CS269929B1 - Method of avidin's insulation - Google Patents

Abstract

The solution is to isolate avidin from waste water produced by lysosyr. This purpose is achieved by avidin is from the waste water produced by lysozyme production in the first phase bound to strongly acidic ionex. This is followed by washing the avidin fraction of ammonium carbonate solution 2.3 to 2.7 wt. and concentration of the solution ultrafiltration with simultaneous removal undesired salts. The purification is then performed on a weakly acidic ion exchanger and after reprocessing thickening and desalting by ultrafiltration is obtained purified avidin freeze-dried.

Description

CS 26992? B1

AND

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a process for isolating avidin from wastewater resulting from the production of isosyme from egg white.

The avidin methods used so far utilize native egg white as the basic raw material. Avidin is about 0.05 wt. the total egg egg white protein, which is 10% by weight with an average total protein content. about 50 mh avidin egg white egg. To obtain purified avidin of high biological activity, it is very easy to apply repeated ion exchange chromatrography or affinity chromatography to provide low yields at high labor and economic cost. The sorbents commonly used are CM-cellulose or AMBERLITE type cation exchangers. The aforementioned disadvantages are eliminated by the process of isolating avidin according to the invention, the principle of which is that the starting material for avidin isolation is wastewater produced by the production of avidin enriched lysozyme in which 1.0 to 2.0% by weight of avidin is present. 200 to 400 mg of avidin in waste water. The use of this effluent will make it possible to reduce the number of purification steps to obtainavidin with high biological activity when the highly acidic SUP-OSTSORB ion exchanger is included in the first stage of purification. Bound avidin is eluted with a solution of ammonium carbonate at a concentration of 2.3 to 2.7% by weight. The avidin fraction obtained is concentrated by ultrafiltration to remove the salts. The avidin fraction concentrate is comprehensively purified on a weak acid ion exchanger, preferably CM-cellulose, CM-32 or CM-52, and freeze-dried after re-concentration and desalting by ultrafiltration.

Avidin is obtained for its ability to bind biotin (Vitamin H, with an extremely high association constant of 10 µM), which predisposes this pair to the introduction of a new generation of protein and nucleic acid detection methods. One of the most effective methods of detection phase analysis in labeled molecule immunochemical analytical methods By using this method, the sensitivity of a commonly used enzyme-linked immunoassay (ELISA) assay can be significantly altered and the isotopic labeling (RIA) method, preferably substantially greater. the stability of conjugates and, last but not least, the elimination of health hazards when working with isotopes are used primarily in clinical biochemistry (diagnostic tests).

The avidin-biotin system is also used in research, for example, in the study of receptors on the surface of biological membranes, in histochemistry, in the isolation of enzyme subunits, nucleic acids and peptides, in the determination of protein structure, and the like. of the initial parameters of 2.0 wt. % total protein and 8 wt. potassium chloride is repeatedly ultra-filtered until the final concentration of potassium chloride is max. 1% by weight, and the volume is concentrated to 100 1. 40 kg of SHP-OSTSORB are added to the resulting solution, the pH is adjusted to 8.5-9 0 and stirred at 4 to 10 ° C for 6 hours. The resulting supernatant is allowed to stand for about 12 hours. The concentrate is separated and the carrier is washed 3 times with ammonium carbonate solution: a concentration of less than 0.5 vol. 80 L for 15 min.

The sorbent-bound avidin is eluted 2 times with 80 l of an ammonium carbonate solution having a concentration of 2.5% by weight. for 60 min. 160 L of the avidin fraction obtained is desalted and concentrated by ultrafiltration. Via a column of 50 L CM-cellulose CM-32 cartridges (WHARMAN - 10 Kg dry cellulose, representing about 10% column capacity), the avidin fraction is allowed to flow at a flow rate of about 200 mL / min. The carrier is washed with 100 l of ammonium carbonate solution at a concentration of 0.3 to 1.0% by weight.

Claims (1)

CS 269929 δΐ The sequential elution of bound proteins follows using 0.5 to 3.0% by weight. a continuous concentration gradient of a solution of ammonium carbonate with a total volume of 150 L (about 3 column volumes of column). After approximately 15% of the total elution volume has flowed out, the avidin solution begins to flow, from which the salts present are ultimately removed by ultrafiltration and dried after lyophilization. The yield from this volume is 90 to 100 g of avidin with a biological activity of 10 to 13 µg of biotin per 1 mg of preparation. OBJECT OF THE INVENTION A method for isolating avidin, characterized in that the feedstock is wastewater produced by the production of lysozyme, enriched with avidin, from which a strongly acidic ion exchanger is weighed in the first phase, followed by washing the avidin fraction with a solution of ammonium carbonate at a concentration of 2.3 to 2.7 wt. and concentrating by ultrafiltration with simultaneous removal of undesired salts, followed by purification on a weakly acidic ion exchanger and, after re-concentration and desalting by ultrafiltration, the purified avidin obtained is freeze-dried.