Microbes and methods for remediation

• US 20030170879A1
• Filed: 12/10/2002
• Published: 09/11/2003
• Est. Priority Date: 01/25/2000
• Status: Active Grant

• Current Assignee: Regents of The University of Minnesota (University of Minnesota)
• Original Assignee: Regents of The University of Minnesota (University of Minnesota)
• Inventors: McTavish, Hugh
• Granted Patent: US 6,919,199 B2
• Time in Patent Office: Days
• US Class Current: 435/262.5
• CPC Class Codes: B09C 1/10 microbiologically, biologically or by using enzymes
• CPC Class Codes: C12N 11/16 Enzymes or microbial cells immobilised on or in a biological cell
• CPC Class Codes: C12N 9/14 Hydrolases (3)

Abstract

The present invention provides methods of using a microbe containing a polypeptide that degrades, preferably detoxifies, a compound that is present in the environment. Preferably, the polypeptide is a hydrolase and the compound is at least one s-triazine. The present invention also provides a microbe containing a polypeptide that degrades, preferably detoxifies, a compound that is present in the environment.

Claims

1. A method for remediating a compound in a sample, the method comprising:

providing a killed microbe comprising a polynucleotide comprising a coding region encoding a polypeptide that degrades a compound; and

contacting a sample comprising the compound with the microbe under conditions effective to decrease the concentration of the compound in the sample relative to the concentration of the compound in a sample not contacted with the microbe.

2. The method of claim 1 wherein the microbe is killed with a cross-linking agent.

3. The method of claim 2 wherein the cross-linking agent is selected from the group consisting of glutaraldehyde, formalin, and iodine.

4. The method of claim 1 wherein the polypeptide is a hydrolase.

5. The method of claim 4 wherein the compound is an s-triazine.

6. The method of claim 5 wherein the nucleotide sequence of the coding region is nucleotides 236 to 1660 of GenBank accession number U55933.

7. The method of claim 5 wherein the complement of the nucleotide sequence of the coding region hybridizes to the nucleotide sequence set forth at nucleotides 236 to 1660 of GenBank accession number U55933 in a solution containing 250 mM Na₂HPO₄, pH 7.4, 2 ml/liter 0.5 M EDTA, pH 8.0, and 10 grams/liter bovine serum albumin at 65°

C. for at least 4 hours, followed by three washes for twenty minutes each at 65°

C. in a solution containing 2×

SSC and 0.1% SDS.

8. The method of claim 5 wherein the amino acid sequence of the polypeptide is the amino acid sequence of GenBank accession number AAC64663, or an active analog or active fragment thereof.

9. The method of claim 1 wherein the sample is selected from the group consisting of soil, water, and a combination thereof.

10. The method of claim 5 wherein the s-triazine is selected from the group of atrazine, desethylatrazine, deisopropylatrazine, desethylhydroxyatrazine, desisopropylhydroxyatrazine, desethyldesisopropylatrazine, simazine, terbuthylazine, melamine, ammelide, ammeline, prometryn, ametryn, propazine, cyanuric acid, terbutryn, cyanazine, propazine, simatone, and cyromazine.

11. The method of claim 5 wherein the s-triazine is atrazine.

12. The method of claim 1 wherein the microbe is attached to a support.

13. The method of claim 1 wherein the compound is detoxified.

14. The method of claim 1 wherein the coding region is an exogenous coding region.

15. The method of claim 1 wherein the microbe is selected from the group consisting of E. coli and P. aeruginosa.

16. The method of claim 1 wherein the microbe is E. coli.

17. The method of claim 1 further comprising measuring the concentration of the compound in the sample after contacting the sample with the microbe.

18. A method for remediating a compound in a sample, the method comprising:

providing killed cross-linked microbes comprising an exogenous polynucleotide comprising a coding region encoding a hydrolase that degrades a compound, wherein the microbes retain at least about 30% hydrolase enzymatic activity compared to the hydrolase enzymatic activity of the microbes that are not killed; and

contacting a sample comprising the compound with the microbes under conditions effective to decrease the concentration of the compound in the sample relative to the concentration of the compound in a sample not contacted with the microbes.

19. The method of claim 18 wherein the microbes are cross-linked with a cross-linking agent selected from the group consisting of glutaraldehyde, formalin, and iodine.

20. The method of claim 18 wherein the sample is selected from the group consisting of soil, water, and a combination thereof.

21. The method of claim 18 wherein the microbes are selected from the group consisting of E. coli and P. aeruginosa.

22. The method of claim 18 further comprising measuring the concentration of the compound in the sample after contacting the sample with the microbes.

