Claims
- 1. A method of detecting an oxidation enzyme comprising the steps of:
(a) contacting the oxidation enzyme with an oxygen donor and a substrate having an aromatic ring to form a compound having a hydroxylated aromatic ring; (b) contacting the compound with a peroxidase enzyme to form a luminescent polymer; and (c) detecting the luminescent polymer.
- 2. The method of claim 1, wherein the oxidation enzyme is a dioxygenase.
- 3. The method of claim 1, wherein the oxidation enzyme is a monooxygenase.
- 4. The method of claim 3, wherein the monooxygenase is a cytochrome P450 oxygenase.
- 5. The method of claim 4, wherein the cytochrome P450 oxygenase is a cytochrome P450cam oxygenase or a function-conservative variant thereof.
- 6. The method of claim 3, wherein the monooxygenase is a methane monooxygenase.
- 7. The method of claim 3, wherein the monooxygenase is a chloroperoxidase.
- 8. The method of claim 3, wherein the monooxygenase is a toluene monooxygenase.
- 9. The method of claim 1, wherein the oxygen donor is molecular oxygen.
- 10. The method of claim 1, wherein the oxygen donor is a peroxide.
- 11. The method of claim 10, wherein the peroxide is selected from hydrogen peroxide and t-butyl peroxide.
- 12. The method of claim 1, wherein the substrate is selected from the group consisting of naphthalene, 3-phenylpropionate, coumarin, benzene, anthracene, benzphetamine, and toluene.
- 13. The method of claim 12, wherein the substrate is selected from the group consisting of naphthalene, 3-phenylpropionate, and coumarin.
- 14. The method of claim 1, wherein the peroxidase enzyme is selected from a horseradish peroxidase, a cytochrome c peroxidase, a tulip peroxidase, a lignin peroxidase, a carrot peroxidase, a peanut peroxidase, and a soybean peroxidase.
- 15. The method of claim 1, wherein the luminescent polymer is a luminescent dimer.
- 16. The method of claim 1, wherein step (b) comprises contacting a chemiluminescent agent with the compound and the peroxidase enzyme, and the luminescent polymer comprises the compound and the chemiluminescent agent.
- 17. The method of claim 16, wherein the chemiluminescent agent is luminol.
- 18. The method of claim 16, wherein the chemiluminescent agent is a 1,2-dioxetane.
- 19. The method of claim 1, wherein the luminescent polymer is fluorescent or chemiluminescent.
- 20. The method of claim 1, wherein the luminescent compound has a detectable color.
- 21. The method of claim 1, wherein the oxidation enzyme is a function-conservative variant of a wild-type oxidation enzyme.
- 22. The method of claim 1, wherein the peroxidase enzyme is a function-conservative variant of a wild-type peroxidase enzyme.
- 23. The method of claim 1, wherein at least one of the oxidation enzyme and the peroxidase enzyme is expressed by a host cell.
- 24. The method of claim 19, wherein both the oxidation enzyme and the peroxidase enzyme are expressed by a host cell.
- 25. A method of detecting an oxidation enzyme comprising the steps of:
(a) contacting the oxidation enzyme with a peroxide and a substrate having an aromatic ring to form a compound having a hydroxylated ring; (b) contacting the compound with a peroxidase enzyme to form a luminescent polymer comprising the compound; and (c) detecting the luminescent polymer.
- 26. The method of claim 25, wherein at least one of the oxidation enzyme and the peroxidase enzyme is expressed by a host cell.
- 27. The method of claim 26, wherein both the oxidation enzyme and the peroxidase enzyme are expressed by a host cell.
- 28. The method of claim 25, wherein step (a) is conducted in the absence of a co-enzyme.
- 29. The method of claim 26, wherein the co-enzyme is selected from NAD+ and NADP+.
- 30. The method of claim 25, wherein the oxidation enzyme is a function-conservative variant of a wild-type oxidation enzyme.
