Process for the preparation of molecularly imprinted polymers useful for separation of enzymes

• US 6,379,599 B1
• Filed: 01/10/2000
• Issued: 04/30/2002
• Est. Priority Date: 01/10/2000
• Status: Expired due to Fees

• Current Assignee: Council of Scientific & Industrial Research
• Original Assignee: Council of Scientific & Industrial Research
• Inventors: Kulkarni, Mohan Gopalkrishna, Lele, Bhalchandra Shripad, Vaidya, Alankar Arun, Mashelkar, Raghunath Anant
• Primary Examiner(s): Lilling, Herbert J.
• Application Number:
  US09/481,650
• Time in Patent Office: 841 Days
• Field of Search: 264/108, 264/220, 264/227, 264/330, 264/331.11, 435/7.1, 435/41, 435/128, 435/134, 435/177, 435/182, 435/814, 526/199, 526/200, 526/201, 526/215, 526/243, 526/317.1, 526/318.43
• US Class Current: 264/220
• CPC Class Codes: B01J 20/268 Polymers created by use of a template, e.g. molecularly imprinted polymers
• CPC Class Codes: C08F 2/44 Polymerisation in the presence of compounding ingredients, e.g. plasticisers, dyestuffs, fillers
• CPC Class Codes: C08F 220/06 Acrylic acid; Methacrylic acid; Metal salts or ammonium salts thereof
• CPC Class Codes: C08F 220/34 Esters containing nitrogen , e.g. N,N-dimethylaminoethyl (meth)acrylate
• CPC Class Codes: C08F 220/56 Acrylamide; Methacrylamide
• CPC Class Codes: C08F 222/102 of dialcohols, e.g. ethylene glycol di(meth)acrylate or 1,4-butanediol dimethacrylate
• CPC Class Codes: C12N 9/2462 Lysozyme (3.2.1.17)
• CPC Class Codes: C12N 9/6427 Chymotrypsins (3.4.21.1; 3.4.21.2); Trypsin (3.4.21.4)
• CPC Class Codes: C12Y 304/21001 Chymotrypsin (3.4.21.1)
• CPC Class Codes: C12Y 304/21004 Trypsin (3.4.21.4)
• CPC Class Codes: C12Y 304/21075 Furin (3.4.21.75)
• CPC Class Codes: Y10S 435/814 Enzyme separation or purification

Abstract

The invention relates to a process for the preparation of molecularly imprinted polymers useful for separation of enzymes, which comprises the steps of reacting a complex of enzyme and affinity monomer, a comonomer and a crosslinker, with a polymerization initiator and a polymerization accelerator at ambient temperature and pressure for a period ranging between 2 to 24 hrs, thereby obtaining a crosslinked polymer, crushing the cross linked polymer obtained to fine particles, adding a solvent and extracting imprinted enzyme from the polymer, obtaining the molecularly imprinted polymer, contacting the imprinted polymer with aqueous solution containing imprinted enzyme or a mixture of imprinted enzyme and other enzymes and isolating the enzyme-adsorbed polymer.

Claims

1. A process for the preparation of a molecularly imprinted polymer useful for separation of enzymes, which comprises the steps of

(i) reacting a complex of enzyme and an affinity monomer that specifically recognizes the enzyme, a comonomer, and a crosslinker, with a polymerization initiator and a polymerization accelerator at ambient temperature and pressure for 2 to 24 hrs., thereby obtaining a crosslinked polymer,

(ii) crushing the crosslinked polymer obtained to fine particles,

(iii) adding a solvent and extracting the enzyme from the polymer, and

(iv) obtaining the molecularly imprinted polymer.

2. A process as claimed in claim 1 wherein the enzyme is selected from the group consisting of trypsin, chymotrypsin, lysozyme and ovalbumin.

3. A process as claimed in claim 1 wherein the complexing affinity monomer is an acryloyl derivative of an enzyme inhibitor selected from the group consisting of N-acryloyl-para-aminobenzamidine, N-acryloyl-4-phenylbutylamine, acryloyl-N-acetylglucosamine, and N-oleoylacrylamide.

4. A process as claimed in claim 1 wherein the enzyme-affinity monomer complex is prepared by contacting aqueous solutions of enzyme and the respective affinity monomer.

5. A process as claimed in claim 1 wherein the affinity monomer is prepared by reacting acryloyl chloride with para-aminobenzamidine, 4-phenylbutylamine, or N-acetylglucosamine, and acrylamide.

6. A process as claimed in claim 1 wherein the ratio of enzyme to affinity monomer is 1:

1 to 1;

10 (w/w).

7. A process as claimed in claim 1 wherein the comonomer is selected from the group consisting of acrylamide, methacrylamide, N-isopropylacrylamide, N-isopropylmethacrylamide, acrylic acid, and methacrylic acid.

8. A process as claimed in claim 1 wherein the amount of comonomer in the polymerization mixture is 5 to 80% by weight of the total feed.

9. A process as claimed in claim 1 wherein the crosslinker is selected from the group consisting of methylene bisacrylamide, N,N-diacryloyl-1,2-diaminoethane, N,N-dimethacryloyl-1,2-diaminoethane, N,N-diacryloyl-1,6-diaminohexane, and ethylene glycol dimethacrylate.

10. A process as claimed in claim 1 wherein the amount of crosslinker is from about 10% to about 90% by weight of the total feed.

11. A process as claimed in claim 1 wherein the polymerization initiator is selected from the group consisting of ammonium persulfate, ceric ammonium sulfate, potassium persulfate, and azobis(isobutyro)nitrile.

12. A process as claimed in claim 1 wherein the amount of polymerization initiator is from about 0.1% to about 1% by weight of the total feed.

13. A process as claimed in claim 1 wherein the polymerization accelerator is ethylenediamine or N,N,N,N-tetramethylethylenediamine.

14. A process as claimed in claim 1 wherein the amount of polymerization accelerator is 1% to 4% by weight of the total feed.

15. A process as claimed in claim 1 wherein the solvent used to extract the enzyme from the imprinted polymer is selected from the group consisting of phenol, chloroform, and glacial acetic acid.

16. A process as claimed in claim 1 wherein the ratio of enzyme to affinity monomer is 1:

1 (w/w).

17. A process as claimed in claim 1 wherein the amount of comonomer in the polymerization mixture is 45 to 55% by weight of the total feed.

18. A process as claimed in claim 1 wherein the amount of crosslinker is from about 30% to about 50% by weight of the total feed.