23. The method of claim 18 wherein the cross-linked microbes are attached to a support.

24. A method for remediating a compound in a sample, the method comprising:

providing killed cross-linked microbes comprising an exogenous polynucleotide comprising a coding region encoding a hydrolase that degrades an s-triazine, wherein the complement of the nucleotide sequence of the coding region hybridizes to the nucleotide sequence set forth at nucleotides 236 to 1660 of GenBank accession number U55933 in a solution containing 250 mM Na₂HPO₄, pH 7.4, 2 ml/liter 0.5 M EDTA, pH 8.0, and 10 grams/liter bovine serum albumin at 65°

C. for at least 4 hours, followed by three washes for twenty minutes each at 65°

C. in a solution containing 2×

SSC and 0.1% SDS, and wherein the microbes retain at least about 30% hydrolase enzymatic activity compared to the hydrolase enzymatic activity of the microbes that are not killed; and

contacting a sample comprising the s-triazine with the microbes under conditions effective to decrease the concentration of the s-triazine in the sample relative to the concentration of the s-triazine in a sample not contacted with the microbes.

25. The method of claim 24 wherein the s-triazine is selected from the group of atrazine, desethylatrazine, deisopropylatrazine, desethylhydroxyatrazine, desisopropylhydroxyatrazine, desethyldesisopropylatrazine, simazine, terbuthylazine, melamine, ammelide, ammeline, prometryn, ametryn, propazine, cyanuric acid, terbutryn, cyanazine, propazine, simatone, and cyromazine.

26. A method for remediating a compound in a sample, the method comprising:

providing killed cross-linked microbes comprising an exogenous polynucleotide comprising a coding region encoding a hydrolase that degrades atrazine, wherein the complement of the nucleotide sequence of the coding region hybridizes to the nucleotide sequence set forth at nucleotides 236 to 1660 of GenBank accession number U55933 in a solution containing 250 mM Na₂HPO₄, pH 7.4, 2 ml/liter 0.5 M EDTA, pH 8.0, and 10 grams/liter bovine serum albumin at 65°

C. for at least 4 hours, followed by three washes for twenty minutes each at 65°

C. in a solution containing 2×

SSC and 0.1% SDS, and wherein the microbes retain at least about 30% hydrolase enzymatic activity compared to the hydrolase enzymatic activity of the microbes that are not killed; and

contacting a sample comprising the atrazine with the microbes under conditions effective to decrease the concentration of the atrazine in the sample relative to the concentration of the atrazine in a sample not contacted with the microbes.

27. A method for remediating a compound in a sample, the method comprising:

providing killed cross-linked microbes comprising an exogenous polynucleotide comprising a coding region encoding a hydrolase that degrades atrazine, wherein the nucleotide sequence of the coding region comprises nucleotides 236 to 1660 of GenBank accession number U55933, and wherein the microbes retain at least about 30% hydrolase enzymatic activity compared to the hydrolase enzymatic activity of the microbes that are not killed; and

contacting a sample comprising the atrazine with the microbes under conditions effective to decrease the concentration of the atrazine in the sample relative to the concentration of the atrazine in a sample not contacted with the microbes.

28. A method for degrading an s-triazine in a sample, the method comprising:

providing a cross-linked microbe comprising a polynucleotide comprising a coding region encoding a hydrolase that degrades an s-triazine; and

contacting a sample comprising the s-triazine with the microbe under conditions effective to decrease the concentration of the s-triazine in the sample relative to the concentration of the s-triazine in a sample not contacted with the microbe.

29. The method of claim 28 wherein the microbe is a prokaryote.

30. The method of claim 29 wherein the prokaryote is selected from the group consisting of E. coli and P. aeruginosa.

31. The method of claim 28 wherein less than about 40% of individual microbes are cross-linked to each other.

32. A method for detoxifying a compound in a sample, the method comprising:

providing killed cross-linked microbes comprising an exogenous polynucleotide comprising a coding region encoding a hydrolase that detoxifies a compound, wherein the microbes retain at least about 30% hydrolase enzymatic activity compared to the hydrolase enzymatic activity of the microbes that are not killed; and

contacting a sample comprising the compound with the microbes under conditions effective to lower the toxicity of the compound in the sample relative to the toxicity of the compound in a sample not contacted with the microbes.

33. The method of claim 32 wherein the microbes are cross-linked with a cross-linking agent selected from the group consisting of glutaraldehyde, formalin, and iodine.

34. The method of claim 32 wherein the sample is selected from the group consisting of soil, water, and a combination thereof.

35. The method of claim 32 wherein the microbes are selected from the group consisting of E. coli and P. aeruginosa.

36. The method of claim 32 further comprising measuring the toxicity of the compound in the sample after contacting the sample with the microbes.