- 31. The method of claim 25, wherein the peroxidase enzyme is a function-conservative variant of a wild-type peroxidase enzyme.
- 32. A method of detecting an oxidation enzyme comprising the steps of:
(a) contacting the oxidation enzyme with a peroxide and a substrate having an aromatic ring to form a compound having a hydroxylated ring; (b) contacting the compound with a peroxidase enzyme to form a luminescent polymer comprising the compound; and (c) detecting the luminescent polymer; wherein the oxidation enzyme is a function-conservative variant of a cytochrome P450 oxidation enzyme.
- 33. The method of claim 32, wherein at least one of the oxidation enzyme and the peroxidase enzyme is expressed by a host cell.
- 34. The method of claim 33, wherein both the oxidation enzyme and the peroxidase enzyme are expressed by a host cell.
- 35. The method of claim 32, wherein the peroxidase enzyme is a function-conservative variant of a wild-type peroxidase enzyme.
- 36. A method of detecting an oxidation enzyme, comprising the steps of:
(a) contacting the oxidation enzyme with an oxygen donor and a substrate having an aromatic ring to form a first compound having a dihydroxylated aromatic ring; (b) contacting the first compound with a dihydrodiol dehydrogenase to form a second compound having a catechol group; (c) contacting the second product with a peroxidase enzyme to form a luminescent polymer comprising the second product; and (d) detecting the luminescent polymer.
- 37. The method of claim 36, wherein the dioxygenase is selected from a toluene dioxygenase and a naphthalene dioxygenase.
- 38. The method of claim 36, wherein the oxygen donor is a peroxide.
- 39. The method of claim 38, wherein the peroxide is selected from hydrogen peroxide and t-butyl peroxide.
- 40. The method of claim 36, wherein the oxygen donor is molecular oxygen.
- 41. The method of claim 36, wherein the oxidation enzyme is a function-conservative variant of a wild-type oxidation enzyme.
- 42. The method of claim 36, wherein the peroxidase enzyme is a function-conservative variant of a wild-type peroxidase enzyme.
- 43. A method of detecting an oxidation enzyme comprising the steps of:
(a) contacting the oxidation enzyme with an oxygen donor and a substrate having an aromatic ring to form a compound having a hydroxylated aromatic ring; (b) contacting the compound with a laccase enzyme to form a luminescent polymer; and (c) detecting the luminescent polymer.
- 44. The method of claim 43, wherein the oxidation enzyme is a dioxygenase.
- 45. The method of claim 43, wherein the oxidation enzyme is a monooxygenase.
- 46. The method of claim 43, wherein the oxygen donor is molecular oxygen.
- 47. The method of claim 43, wherein the oxygen donor is a peroxide.
- 48. The method of claim 43, wherein the substrate is selected from the group consisting of naphthalene, 3-phenylpropionate, coumarin, benzene, anthracene, benzphetamine, and toluene.
- 49. The method of claim 43, wherein the laccase enzyme is selected from a horseradish peroxidase, a cytochrome c peroxidase, a tulip peroxidase, a lignin peroxidase, a carrot peroxidase, a peanut peroxidase, and a soybean peroxidase.
- 50. The method of claim 43, wherein the luminescent polymer is a luminescent dimer.
- 51. The method of claim 43, wherein step (b) comprises contacting a chemiluminescent agent with the compound and the laccase enzyme, and the luminescent polymer comprises the compound and the chemiluminescent agent.
- 52. The method of claim 43, wherein the luminescent polymer is fluorescent or chemiluminescent.
- 53. The method of claim 43, wherein the luminescent compound has a detectable color.
- 54. The method of claim 43, wherein the oxidation enzyme is a function-conservative variant of a wild-type oxidation enzyme.
- 55. The method of claim 43, wherein the laccase enzyme is a function-conservative variant of a wild-type laccase enzyme.
