Red absorbing combination of alcohol oxidase and an azide compound

• US 4,430,427 A
• Filed: 11/04/1980
• Issued: 02/07/1984
• Est. Priority Date: 11/04/1980
• Status: Expired due to Term

• Current Assignee: Moldowan Laboratories Incorporated
• Original Assignee: Phillips Petroleum Company (ConocoPhillips)
• Inventors: Hopkins, Thomas R.
• Primary Examiner(s): NAFF, DAVID M
• Application Number:
  US06/203,923
• Time in Patent Office: 1,190 Days
• Field of Search: 435/25, 435/4, 435/190, 435/188, 435/189, 435/174, 435/177, 435/180, 435/814, 435/815, 435/816
• US Class Current: 435/25
• CPC Class Codes: C12N 9/0006 acting on CH-OH groups as donors (1.1)
• CPC Class Codes: C12Q 1/26 involving oxidoreductase
• CPC Class Codes: Y10S 435/815 by sorption
• CPC Class Codes: Y10S 435/816 by solubility

Abstract

Combining active alcohol oxidase with sufficient amount of an azide compound has been found to form a red absorbing combination. Formation of the red absorbing combination enables determining the presence of active alcohol oxidase by adding the azide compound to a preparation and observing the resultant color. Additionally, alcohol oxidase may be purified by adding the azide compound to a preparation containing active alcohol oxidase in an amount effective to produce a red absorbing complex and separating the red absorbing complex from the preparation.

Claims

1. A composition of matter comprising active alcohol oxidase;

and

an azide compound selected from the group of compounds having the formula R"(N₃)_x wherein R" is a metal atom, a hydrogen atom, or the ammonium radical, and N₃ is the moiety N═

N═

N,

the azide compound being present in an amount effective to form a red absorbing combination with active alcohol oxidase.

2. A composition as in claim 1 wherein:

the azide is selected from the group consisting of azide salts of electropositive metals.

3. A composition as in claim 1 wherein:

the azide is present in an amount in the range of about 1 mole azide/mole alcohol oxidase to about 5.0×

10⁷ moles azide/mole alcohol oxidase.

4. A composition as in claim 1 wherein:

the azide is present in an amount in the range of about 8 moles azide/mole alcohol oxidase to about 5.0×

10⁷ moles azide/mole alcohol oxidase.

5. A composition as in claim 1 wherein:

the azide is present in an amount in the range of about 10 moles azide/mole alcohol oxidase to about 2000 moles azide/mole alcohol oxidase.

6. A composition as in claim 1 wherein:

the alcohol oxidase is isolated from genus Pichia; and

the azide is present in an amount in the range of about 0.07 mg azide/gram alcohol oxidase to about 80,000 mg azide/gram alcohol oxidase.

7. A composition as in claim 1 wherein:

the alcohol oxidase is isolated from genus Pichia; and

the azide is present in an amount in the range of about 0.1 mg azide/gram alcohol oxidase to about 80,000 mg azide/gram alcohol oxidase.

8. A composition as in claim 1 wherein:

the alcohol oxidase is isolated from genus Pichia; and

the azide is present in an amount in the range of about 0.7 mg azide/gram alcohol oxidase to about 135 mg azide/gram alcohol oxidase.

9. A composition as in claim 1 wherein:

the alcohol oxidase has a specific activity in the range of about 10-12 Enzyme Units per mg protein; and

the azide is present in an amount in the range of about 7×

10₆^- mg azide (N₃)/Enzyme Unit to about 8 mg azide (N₃)/Enzyme Unit.

10. A composition as in claim 1 wherein:

the alcohol oxidase has a specific activity in the range of about 10-12 Enzyme Units per mg protein; and

the azide is present in an amount in the range of about 7×

10^-5 mg azide (N₃)/Enzyme Unit to about 8 mg azide (N₃)/Enzyme Unit.

11. A composition as in claim 1 wherein:

the alcohol oxidase has a specific activity in the range of about 10-12 Enzyme Units per mg protein; and

the azide is present in an amount in the range of about 7×

10^-4 mg azide (N₃)/Enzyme Unit to about 1.35×

10^-2 mg azide (N₃)/Enzyme Unit.

