Immobilization of biocatalysts on granular carbon

• US 4,438,196 A
• Filed: 09/28/1982
• Issued: 03/20/1984
• Est. Priority Date: 09/28/1982
• Status: Expired due to Term

• Current Assignee: Genencor International Incorporated (International Flavors & Fragrances Incorporated)
• Original Assignee: Miles Laboratories, Inc. (Bayer AG)
• Inventors: Lantero, Oreste J. Jr.
• Primary Examiner(s): Naff, David M.
• Application Number:
  US06/425,942
• Time in Patent Office: 539 Days
• Field of Search: 435/174, 435/177, 435/176, 435/180, 435/181, 435/95, 435/96, 435/98, 435/99
• US Class Current: 435/96
• CPC Class Codes: C12N 11/14 Enzymes or microbial cells immobilised on or in an inorganic carrier

Abstract

Disclosed is a process whereby enzymes are immobilized on activated granular carbon. The process involves treating the carbon with a polyamine compound having pendant amino groups to cause the polyamine to adhere to the carbon leaving pendant amine groups free to further react. The free amine groups are derivatized by treatment with a difunctional compound having amine reactive moieties, so that free amine groups of the enzyme can be covalently bound to the polyamine via the amine reactive compound. By this method, enzymes can be immobilized onto granular carbon which provides a support having excellent physical properties.

Claims

1. A method of preparing an immobilized enzyme conjugate which comprises the steps of:

(a) contacting porous, granular, activated carbon having a particle size of from 12 to 40 mesh on the U.S. sieve series, pore dimensions of from 35 to 1,000 Å

in radius and a surface area of from 200 to 600 m² /gm with a solution of a polyamine compound having pendant amine groups to cause the polyamine to attach itself to the carbon both by absorption to its surface and by entrapment in the pores thereof;

(b) removing the water and any unattached polyamine dispersed therein from contact with the carbon and contacting it with an aqueous solution of an amine reactive material which is a multifunctional aldehyde, a multifunctional organic halide, a multifunctional anhydride, a multifunctional azo compound, a multifunctional isothiocyanate or a multifunctional isocyanate to cause one of the reactive groups to react with the pendant amine groups and leave an amine reactive moiety available for further reaction;

(c) removing the water and any unreactive amine reactive material dissolved therein from contact with the carbon and contacting the carbon with an aqueous solution of the enzyme to be immobilized to cause the amine groups of the enzyme to react with the unreacted amine reactive moiety by the formation of covalent bonds therebetween to thereby immobilize the enzyme.

2. The method of claim 1 wherein the polyamine is selected from the group of polyethylenediamine, a polyethylenimine, polyhexamethylene-diamine, polymethylenedicyclohexylamine, polymethylenedianiline, polytetraethylenepentamine, and polyphenylenediamine.

3. The method of claim 2 wherein the polyethylenimine is polydiethylenetriamine, polytriethylenetetramine, polypentaethylene-hexamine or polyhexamethylenediamine.

4. The method of claim 1 wherein the polyamine is a copolymer of an epihalohydrin and an alkylene polyamine.

5. The method of claim 1 wherein the polyamine has a molecular weight range of from 500 to 100,000.

6. The method of claim 1 wherein the amine reactive material is bis-diazobenzidine-2,2'"'"'-disulfonic acid;

4,4'"'"'-difluoro-3,3'"'"'-dinitrodiphenylsulfone;

diphenyl-4,4'"'"'-dithiocyanate-2,2'"'"'-disulfonic acid;

3-methoxydiphenylmethane-4,4'"'"'-diisocyanate;

toluene-2-isocyanate-4-isothiocyanate, toluene-2,4-diisothiocyanate;

diazobenzidine;

diazobenzidine-3,3'"'"'-dianisidine;

N,N'"'"'-hexamethylene bisiodoacetamide;

hexamethylene diisocyanate;

cyanuric chloride or 1,5-difluoro-2,4-dinitrobenzene.