37. The method of claim 32 wherein the cross-linked microbes are attached to a support.

38. A method for detoxifying an s-triazine in a sample, the method comprising:

providing killed cross-linked microbes comprising an exogenous polynucleotide comprising a coding region encoding a hydrolase that detoxifies an s-triazine, wherein the complement of the nucleotide sequence of the coding region hybridizes to the nucleotide sequence set forth at nucleotides 236 to 1660 of GenBank accession number U55933 in a solution containing 250 mM Na₂HPO₄, pH 7.4, 2 ml/liter 0.5 M EDTA, pH 8.0, and 10 grams/liter bovine serum albumin at 65°

C. for at least 4 hours, followed by three washes for twenty minutes each at 65°

C. in a solution containing 2×

SSC and 0.1% SDS, and wherein the microbes retain at least about 30% hydrolase enzymatic activity compared to the hydrolase enzymatic activity of the microbes that are not killed; and

contacting a sample comprising the s-triazine with the microbes under conditions effective to lower the toxicity of the s-triazine in the sample relative to the toxicity of the s-triazine in a sample not contacted with the microbes.

39. A cross-linked microbe comprising a polynucleotide comprising a coding region encoding a polypeptide that degrades an s-triazine, wherein the microbe has been killed.

40. The microbe of claim 39 wherein the microbe is isolated.

41. The microbe of claim 39 wherein the nucleotide sequence of the coding region is nucleotides 236 to 1660 of GenBank accession number U55933.

42. The microbe of claim 39 wherein the complement of the nucleotide sequence of the coding region hybridizes to the nucleotide sequence set forth at nucleotides 236 to 1660 of GenBank accession number U55933 in a solution containing 250 mM Na₂HPO₄, pH 7.4, 2 ml/liter 0.5 M EDTA, pH 8.0, and 10 grams/liter bovine serum albumin at 65°

C. for at least 4 hours, followed by three washes for twenty minutes each at 65°

C. in a solution containing 2×

SSC and 0.1% SDS.

43. The method of claim 39 wherein the amino acid sequence of the polypeptide is the amino acid sequence of GenBank accession number AAC64663, or an active analog or active fragment thereof.

44. The microbe of claim 39 wherein the s-triazine is selected from the group of atrazine, desethylatrazine, deisopropylatrazine, desethylhydroxyatrazine, desisopropylhydroxyatrazine, desethyldesisopropylatrazine, simazine, terbuthylazine, melamine, ammelide, ammeline, prometryn, ametryn, propazine, cyanuric acid, terbutryn, cyanazine, propazine, simatone, and cyromazine.

45. The microbe of claim 39 wherein the s-triazine is atrazine.

46. The microbe of claim 39 wherein the microbe is killed with a cross-linking agent.

47. The method of claim 46 wherein the cross-linking agent is selected from the group consisting of glutaraldehyde, formalin, and iodine.

48. Killed cross-linked microbes comprising a polynucleotide comprising a coding region encoding a hydrolase that degrades a compound, wherein the cross-linked microbes retain at least about 30% hydrolase enzymatic activity compared to the hydrolase enzymatic activity of the microbes that are not killed.

49. Killed cross-linked microbe comprising a polynucleotide comprising a coding region encoding a hydrolase that degrades an s-triazine, wherein the complement of the nucleotide sequence of the coding region hybridizes to the nucleotide sequence set forth at nucleotides 236 to 1660 of GenBank accession number U55933 in a solution containing 250 mM Na₂HPO₄, pH 7.4, 2 ml/liter 0.5 M EDTA, pH 8.0, and 10 grams/liter bovine serum albumin at 65°

C. for at least 4 hours, followed by three washes for twenty minutes each at 65°

C. in a solution containing 2×

SSC and 0.1% SDS, and wherein the microbes retain at least about 30% hydrolase enzymatic activity compared to the hydrolase enzymatic activity of the microbes that are not killed.

50. Killed cross-linked microbes comprising a polynucleotide comprising a coding region encoding a hydrolase that degrades atrazine, wherein the complement of the nucleotide sequence of the coding region hybridizes to the nucleotide sequence set forth at nucleotides 236 to 1660 of GenBank accession number U55933 in a solution containing 250 mM Na₂HPO₄, pH 7.4, 2 ml/liter 0.5 M EDTA, pH 8.0, and 10 grams/liter bovine serum albumin at 65°

C. for at least 4 hours, followed by three washes for twenty minutes each at 65°

C. in a solution containing 2×

SSC and 0.1% SDS, and wherein the microbes retain at least about 30% hydrolase enzymatic activity compared to the hydrolase enzymatic activity of the microbes that are not killed.

51. Killed cross-linked microbes comprising a polynucleotide comprising a coding region encoding a hydrolase that degrades atrazine, wherein the nucleotide sequence of the coding region comprises nucleotides 236 to 1660 of GenBank accession number U55933, and wherein the microbes retain at least about 30% hydrolase enzymatic activity compared to the hydrolase enzymatic activity of the microbes that are not killed.