12. A method of purifying an active alcohol oxidase comprising:

adding an azide compound selected from the group of compounds having the formula R"(N₃)_x wherein R" is a metal atom, a hydrogen atom, or the ammonium radical, and N₃ is the moiety N═

N═

N to a preparation comprising the active alcohol oxidase in an amount effective for producing a red absorbing complex; and

separating the red absorbing complex from the preparation.

13. A method as in claim 12 wherein adding an azide comprises:

adding an amount of sodium azide in the range of about 1 mole azide/mole alcohol oxidase to about 5.0×

10⁷ moles azide/mole alcohol oxidase.

14. A method as in claim 12 wherein adding an azide comprises:

adding an amount of sodium azide in the range of about 8 moles azide/mole alcohol oxidase to about 5.0×

10⁷ moles azide/mole alcohol oxidase.

15. A method as in claim 12 wherein adding an azide comprises:

adding an amount of sodium azide in the range of about 10 moles azide/mole alcohol oxidase to about 2000 moles azide/mole alcohol oxidase.

16. A method as in claim 12 wherein adding an azide comprises:

adding an amount of sodium azide in the range of about 0.07 mg azide/gram alcohol oxidase to about 80,000 mg azide/gram alcohol oxidase; and

wherein

the alcohol oxidase is isolated from genus Pichia.

17. A method as in claim 12 wherein adding an azide comprises:

adding an amount of sodium azide in the range of about 0.1 mg azide/gram alcohol oxidase to about 80,000 mg azide/gram alcohol oxidase; and

wherein

the alcohol oxidase is isolated from genus Pichia.

18. A method as in claim 12 wherein adding an azide comprises:

adding an amount of sodium azide in the range of about 0.7 mg azide/gram alcohol oxidase to about 135 mg azide/gram alcohol oxidase; and

wherein

the alcohol oxidase is isolated from genus Pichia.

19. A method as in claim 12 wherein adding an azide comprises:

adding an amount of sodium azide in the range of about 7×

10^-6 mg azide (N₃)/Enzyme Unit to about 8 mg azide (N₃)/Enzyme Unit; and

wherein

the alcohol oxidase has a specific activity in the range of about 10-12 Enzyme Units per mg protein.

20. A method as in claim 12 wherein adding an azide comprises:

adding an amount of sodium azide in the range of about 7×

10^-5 mg azide (N₃)/Enzyme Unit to about 8 mg azide (N₃)/Enzyme Unit; and

wherein

the alcohol oxidase has a specific activity in the range of about 10-12 Enzyme Units per mg protein.

21. A method as in claim 12 wherein adding an azide comprises:

adding an amount of sodium azide in the range of about 7×

10^-4 mg azide (N₃)/Enzyme Unit to about 1.35×

10^-2 mg azide (N₃)/Enzyme Unit; and

wherein

the alcohol oxidase has a specific activity in the range of about 10-12 Enzyme Units per mg protein.

22. The method of claim 12 in which the red absorbing complex is separated from the preparation by the use of differential solubility using ammonium sulfate or polyethylene glycol.

23. The method of claim 12 in which the red absorbing complex is separated from the preparation by specific precipitation.

24. The method of claim 12 in which the red absorbing complex is separated from the preparation by column chromatography.

25. The method of claim 12 in which the red absorbing complex is separated from the preparation by preparative electrophoresis.

26. The method of claim 12 in which the red absorbing complex is separated from the preparation by preparative ultracentrifugation.

27. The method of claim 12 in which the red absorbing complex is separated from the preparation by dialysis to crystallization.

28. A method of determining the presence of active alcohol oxidase comprising:

observing the color of a composition of matter comprising an alcohol oxidase and an azide compound selected from the group of compounds having the formula R"(N₃)_x wherein R" is a metal atom, a hydrogen atom, or the ammonium radical, and N₃ is the moiety

space="preserve" listing-type="equation">N═

N═

N

the azide compound being present in an amount effective to form a red absorbing combination with active alcohol oxidase.