Isolated and purified DNA molecule and protein for the degradation of triazine compounds

Abstract

Abstract of the Disclosure An isolated and purified DNA molecule, and an isolated and purified protein, that are involved in the degradation of s-triazine compounds (e.g., atrazine) are provided. A method for the purification of this protein is also provided.

What is claimed is:  
     
         1 . An isolated and purified DNA molecule encoding 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.  
     
     
         2 . The isolated and purified DNA molecule of  claim 1  encoding the atrazine chlorohydrolase having an amino acid sequence shown in FIG. 7 (SEQ ID NO:2).  
     
     
         3 . The isolated and purified DNA molecule of  claim 1  having the nucleotide sequence shown in FIG. 6 (SEQ ID NO:1) beginning at position 236 and ending at position 1655.  
     
     
         4 . The isolated and purified DNA molecule of  claim 1  having the nucleotide sequence shown in FIG. 6 (SEQ. ID NO:1).  
     
     
         5 . An isolated and purified protein having a molecular weight of about 245 kilodaltons that converts atrazine to hydroxyatrazine.  
     
     
         6 . The isolated and purified protein of  claim 5  which is a homotetramer.  
     
     
         7 . The isolated and purified protein of  claim 5  which has the amino acid sequence shown in FIG. 7 (SEQ. ID NO:2).  
     
     
         8 . The isolated and purified protein of  claim 7  bound to an immobilization support.  
     
     
         9 . An isolated and purified protein encoded by the DNA molecule of  claim 1 .  
     
     
         10 . An isolated and purified protein encoded by the DNA molecule of  claim 3 .  
     
     
         11 . A polyclonal antibody preparation produced from the isolated and purified protein of  claim 5 .  
     
     
         12 . A polyclonal antibody preparation produced from the isolated and purified protein of  claim 7 .  
     
     
         13 . A vector comprising the DNA molecule of  claim 1 .  
     
     
         14 . The vector of  claim 13  wherein the DNA molecule of  claim 1  is derived from a Pseudomonas strain.  
     
     
         15 . A non-Pseudomonas bacterial cell comprising the vector of  claim 14 .  
     
     
         16 . An isolated and purified oligonucleotide of about 7-300 nucleotides which hybridizes 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.  
     
     
         17 . 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.  
     
     
         18 . The method of  claim 17  wherein ammonium sulfate is added in an amount of no greater than about 20% of saturation.  
     
     
         19 . A method for the degradation of compounds have the following general formula:  
       
         
           
           
               
               
           
         
         wherein R 1 ═Cl, R 2 ═NR 4 R 5  (wherein R 4  and R 5  are each independently H or a C 1−3  alkyl group), and R 3  ═NR 6 R 7  (wherein R 6  and R 7  are each independently H or a C 1−3  alkyl group), with the proviso that at least one of R 2  or R 3  is an alkylamino group; said method comprising adding atrazine chlorohydrolase to a sample containing said compound.  
       
     
     
         20 . The method of  claim 19  wherein the sample is a soil sample.  
     
     
         21 . The method of  claim 20  wherein the soil sample is contaminated with a nitrogen-containing fertilizer.  
     
     
         22 . The method of  claim 19  wherein the step of adding atrazine chlorohydrolase comprises adding a recombinant bacterium that expresses atrazine chlorohydrolase.  
     
     
         23 . The method of  claim 19  wherein the step of adding atrazine chlorohydrolase comprises adding the bacterial cell of  claim 15 .  
     
     
         24 . An isolated and purified protein that converts atrazine to hydroxyatrazine, wherein the protein comprises an amino acid sequence encoded by a DNA molecule having a compliment that hybridizes to a 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.  
     
     
         25 . An isolated and purified protein and biologically active derivatives thereof that convert atrazine to hydroxyatrazine, wherein the protein comprises an amino acid sequence encoded by a DNA molecule having a compliment that hybridizes to a 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.  
     
     
         26 . An isolated and purified protein that converts atrazine to hydroxyatrazine, wherein the protein comprises an amino acid sequence having greater than about 80% sequence identity to the amino acid sequence depicted at SEQ ID NO:2.  
     
     
         27 . An isolated and purified protein and biologically active derivatives thereof that convert atrazine to hydroxyatrazine, wherein the protein comprises an amino acid sequence having greater than about 80% sequence identity to the amino acid sequence depicted at SEQ ID NO:2.