7. The method of claim 1 or 2 wherein the amine reactive material is glutaraldehyde.

8. The method of claim 1 wherein the enzyme is glucoamylase.

9. An immobilized enzyme conjugate comprising porous, granular, activated carbon having a particle size of from 12 to 40 mesh on the U.S. sieve series, pore dimensions of from 35 to 1,000 Å

in radius and a surface area of from 200 to 600 m² /gm as a support having attached thereto the reaction product of a polyamine compound having pendant amine groups, an amine reactive material which is a multifunctional aldehyde, a multifunctional organic halide, a multifunctional anhydride, a multifunctional azo compound, a multifunctional isothiocyanate or a multifunctional isocyanate whose unreacted amine reactive groups have been reacted with free amine groups of the enzyme to bind it thereto.

10. The conjugate of claim 9 wherein the polyamine is selected from the group of polyethylenediamine, a polyethylenimine, polyhexamethylenediamine, polymethylenedicyclohexylamine, polymethylenedianiline, polytetraethylenepentamine and polyphenylenediamine.

11. The conjugate of claim 10 wherein the polyethylenimine is polydiethylenetriamine, polytriethylenetetramine, polypentaethylene-hexamine or polyhexamethylenediamine.

12. The conjugate of claim 9 wherein the polyamine is a copolymer of an epihalohydrin and an alkylene polyamine.

13. The conjugate of claim 9 wherein the polyamine has a molecular weight of from 500 to 100,000.

14. The conjugate of claim 9 wherein the amine reactive material is bis-diazobenzidine-2,2'"'"'-disulfonic acid;

4,4'"'"'-difluoro-3,3'"'"'-dinitrodiphenylsulfone;

diphenyl-4,4'"'"'-dithiocyanate-2,2'"'"'-disulfonic acid;

3-methoxydiphenylmethane-4,4'"'"'-diisocyanate;

toluene-2-isocyanate-4-isothiocyanate, toluene-2,4-diisothiocyanate;

diazobenzidine;

diazobenzidine-3,3'"'"'-dianisidine;

N,N'"'"'-hexamethylene bisiodoacetamide;

hexamethylene diisocyanate;

cyanuric chloride or 1,5-difluoro-2,4-dinitrobenzene.

15. The conjugate of claim 9 or 10 wherein the amine reactive material is glutaraldehyde.

16. The conjugate of claim 9 wherein the enzyme is glucoamylase.

17. A method of preparing an immobilized glucoamylase conjugate, and saccharifying starch, which comprises the steps of:

(a) contacting porous, granular, activated carbon having a pore volume of about 0.95 ml/gm, an average pore diameter of about 200 angstroms and an average surface area of about 600 m² /gm with a solution of a polyamine compound having pendant amine groups to cause the polyamine to attach itself to the carbon both by absorption to the surface and by entrapment in the pores thereof;

(b) removing the water and any unattached polyamine dispersed therein from contact with the carbon and contacting it with an aqueous solution of an amine reactive material which is a multifunctional aldehyde, a multifunctional organic halide, a multifunctional anhydride, a multifunctional azo compound, a multifunctional isothiocyanate or a multifunctional isocyanate to cause one of the amine reactive groups to react with the pendant amine groups and leave an amine reactive moiety available for further reaction;

(c) removing the water and any unreacted amine reactive material dissolved therein from contact with the carbon and contacting the carbon with an aqueous solution of the enzyme to be immobilized to cause the amine groups of the glucoamylase to react with the unreacted amine reactive moiety by the formation of covalent bonds therebetween to thereby immobilize the glucoamylase; and

(d) contacting a bed of the immobilized glucoamylase with liquid starch to thereby accomplish the saccharification of the starch.

18. The method of claim 17 wherein after sufficient starch has been saccharified to wholly or partially inactive the glucoamylase, the bed of immobilized glucoamylase is washed with dilute hydrochloric acid to thereby restore the granular, activated carbon for reuse.