind will be well understood by those skilled in the art.
The purified enzyme is visibly yellow and shows a visible absorption

REFERENCES:
Chemical Abstracts, vol. 109, No. 7, Aug. 15, 1988 Columbus, Ohio, US; abstract No. 47787, Posadas Del Rio, Francisco A. et al: "Biotransformation of organic nitrate esters in vitro by human liver, kidney, intestine, and blood serum" XP002018204 see abstract & Drug Metab. Dispos. (1988), 16(3), 477-81 CODEN: DMDSAI;ISSN: 0090-9556.
Chemical Abstracts, vol. 125, No. 3, Jul. 15, 1996 Columbus, Ohio, US; abstract No. 27992, Binks, Peter R. et al: "Degradation of pentaerythritol tetranitrate by Enterobacter cloacae PB2" XP002018205 see abstract & Appl. Environ. Microbiol. (1996), 62(4), 1214-19 CODEN: AEMIDF;ISSN: 0099-2240.

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