Chemistry: molecular biology and microbiology – Process of utilizing an enzyme or micro-organism to destroy... – Destruction of hazardous or toxic waste
Reexamination Certificate
1995-10-23
2001-09-04
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Process of utilizing an enzyme or micro-organism to destroy...
Destruction of hazardous or toxic waste
C435S069100, C435S091100, C435S091400, C435S252300, C435S122000, C530S350000, C536S024300
Reexamination Certificate
active
06284522
ABSTRACT:
BACKGROUND OF THE INVENTION
Atrazine [2-chloro-4-(ethylamino)-6-(isopropylamino)-1,3,5-triazine)] is a widely used s-triazine (i.e., symmetric triazine) herbicide for the control of broad-leaf weeds. Approximately 800 million pounds were used in the United States between 1980 and 1990. As a result of this widespread use, for both selective and nonselective weed control, atrazine and other s-triazine derivatives have been detected in ground and surface water in several countries.
Numerous studies on the environmental fate of atrazine have shown that atrazine is a recalcitrant compound that is transformed to CO
2
very slowly, if at all, under aerobic or anaerobic conditions. It has a water solubility of 33 mg/l at 27° C. Its half-life (i.e., time required for half of the original concentration to dissipate) can vary from about 4 weeks to 57 weeks if in soils at low concentration (i.e., less than about 2 parts per million (ppm)). High concentrations of atrazine, such as those occurring in spill sites, have been reported to dissipate even more slowly.
As a result of its widespread use, atrazine is often detected in ground water and soils in concentrations exceeding the maximum contaminant level (MCL) of 3 &mgr;g/l (i.e., 3 parts per billion (ppb)), a regulatory level that took effect in 1992. Point source spills of atrazine have resulted in levels as high as 25 ppb in some wells. Levels of up to 40,000 mg/l (i.e., 40,000 parts per million (ppm)) atrazine have been found in the soil of spill sites more than ten years after the spill incident. Such point source spills and subsequent runoff can cause crop damage and ground water contamination.
There have been numerous reports of the isolation of s-triazine-degrading microorganisms (see, e.g., Behki et al.,
J. Agric. Food Chem
., 34, 746-749 (1986); Behki et al.,
Appl. Environ. Microbiol
., 59, 1955-1959 (1993); Cook,
FEMS Microbiol. Rev
., 46, 93-116 (1987); Cook et al.,
J. Agric. Food Chem
., 29, 1135-1143 (1981); Erickson et al.,
Critical Rev. Environ. Cont
., 19, 1-13 (1989); Giardina et al.,
Agric. Biol. Chem
., 44, 2067-2072 (1980); Jessee et al.,
Appl. Environ. Microbiol
., 45, 97-102 (1983); Mandelbaum et al.,
Appl. Environ. Microbiol
., 61, 1451-1457 (1995); Mandelbaum et al.,
Appl. Environ. Microbiol
., 59, 1695-1701 (1993); Mandelbaum et al.,
Environ. Sci. Technol
., 27, 1943-1946 (1993); Radosevich et al.,
Appl. Environ. Microbiol
., 61, 297-302 (1995); and Yanze-Kontchou et al.,
Appl. Environ. Microbiol
., 60, 4297-4302 (1994)). Many of the organisms described, however, failed to mineralize atrazine (see, e.g., Cook,
FEMS Microbiol. Rev
., 46, 93-116 (1987); and Cook et al.,
J. Agric. Food Chem
., 29, 1135-1143 (1981)). While earlier studies have reported atrazine degradation only by mixed microbial consortia, more recent reports have indicated that several isolated bacterial strains can degrade atrazine. For example, we previously reported the isolation of a pure bacterial culture, identified as Pseudomonas sp. strain ADP (Mandelbaum et al.,
Appl. Environ. Microbiol
., 61, 1451-1457 (1995); Mandelbaum et al.,
Appl. Environ. Microbiol
., 59, 1695-1701 (1993); and Mandelbaum et al.,
Environ. Sci. Technol
., 27, 1943-1946 (1993)), which degraded a high concentration of atrazine (>1,000 &mgr;g/ml) under growth and non-growth conditions. See also, Radosevich et al.,
Appl. Environ. Microbiol
., 61, 297-302 (1995) and Yanze-Kontchou et al.,
Appl. Environ. Microbiol
., 60, 4297-4302 (1994). Pseudomonas sp. strain ADP (Atrazine Degrading Pseudomonas) uses atrazine as a sole source of nitrogen for growth. The organism completely mineralizes the s-triazine ring of atrazine under aerobic growth conditions. That is, this bacteria is capable of degrading the s-triazine ring and mineralizing organic intermediates to inorganic compounds and ions (e.g., CO
2
).
