Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
2000-11-27
2003-02-25
Nashed, Nashaat T. (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S195000, C435S252300, C435S252330, C435S254110, C435S320100, C435S262500, C536S023200, C536S023500, C424S094600
Reexamination Certificate
active
06524834
ABSTRACT:
The present invention relatesto a diisopropyl-fluorophosphatase (enzyme classification EC 3.1.8.2., called DFPase hereinafter) from
Loligo vulgaris,
to the base sequence encoding the enzyme, and to the use of the enzyme and a method for its production in transformed cells.
The term DFPase has a wide scope in the description of a class of enzymes able to hydrolyse diisopropyl fluorophosphate (DFP) and other organophosphorus compounds with a similar structure. The group of these organophosphorus compounds can be traced back to compounds of the following basic structure:
In this, Z represents oxygen or sulphur, Y represents a group which may, as H—Y compound, have acidic properties. These are, inter alia, anhydride groups (deprotonated acid groups)(in particular an F or CN group) or ester groups such as a thioester, an enol ester or a p-nitrophenyl ester group. The groups X
1
and X
2
may be straight- or branched-chain or cyclic alkoxy, alkyl, aryl, alkylamino or dialkylamino groups containing 1 to 15 carbon atoms. A large number of such compounds are or were used as insecticides. Other compounds are covered by the term of so-called nerve gases. The nerve gases of this class of compounds include, inter alia, DFP, tabun, soman, sarin, thylsarin and cyclosarin.
Reducing the stocks of these highly toxic compounds in the world is an increasing problem. In addition, it is necessary to find environmentally acceptable methods for decontaminating already contaminated areas of the environment.
The most important method to date for destroying large amounts of these substances comprises incineration at high temperatures.
In addition, for example, U.S. Pat. No. 4,666,696 deals with the destruction of nerve gases and other cholinesterase inhibitors by reduction with molten aluminium.
Further approaches to solving this problem comprise, for example, the use of physical methods. Thus, for example, U.S. Pat. No. 5,550,311 describes the thermal decomposition of toxic compounds (nerve gases) in an aqueous stream. Another approach is described in U.S. Pat. No. 5,648,591, in which chemical weapons such as, for example, sarin are broken down by activation in mechanical mills. All these methods have severe disadvantages in relation to their economic efficiency and their limited range of applications. Thus, for example, it is necessary in all these methods to feed the appropriate toxic substances into particular apparatuses, which makes, for example, use in the field difficult if not impossible.
Biological disposal of chemical weapons stocks and decontamination by microorganisms or enzymes which can be produced in large quantities might provide a simplification or a more effective approach to solving these problems.
The present invention therefore contrasts with the decontamination methods described above and builds on investigations by Aldrige as long ago as 1953. Aldridge observed that various tissues from different organisms contain enzymes which hydrolyse paraoxon, a cholinesterase inhibitor. Later investigations in 1966 by Hoskin and co-workers showed that the squid
Loligo pealii
contains a DFP-cleaving enzyme. Numerous investigations in this area have followed to the present day, but it has not yet been possible to sequence or even isolate a recombinant protein. The enzyme investigated by Hoskin could not be accurately characterized in terms of its molecular weight. Whereas Hoskin assumed a molecular weight of 26,600 Da, other research groups, for example Kopec-Smyth et al. (1993), regarded it as probable that the 26.6 kDa protein is only a fragment of a very unstable 42 kDa protein. A short amino acid sequence of a peptide fragment of this DFPase was published in 1993 by Ward and Deschamps. The sequence section described therein is not found in the claimed amino acid sequence of the present invention, for which reason it must be assumed that the DFPase from
Loligo pealii
differs distinctly from the DFPase of the present invention.
Similar proteins have been identified in a wide variety of organisms apart from
Loligo pealii.
Thus, they have been found inter alia in thermophilic or halophilic bacteria, in
E. coli, Proteus vulgaris, Saccharomyces cerevisiae, Pseudomonas diminuta,
Flavobacterium and the eukaryotic unicellular organism
Tetrahymena thermophilia,
and in insects and invertebrates. Detection has also been possible in mammals such as mice, rats, rabbits, pigs and humans. Despite the efforts of a large number of research groups concerned with enzymes which resemble in their specificity the DFPase from
Loligo vulgaris
of the present invention, none of these groups has succeeded incompletely elucidating the amino acid sequence, and the nucleotide sequence underlying the latter, of an enzyme corresponding to the present invention, in transforming the corresponding nucleotide sequence using a vector into a host cell, and thus in making industrial production possible in excellent purity. Although Dierl (1995, thesis, Johann Wolfgang Goethe University, Frankfurt/Main) was able during his research work to elucidate a partial sequence of the DFPase from
Loligo vulgaris,
no details of the amino acid sequence or nucleotide sequence are to be found in this study. The partial information provided by Dierl is incomplete and reproducibility of the results presented therein is impossible simply because of the unavailability to the public of the cDNA gene bank on which the study is based but which is not accessible to the public.
One of the objects of the present invention is to provide the complete and functioning DFP-cleaving enzyme from
Loligo vulgaris
and the nucleotide sequence encoding the enzyme in order to make industrial production of a genetic engineering type possible. A further object was to provide a DFPase which can be isolated on an industrial scale without loss of stability by fractional ammonium sulphate precipitation and which has its activity optimum at a neutral pH of about 7.5 and room temperature (about 25° C.). It was intended thereby not least to achieve the object of environmentally friendly, energy-saving disposal of nerve gases and insecticides. A further object to be achieved was to provide a storage- and solvent-stable DFPase. Thus, it was intended, for example, that a concentrated solution be stable with negligible loss of activity at 4° C. for a lengthy period. It was also intended that various solvents or solvent-containing aqueous media (for example 10% strength aqueous ethanolic solution) have no effect on the activity of the enzyme. A further object to be achieved was to provide a DFPase which is active in a wide variety of buffer systems. The provision of the DFPase was further intended to make it possible to decontaminate, detoxify and moreover detect acetylcholinesterase inhibitors which serve as substrate of the DFPase. The provision of such an enzyme is intended not only to make the industrial destruction of appropriate nerve gases or insecticides possible but also to provide the possibility of decontaminating contaminated habitats (soils, watercourses, etc.). A cloning in plants is also possible thereby. It is further conceivable to employ enzyme for producing medicinal products for detoxifying or treating humans and animals. This might involve the enzyme being employed, for example, locally in the form of a skin cream, parenterally in the form of a solution for infusion or inhalation, or else orally.
The said objects have been achieved by providing a DFPase of the following amino acid sequence (SEQ ID NO: 1):
Met Glu Ile Pro Val Ile Glu Pro Leu Phe Thr Lys Val Thr Glu Asp
1 5 10 15
Ile Pro Gly Ala Glu Gly Pro Val Phe Asp Lys Asn Gly Asp Phe Tyr
20 &em
Dierl Stefan
Ruterjans Heinz
Nashed Nashaat T.
Roche Diagnostics GmbH
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