Immobilization of proteins on inorganic supports

• US 4,206,286 A
• Filed: 11/14/1977
• Issued: 06/03/1980
• Est. Priority Date: 11/14/1977
• Status: Expired due to Term

• Current Assignee: Technicon Instruments Corporation
• Original Assignee: Technicon Instruments Corporation
• Inventors: Keyes, Melvin H.
• Primary Examiner(s): Naff, David M.
• Application Number:
  US05/850,890
• Time in Patent Office: 932 Days
• Field of Search: 195/63, 195/68, 195/DIG. 4, 260/112 R, 435/176, 435/190
• US Class Current: 435/176
• CPC Class Codes: C12N 11/14 Enzymes or microbial cells immobilised on or in an inorganic carrier

Abstract

A method of producing an immobilized protein composite, i.e. enzymes such as glucose oxidase, on an alumina support, comprising modifying some of the free functional groups of the protein such as amino groups, by reaction with an acid or anhydride, depositing the modified protein on a porous inorganic support at controlled pH and temperature, and cross-linking remaining free functional groups of the protein with adjacent deposited protein functional groups to form interprotein peptide bonds. The resultant composite has long-term continuous use.

Claims

1. A method for preparing an immobilized composite from a protein having free amino-functional and carboxyl-functional groups, comprising modifying said protein by reacting at least some of said amino-functional groups with a modifying agent selected from a group consisting of:

succinic anhydride, succinic acid, malic acid, and glutaric anhydride, to produce a modified protein while leaving some of said amino-functional groups unmodified, depositing said modified protein on the surface of a porous, inorganic support having a high surface area by maintaining said composite within a pH and temperature range which facilitates deposition of said protein, and directly reacting said unmodified groups of said deposited protein with free functional groups in the presence of a carbodiimide agent to form peptide bonds between said deposited proteins, whereby an immobilized protein-support composite is prepared.

2. The method according to claim 1, wherein said support is -60+70 mesh, U.S. Standard Sieve, having about 0.1 micron diameter pores.

3. The method according to claim 1, wherein said support is -60+70 mesh, U.S. Standard Sieve, porous alumina having about 0.1 micron diameter pores.

4. The method according to claim 1, wherein said carbodiimide is 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide HCl.

5. The method according to claim 1, wherein said protein is an enzyme.

6. The method according to claim 1, wherein said protein is glucose oxidase.

7. The method according to claim 1, wherein said inorganic support is alumina.

8. The method according to claim 7, wherein said alumina is activated prior to the deposition of said protein by contact with aqueous hydrochloric acid for a sufficient period of time.

9. The method according to claim 1, wherein said pH range is about 3.5 to about 9.

10. The method according to claim 1, wherein said pH is in the range of about 5 to about 8.

11. The method according to claim 1, wherein said temperature is in the range between 5°

and 50°

C.

12. The method according to claim 1, wherein said temperature is in the range between 15°

and 25°

C.

13. The method according to claim 1, wherein said carbodiimide is 1-cyclohexyl-3-(2-murpholinethyl)-carbodiimide metho-p-toluene sulfonate.

14. An immobilized protein composite made in accordance with the method of claim 1.

15. The immobilized composite of claim 14, wherein said protein is an enzyme.

16. The immobilized composite of claim 14, wherein said protein is glucose oxidase.

17. An immobilized composite of claim 14, wherein said support is alumina.