Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving hydrolase
Reexamination Certificate
1998-02-23
2001-06-19
Smith, Lynette R. F. (Department: 1645)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving hydrolase
C530S344000, C530S350000, C435S006120, C435S032000, C435S228000, C435S106000, C514S002600
Reexamination Certificate
active
06248551
ABSTRACT:
This invention relates to Helicobacter species aliphatic amidase AmiE polypeptides, the DNA encoding those polypeptides, and transformed microorganisms capable of expressing those polypeptides. In addition, this invention relates to the use of Helicobacter sp. particularly
Helicobacter pylori
) amidase AmiE polypeptides and antibodies specific for those polypeptides in immunogenic, therapeutic and diagnostic application.
BACKGROUND OF THE INVENTION
An aliphatic amidase is an acylamide amidohydrolase (E.C. 3.5.1.4) (Merck Index). It hydrolyses short-chain aliphatic amides (C1-C4 such as acrylamide, acetamide, propionamide or isobutyramide) to produce ammonia and the corresponding organic acid. In addition, an aliphatic amidase possesses acyl transferase activity, i.e., it is able to transfer the acyl group of amides to hydroxylamine to form an acyl hydroxamate plus ammonia.
Aliphatic amidases have been identified in
Pseudomonas aeruginosa
(Brammar et al., 1987) and Rhodococcus sp. R312 (previously named Brevibacterium sp. R312; Soubrier et al., 1992). Other aliphatic amidases have been identified in
Methylophilus methylotrophus
(Silman et al., 1991), Arthrobacter sp. J-1 (Asano et al., 1982), and
Alcaligenes eutrophus
(Friedrich and Mitrenga, 1981). However, no molecular characterization of these latter three enzymes has been reported.
Aliphatic amidases are cytoplasmic enzymes; they have very similar enzymatic properties and molecular masses (38.4 kDa for
P. aeruginosa;
38.2 kDa for Rhodococcus sp. R312; 37.8 kDa for
M. methylotrophus;
and 39 kDa for Arthrobacter sp. J-1), and have either a tetra-, hexa-, or octameric structure. Some of these amidases have been shown to be inducible by their amide substrate. Database searches with the amino acid sequences of these aliphatic amidases indicates that they are more closely related to nitrilases (which catalyze the direct cleavage of nitrites to ammonia and to the corresponding acid) than to the nitrile hydratases (which hydrolyze nitrites to produce amides) or amidases from other classes (Novo et al., 1995).
The prevailing theory on the physiological role of the aliphatic amidases is that hydrolysis of amides supplies carbon and nitrogen sources to the bacteria. Curiously, Helicobacter sp. possess a very potent urease, which should be sufficient for nitrogen supply in this genus of bacteria. However, Helicobacter sp. are not the only bacteria possessing both urease and amidase, since this is also the case for
P. aeruginosa, M. methylotrophus,
and
A. eutrophus.
Acrylamide, an aliphatic amide, is extensively used in a great number of industrial processes. Global production of acrylamide has been estimated to be over 200,000 tons. Widespread use and indiscriminate discharge of acrylamide have resulted in the contamination of terrestrial and aquatic ecosystems throughout the world. Other aliphatic amides are either active ingredients or metabolites of herbicide degradation (Roberts, 1984). Elimination of acrylamide and other toxic aliphatic amide by-products by an aliphatic amidase would be of great importance because these substances pose serious health hazards for humans and animals (Nawaz et al., 1994, 1996) (Nagasawa and Yamada, 1989).
Helicobacter pylori
has become identified as a primary cause of chronic gastroduodenal disorders, such as gastritis, dyspepsia, and peptic ulcers, in humans.
H. pylori
can be successfully eradicated (80% to 90%) by a treatment combining two antibiotics with a proton pump inhibitor. However, few antibiotics are active against
H. pylori,
and antibiotic resistant strains (e.g., to metronidazole or clarythromycin) have begun to appear. Like
H. pylori, Helicobacter heilmanii
has been identified as the cause of gastric ulcers in pigs. Porcine gastric ulcers lead to lower weight pigs and consequently, less food product production. Due to the presence of numerous urea positive bacteria in the porcine gastrointestinal tract, methods that are not based on urease are preferred for detecting, treating or preventing Helicobacter infections in pigs.
Thus, a need exists for an effective method of diagnosing, preventing, and treating gastrointestinal disorders caused by Helicobacter sp., particularly
H. pylori
and
H. heilmanii.
SUMMARY OF THE INVENTION
This invention provides polynucleotides corresponding to Helicobacter species aliphatic amidase amiE. More particularly, this invention provides polynucleotides selected from the group consisting of:
(a) all or part of the DNA sequence encoding Helicobacter sp. and particularly,
Helicobacter pylori
aliphatic amidase amiE (depicted in FIG.
5
);
(b) all or part of the DNA sequence depicted in
FIG. 4
;
(c) a DNA sequence which hybridizes with all or part of DNA sequence (a) or (b) under stringent conditions and encodes a polypeptide having the biological or immunological properties of Helicobacter sp. and particularly,
Helicobacter pylori
aliphatic amidase or a fragment thereof; and
(d) an analog of DNA sequence (a), (b), or (c) resulting from the degeneracy of the genetic code.
Other aspects of this invention include polypeptides encoded by the polynucleotides of this invention; antibodies to those polypeptides; immunogenic, pharmaceutical, and therapeutic compositions comprising the polypeptides and antibodies of the invention; methods of using the polypeptides and antibodies of the invention to detect, treat or prevent Helicobacter sp. infections in man and animals; detection kits comprising the polypeptides and antibodies of the invention; processes for producing polypeptides according to the invention and intermediates useful in their production.
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Stedman'
De Reuse Hilde
Labigne Agnes
Skouloubris Stephane
Finnegan Henderson Farabow Garrett & Dunner L.L.P.
Institut Pasteur
Portner Ginny Allen
Smith Lynette R. F.
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