Separation of proteins

• US 5,728,559 A
• Filed: 12/01/1995
• Issued: 03/17/1998
• Est. Priority Date: 07/09/1993
• Status: Expired due to Term

• Current Assignee: Novozymes A/S (Novozymes)
• Original Assignee: Novo Nordisk A/S
• Inventors: Rancke-Madsen, Anders, Laustsen, Mads Aage, Nilsson, Birgitte Mahler
• Primary Examiner(s): Lankford, Jr., Leon B.
• Application Number:
  US08/557,056
• Time in Patent Office: 837 Days
• Field of Search: 435/232, 435/816, 435/814, 435/815, 435/226, 435/219, 435/183
• US Class Current: 435/183
• CPC Class Codes: C07K 1/306 by crystallization
• CPC Class Codes: C12N 9/00 Enzymes; Proenzymes; Compositions thereof preparations containing enzymes for cleaning teeth A61K8/66, A61Q11/00; medicinal preparations containing enzymes or proenzymes A61K38/43; enzyme containing detergent compositions C11D; enzymes with nucleic acid structure, e.g. ribozymes, C12N15/113; Processes for preparing, activating, inhibiting, separating or purifying enzymes preparation of malt C12C1/00
• CPC Class Codes: Y10S 435/814 Enzyme separation or purification

Abstract

The present invention relates to a method of separating a protein, in particular an enzyme, from an aqueous solution of proteins, comprising (a) providing an aqueous mixture of proteins with a salt concentration at or below 1.5 Molar, to which a water soluble polymer has been added, and (b) recovery of the protein on crystalline form.

Claims

1. A method for obtaining a crystallized microbial protein from an aqueous mixture of proteins, said method comprising of the steps of:

(a) obtaining a microbial culture broth having a mixture of proteins, comprising a desired protein selected from the group consisting of an oxidoreductase, a protease, a lipase, a cellulase and an amylase;

(b) adjusting the salt concentration of said protein mixture at or below 1.5 Molar;

(c) adding a water soluble polymer to the protein mixture of step (b);

(d) stirring the polymer-containing protein mixture of step (c) for up to 48 hrs; and

(e) recovering the desired protein in crystalline form from the stirred mixture of step (d).

2. A method according to claim 1, which method further comprises concentration of the protein mixture prior to adding the water soluble polymer.

3. The method according to claim 2, wherein the protein mixture is concentrated by ultrafiltration, diafiltration, dialysation, evaporation or precipitation.

4. A method according to claim 2, in which the protein mixture is concentrated to a protein content of from 0.1 to 25% w/w.

5. A method according to claim 1, in which the water soluble polymer is a glycol or a mixture of glycols.

6. A method according to claim 5, in which the glycol is polyethylene glycol or polypropylene glycol.

7. A method according to claim 6, in which the polyethylene glycol has a MW of from 200 to 10000.

8. A method according to claim 5, in which the glycol is added in concentrations of 1-50% w/w.

9. A method according to claim 1, in which a salt is added to the mixture of proteins in a concentration of up to 1.5 Molar.

10. A method according to claim 9, in which the added salt is a salt of Magnesium, Calcium, Sodium, Potassium or Ammonium.

11. A method according to claim 10, in which the anion of the salt is selected from the group consisting of Chloride, Formiate, Acetate and Sulphate.

12. A method according to claim 1, in which the pH of the solution is adjusted to the optimum of the crystallization.

13. A method according to claim 12, in which pH is adjusted to pI±

1.

14. A method according to claim 9, wherein a salt is added to the mixture of proteins in a concentration of up to 1.5 Molar, and wherein the pH of the solution is adjusted to the optimum of the crystallization.

15. The method of claim 4, wherein the protein mixture is concentrated to a protein content of from 0.5 to 15% w/w.

16. The method of claim 15, wherein the protein mixture is concentrated to a protein content of from 1 to 10% w/w.

17. A method according to claim 8, in which the glycol is added in concentrations of 2-40% w/w.

18. A method according to claim 9, in which a salt is added in a concentration of up to 1.0M.

19. A method according to claim 14, in which a salt is added to a concentration of up to 1.0M.