Chemistry: molecular biology and microbiology – Micro-organism – per se ; compositions thereof; proces of... – Bacteria or actinomycetales; media therefor
Reexamination Certificate
1998-09-17
2001-07-24
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, per se ; compositions thereof; proces of...
Bacteria or actinomycetales; media therefor
C435S069100, C435S093000, C435S183000, C435S195000, C435S320100, C435S252330, C536S023200
Reexamination Certificate
active
06265201
ABSTRACT:
BACKGROUND OF THE INVENTION
More than 8 million organic compounds are known and many are thought to be biodegradable by microorganisms, the principle agents for recycling organic matter on Earth. In this context, microbial enzymes represent the greatest diversity of novel catalysts. This is why microbial enzymes are predominant in industrial enzyme technology and in bioremediation, whether used as purified enzymes or in whole cell systems.
There is increased interest in engineering bacterial enzymes for improved industrial performance. For example, site directed mutagenesis of subtilisin has resulted in the development of enzyme variants with improved properties for use in detergents. Most applied enzymes, particularly those used in biodegrading pollutants, however, are naturally evolved. That is, they are unmodified from the form in which they were originally present in a soil bacterium.
For example, most bioremediation is directed against petroleum hydrocarbons, pollutants that are natural products and thus have provided selective pressure for bacterial enzyme evolution over millions of years. Synthetic compounds not resembling natural products are more likely to resist biodegradation and hence accumulate in the environment. This changes over a bacterial evolutionary time scale; compounds considered to be non-biodegradable several decades ago, for example PCBs and tetrachloroethylene, are now known to biodegrade. This is attributed to recent evolution and dispersal of the newly evolved gene(s) throughout microbial populations by mechanisms such as conjugative plasmids and transposable DNA elements.
A better understanding of the evolution of new biodegradative enzymes will reveal how nature cleanses the biosphere. Furthermore, the ability to emulate the process in the laboratory may shave years off the lag period between the introduction of a new molecular compound into the environment and the development of a dispersed microbial antidote that will remove it.
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-containing compounds have been detected in ground and surface water in several countries.
Numerous studies on the enviromnental 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 about 57 weeks when present at a low concentration (i.e., less than about 2 parts per million (ppm)) in soil. 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/i (i.e., 40,000 parts per million (ppm)) atrazine have been found in the soil at 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 on 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. Azric. 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. In fact, research groups have identified atrazine-degrading bacteria classified in different genera from several different locations in the U.S. (e.g., Minnesota, Iowa, Louisiana, and Ohio) and Switzerland (Basel).
An atrazine-degrading 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); de Souza et al.,
J. Bact.,
178, 4894-4900 (1996); and Mandelbaum et al.,
Environ. Sci. Technol.,
27, 1943-1946 (1993)), was isolated and was found to degrade atiazine at concentrations greater than about 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 Pseudononas) 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
).
The genes that encode the enzymes for MELAMINE (2,4,6-triamino-s-triazine) metabolism have been isolated from a Pseudomonas sp. strain. The genes that encode atrazine degradation activity have been isolated from Rhodococcus sp. strains; however, the reaction results in the dealkylation of atrazine. In addition, the gene that encodes atrazine dechlorination has been isolated from a Pseudomonas sp. strain. See, for example, de Souza et al.,
Appl. Environ. Microbiol.,
61, 3373 (1995). The protein expressed by the gene disclosed by de Souza et al., degrades atrazine, for example, at a V
max
of about 2.6 &mgr;mol of hydroxyatrazine per min per mg protein. Although this is significant, it is desirable to obtain genes and the proteins they express that are able to dechlorinate triazine-containing compounds with chlorine moieties at an even higher rate and/or under a variety of conditions, such as, but not limited to, conditions of high temperature (e.g., at least about 45° C. and preferably at least about 65° C.), various pH conditions, and/or under conditions of high salt content (e.g., about 20-30 g/L), or under other conditions in which the wild type enzyme is not stable, efficient, or active. Similarly, it is desirable to obtain genes and proteins encoded by these genes that degrade triazine-containing compounds such as those triazine containing compounds available under the trade names; “AMETRYN”, “PROMETRYN”, “CYANAZINE”, “MELAMINE”, “SIMAZINE”, as well as TERBUTHYLAZINE and desethyldesisopiopylatriazine. It is also desirable to identify proteins expressed in organisms that degrade triazine-containing compounds in the presence of other nitrogen sources such as ammonia and nitrate.
SUMMARY OF THE INVENTION
The present invention provides isolated and purified DNA molecules that encode atrazine degrading enzymes similar to, but having different catalytic activities from a wild type (i.e. from an isolated but naturally occurring atrazine chlo
de Souza Mervyn L.
Minshull Jeremy S.
Sadowsky Michael J.
Wackett Lawrence P.
Hutson Richard
Mueting Raasch & Gebhardt, P.A.
Prouty Rebecca E.
Regents of the University of Minnesota
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