Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Casein or caseinate
Patent
1995-07-12
1998-07-14
Robinson, Douglas W.
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Casein or caseinate
530362, 530363, 530366, 530385, 530386, 5303905, 530416, 530427, 2101982, 210635, 210656, 210660, C07K 1600, B01D 1508
Patent
active
057805938
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
This invention relates to a method of isolating a biomolecule such as a protein or peptide from a medium containing biomolecules by ion exchange; a method of isolating phosphopeptides from casein hydrolysates; and the use of a biomolecule isolated according to such methods in the production of a food, a feed, a health care product, a cosmetic, or a pharmaceutical.
1. The Technical Field
Isolation or fractionation of biomolecules in a medium containing biomolecules by ion exchange usually involves the binding of the biomolecule to an ion exchanger at an appropriate pH and low salt concentration e.g. less than 0.1M salt of the medium. Following wash out of unbound components, the bound biomolecules are eventually eluted by a suitable eluant.
In general, elution of biomolecules bound to the ion exchanger may be achieved by changing the pH and/or increasing the salt concentration of the medium whereby the equilibrium reaction of bound/unbound biomolecules is shifted to unbound biomolecules. Especially for proteins and peptides to be used in food, feed, health care, cosmetic, or pharmaceutical products, it is of great importance that the biomolecules are eluted from the ion exchanger under conditions which minimize the destruction of their biological and physico-chemical properties. At the same time, it is highly desirable to keep a low salt concentration in the eluate whereby desalination of the eluate, losses of the isolated biomolecule and increased costs of the final product can be avoided.
Biomolecules of interest include as mentioned proteins and peptides, particularly casein phosphopeptides which have attracted increasing attention as important biological molecules due to their calcium binding properties.
2. Prior Art Disclosure
European Patent Application No. 0 476 199 discloses a method of separating phosphopeptides from an aqueous medium containing casein hydrolysates wherein the pH of the medium is adjusted to 4.6 and the solids therefrom are separated; the pH of the medium is adjusted to a value larger than 4.0, preferably larger than 6.0; the medium is subjected to ion exchange, thereby saturating the resin, and the unbound material is collected; the bound phosphopeptides are eluted from the ion exchange resin with a suitable eluant; and the obtained product is dried.
The preferred ion exchanger resin is an anion exchanger. As a suitable eluant there is suggested an aqueous medium having a pH less than 4.0, or an aqueous medium having a pH larger than 4.0 and a salt concentration larger than 100 mM, preferably 500 mM; said salt exemplified by sodium chloride. As an example of an eluant having a pH less than 4.0, an aqueous medium having a pH of 1.0 is given. However, no indication is provided about the substance used to obtain said pH. After elution the eluate is adjusted to pH 7.0 and subsequently dried.
Thus, eluting the anion exchange resin is either performed with an acidic eluant, which provides a low pH in the eluate, or eluting the anion exchanger with a neutral or basic eluant also containing high concentrations of salt, which provides too much salt in the eluate.
A major drawback of this method is the high contents of salt in the final product. In the case that hydrochloric acid is used to achieve a pH of 1.0 in the eluant, the subsequent neutralization to pH 7.0 with sodium hydroxide results in a sodium chloride concentration of at least 0.1M in the eluated aqueous medium before the drying step. As such aqueous eluates rarely contains more than a few percent of product, drying of the eluate will result in an unacceptable high contents of salt in the dried product. Alternatively the eluate may be desalinated before drying but this decreases the yield of separated phosphopeptides and increases the costs of the final product. Furthermore, the use of low pH for elution may generally lead to partial destruction of the biological and physico-chemical properties of the eluted proteins and peptides.
Juillerat et al., Journal of Dairy Research, Vol. 56, 603-611 (1989),
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Juillerat et al, Journal of Dairy Research, vol. 56, pp. 603-611, 1989.
"The Separation and Amino Acid Composition of a Pure Phosphopeptone Prepared from B-Casein by the Action of Trypsin.sup.2 ", R.F. Peterson et al., Amino Acid Composition of a Pure Phosphopeptone, Jan. 5, 1958, vol. 80, pp. 95-99.
"Separation chromatographique de peptides issus de 1 'hydrolyse enzymatique de proteines de lactoserum et de caseines", Frederique Touraine et al., Le Lait, 1987, 67 (4). 419-435 (Summary in English).
"Tryptic phosphopeptides from whole casein", Marcel A. Juillerat et al., Journal of Dairy Research (1980) 56, 603-611.
"Glutamine purification -from Corynebacterium sp. fermentation broth by cation exchange chromatography, anion exchange chromatography and precipitation", & KR 8912915, 890907, Dialog Information Services, File 357, Biotechnology abstracts, Dialog acc. No. 142332 (Miwon-Foods).
Aagesen Margit Irene
Elleg.ang.rd Katrine Hvid
Gammelg.ang.rd-Larsen Claus
Lihme Allan Otto Fog
Kem-En-Tec A/S
MD Foods amba
Mohamed Abdel A.
Robinson Douglas W.
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