Proteins in a porous support

• US 20040106178A1
• Filed: 02/21/2001
• Published: 06/03/2004
• Est. Priority Date: 02/21/2001
• Status: Active Grant

• Current Assignee: Battelle Memorial Institute
• Original Assignee: Battelle Memorial Institute
• Inventors: Liu, Jun, Ackerman, Eric
• Granted Patent: US 7,846,684 B2
• Time in Patent Office: Days
• US Class Current: 435/182
• CPC Class Codes: C12N 11/14 Enzymes or microbial cells immobilised on or in an inorganic carrier
• CPC Class Codes: C12N 9/16 acting on ester bonds (3.1)

Abstract

A protein system is described in which a protein is bound within a matrix material that has pores that are sized to achieve excellent properties such as: activity, protein density, and stability. In a preferred embodiment, the pore sizes range from 50 to 400 Å. One protein that has demonstrated surprisingly good results in this system is OPH. This protein is known to degrade organophosphorus compounds such as are found in chemical weapons and pesticides. Novel methods of forming the protein system and methods of making OPH are also described.

Claims

1. A protein system comprising:

a porous matrix material having a pore volume wherein at least 90% of the pore volume is composed of pores having sizes in the range of 50 to 400 Å

, and

further comprising a chemically-active protein bonded to the matrix material.

2. The system of claim 1 wherein the protein system comprises 0.01 to 1 mmol of said protein per gram of matrix material and wherein said protein in the protein system exhibits an activity of at least 65% that of the activity of the protein in the active state.

3. The system of claim 1 wherein said protein occupies between 5 and 40% of the average pore volume.

4. The system of claim 3 wherein the protein system comprises 0.01 to 1 mmol of said protein per gram of matrix material and wherein at least 90% of the pore volume is composed of pores having sizes in the range of 100 to 200 Å

.

5. The system of claim 3 wherein said protein is an enzyme.

6. The system of claim 5 wherein the enzyme has a volume in the range of 0.5×

10⁵ Å

³ to 3×

10⁵ Å

³.

7. The system of claim 6 wherein said enzymes have activities of at least 50% that of the active state.

8. The system of claim 7 wherein said enzyme is OPH having an activity of 60 to 95% that of the active state.

9. The system of claim 6 wherein the volume of the protein is in the range of 10 to 25% of the average pore volume.

10. The system of claim 6 wherein the surface area of the porous matrix material is at least 700 m²/g.

11. The system of claim 5 wherein the enzyme is OPH, and wherein said system comprises between 5 and 25 mg OPH per cubic centimeter.

12. The system of claim 5 wherein the enzyme is OPH, and having a V_max of 0.15 to 0.66.

13. The system of claim 5 wherein the system retains about 10% of its activity after 24 hours under alkaline conditions.

14. The system of claim 2 wherein the matrix is a SAMMS.

15. A chemical process catalyzed by the system of claim 1.

16. A protein system comprising:

a porous matrix material being sized such that the protein system comprises 0.01 to 1 mmol of protein per gram of matrix material and wherein said protein in the protein system exhibits an activity of at least 65% that of the activity of the protein in the active state.

17. The protein system of claim 16 wherein the porous matrix material has a pore volume wherein at least 90% of the pore volume is composed of pores having sizes in the range of 50 to 400 Å

.

18. The protein system of claim 17 wherein said protein occupies between 5 and 40% of the average pore volume.

19. The protein system of claim 17 wherein said enzyme is OPH.

20. The protein system of claim 17 wherein the matrix is a mesoporous oxide material.

21. A chemical process catalyzed by the system of claim 16.

22. A method of forming a protein system comprising the steps of:

providing a porous matrix material having a pore volume wherein at least 90% of the pore volume is composed of pores having sizes in the range of 50 to 400 Å

, and

reacting the porous matrix material with a protein so that said protein chemically bonds to the porous matrix material.

23. The method of claim 22 wherein the porous matrix material comprises surface hydroxyls and further comprising the step of reacting said surface hydroxyls with a coupling agent to form a functionalized monolayer.

24. The method of claim 23 wherein said functionalized monolayer comprises reactive moieties selected from the group consisting of mercapto, amino, carboxyl, hydroxyl, and azido.

25. The method of claim 23 wherein the coupling agent comprises mercaptopropyltrimethoxysilane.

26. The method of claim 23 wherein the coupling agent has a chain length of 2 to 20 atoms.

27. The method of claim 22 wherein the porous matrix material, prior to the step of reacting, has a surface area of at least 900 m²/g.

28. A protein system made by the method of claim 22.

29. A method for producing OPH comprising:

transfecting a host cell with said vector comprising a sequence encoding OPH, said sequence being operably linked to a T7 expression control sequence, culturing said transfected host cell under conditions permitting expression under the control of said expression control sequence, and purifying said OPH from the cell or the medium of the cell.

30. The method of claim 29 wherein the vector is provided with the sequence encoding OPH operably linked to the T7 expression control sequence.

31. The method of claim 29 wherein the OPH has an activity of about 13,000 units/mg.

32. The method of claim 29 wherein said vector is a plasmid.

33. The method of claim 30 wherein the vector is a plasmid.

34. The method of claim 29 wherein said host cell is a prokaryotic cell.

35. The method of claim 34 wherein said prokaryotic cell is a bacterium.

36. The method of claim 35 wherein said bacteria is Escherichia coli.

37. The method of claim 29 wherein said host cell is a eukaryotic cell.

38. The method of claim 37 wherein said eukaryotic cell is a yeast cell.

39. The method of claim 38 wherein said yeast cell is Pichia pastoris.