- 56. The method of claim 43, wherein at least one of the oxidation enzyme and the laccase enzyme is expressed by a host cell.
- 57. A method of detecting an oxidation enzyme comprising the steps of:
(a) contacting the oxidation enzyme with a peroxide and a substrate having an aromatic ring to form a compound having a hydroxylated ring; (b) contacting the compound with a laccase enzyme to form a luminescent polymer comprising the compound; and (c) detecting the luminescent polymer.
- 58. The method of claim 57, wherein at least one of the oxidation enzyme and the laccase enzyme is expressed by a host cell.
- 59. The method of claim 57, wherein step (a) is conducted in the absence of a co-enzyme.
- 60. The method of claim 57, wherein the oxidation enzyme is a function-conservative variant of a wild-type oxidation enzyme.
- 61. The method of claim 57, wherein the laccase enzyme is a function-conservative variant of a wild-type laccase enzyme.
- 62. A method of detecting an oxidation enzyme comprising the steps of:
(a) contacting the oxidation enzyme with a peroxide and a substrate having an aromatic ring to form a compound having a hydroxylated ring; (b) contacting the compound with a laccase enzyme to form a luminescent polymer comprising the compound; and (c) detecting the luminescent polymer; wherein the oxidation enzyme is a function-conservative variant of a cytochrome P450 oxidation enzyme.
- 63. The method of claim 62, wherein at least one of the oxidation enzyme and the laccase enzyme is expressed by a host cell.
- 64. The method of claim 62, wherein the laccase enzyme is a function-conservative variant of a wild-type laccase enzyme.
- 65. A method of detecting an oxidation enzyme, comprising the steps of:
(a) contacting the oxidation enzyme with an oxygen donor and a substrate having an aromatic ring to form a first compound having a dihydroxylated aromatic ring; (b) contacting the first compound with a dihydrodiol dehydrogenase to form a second compound having a catechol group; (c) contacting the second product with a laccase enzyme to form a luminescent polymer comprising the second product; and (d) detecting the luminescent polymer.
- 66. The method of claim 65, wherein the dioxygenase is selected from a toluene dioxygenase and a naphthalene dioxygenase.
- 67. The method of claim 65, wherein the oxygen donor is a peroxide.
- 68. The method of claim 65, wherein the oxygen donor is molecular oxygen.
- 69. The method of claim 65, wherein the oxidation enzyme is a function-conservative variant of a wild-type oxidation enzyme.
- 70. The method of claim 65, wherein the laccase enzyme is a function-conservative variant of a wild-type laccase enzyme.
Parent Case Info
[0001] This application is a continuation of U.S. application Ser. No. 09/661,093, filed on Sep. 13, 2000, which is a continuation-in-part of U.S. application Ser. No. 09/246,451, which claims priority under 35 U.S.C. §119 from U.S. application Ser. No. 60/094,403., filed on Jul. 28, 1998; U.S. Ser. No. 60/106,840 filed on Nov. 3, 1998; U.S. Ser. No. 60/086,206 filed May 21, 1998; and U.S. Ser. No. 60/106,834 filed on Nov. 3, 1998. The above-identified prior applications are all incorporated herein by reference in their entirety.
Government Interests
[0002] The Government has certain rights to this invention pursuant to Grant No. N0014-96-1-0340, awarded by the United States Navy.
Provisional Applications (4)
|
Number |
Date |
Country |
|
60094403 |
Jul 1998 |
US |
|
60106840 |
Nov 1998 |
US |
|
60086206 |
May 1998 |
US |
|
60106834 |
Nov 1998 |
US |
Continuations (1)
|
Number |
Date |
Country |
Parent |
09661093 |
Sep 2000 |
US |
Child |
10453104 |
Jun 2003 |
US |
Continuation in Parts (1)
|
Number |
Date |
Country |
Parent |
09246451 |
Feb 1999 |
US |
Child |
09661093 |
Sep 2000 |
US |