Little information is available concerning the genes and enzymes involved in the metabolism of s-triazine compounds. Although genes that encode the enzymes for melamine (2,4,6-triamino-s-triazine) metabolism have been isolated from a Pseudomonas sp. strain, and that encode atrazine degradation activity from Rhodococcus sp. strains, to date there have been no reports identifying the genes encoding atrazine dechlorination.
SUMMARY OF THE INVENTION
The present invention provides an isolated and purified DNA molecule that encodes atrazine chlorohydrolase. The DNA molecule hybridizes to DNA complementary to DNA having the sequence shown in
FIG. 6
(SEQ ID NO:1), beginning at position 236 and ending at position 1655, under the stringency conditions of hybridization in buffer containing 0.25 M Na
2
HPO
4
, 7% SDS, 1% BSA, 1.0 mM EDTA at 65° C., followed by washing with 0.1% SDS and 0.1×SSC at 65° C. Preferably, the present invention provides an isolated and purified DNA molecule encoding the atrazine chlorohydrolase having an amino acid sequence shown in
FIG. 7
(SEQ ID NO:2). Preferably, the DNA molecule has the nucleotide sequence shown in
FIG. 6
(SEQ ID NO:1) beginning at position 236 and ending at position 1655. The present invention also provides a vector comprising the DNA molecule described herein, a transformed cell line, and isolated and purified oligonucleotides of about 7-300 nucleotides.
The present invention also provides an isolated and purified protein having a molecular weight of about 245 kilodaltons that converts atrazine to hydroxyatrazine. Preferably, this protein has the amino acid sequence shown in
FIG. 7
(SEQ. ID NO:2). Also provided is an isolated and purified preparation of polyclonal antibodies produced from this isolated and purified protein.
The present invention also provides a method for the purification of atrazine chlorohydrolase in at least about 90% yield consisting of a step of adding ammonium sulfate to an aqueous cell-free extract of an atrazine chlorohydrolase-containing bacterium. This ammonium sulfate is present in an amount of no more than about 20% of saturation. Finally, the present invention provides a method for degrading s-triazine compounds.
REFERENCES:
patent: 4745064 (1988-05-01), Cook et al.
patent: 5429949 (1995-07-01), Radosevich et al.
patent: 5508193 (1996-04-01), Mandelbaum et al.
patent: 0 141 784 (1984-10-01), None
patent: WO 95/01437 (1995-01-01), None
de Souza et al., “Atrazine Chlorohydrolase from Pseudomonas sp. Strain ADP: Gene Sequence, Enzyme Purification, and Protein Characterization,”Journal of Bacteriology, 178(16), 4894-4900 (1996).
EMBL Database entry TT3ITRA, Accession No. M13165, Jul. 16, 1988 Sequence: Huang et al., “Analysis of Tn3 sequences required for transposition and immunity,”Gene, 41, 23-31 (1986).
Shao et al., “Cloning and Expression of the s-Traizine Hydrolase Gene (trzA) fromRhodococcus corallinusand Development of Rhodococcus Recombinant Strains Capable of Dealkylating and Dechlorinating the Herbicide Atrazine,”Journal of Bacteriology, 177(20), 5748-5755 (1995).
EMBL Database Entry RCTRZA Accession No. L16534; Oct. 2, 1993, Shao et al.,Rhodococcus corallinus(NRRL 15444B) N-ethylammeline chlorohydrolase (trzA) gene, complete cds.
de Souza et al., “Cloning, Characterization, and Expression of a Gene Region from Pseudomonas sp. Strain ADP Involved in the Dechlorination of Atrazine,”Applied and Environmental Microbiology, 61(9) 3373-3378 (1995).
U.S. Department of Agriculture—BARD Program, Grant No. 94-34339-112, obtained from the Departartment of BARD (1994) Abstract only.
Armstrong et al., “Adsorption Catalyzed Chemical Hydrolysis of Atrazine,”Environ. Sci. Technol., 2(9), 683-689 (1968).
Behki et al., “Metabolism of the Herbicide Atrazine by Rhodococcus Strains,”Applied and Environmental Microbiology, 59(6), 1955-1959 (1993).
Behki et al., “Degradation of Atrazine by Pseudomonas: N-Dealkylation and Dehalogenation of Atrazine and Its Metabolites,”J. Agric. Food Chem., 34, 746-749 (1986).
Bergman et al., “Determination of Trace Amounts in Chlorine in Naphtha,”Anal. Chem., 29(2), 241-243 (1957).
Chang et al., “Construction and Characterization of Amplifiable Mul
de Souza Mervyn L.
Sadowsky Michael J.
Wackett Lawrence P.
Hutson Richard
Mueting Raasch & Gebhardt, P.A.
Prouty Rebecca E.
Regents of the University of Minnesota
LandOfFree
Isolated and purified DNA molecule and protein for the... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Isolated and purified DNA molecule and protein for the..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Isolated and purified DNA molecule and protein for the... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2538563