DE2248843B2 - PROCESS FOR THE PRODUCTION OF LYSOZYME, FROM PROTEIN - Google Patents

Description

The invention relates to a method for obtaining lysozyme from protein.

The isolation of lysozyme was carried out by Fleming because of the strong unspecific immunological effect of this compound to its extensive Use as a medicament for the treatment of viral and bacterial infections in humans. Lysotymium was a. also as an anti-inflammatory agent and as an agent to stimulate leukocyte formation used. For parenteral administration, therefore, constantly increasing amounts of lysozyme mi 'were higher Activity and great purity are required.

In GB-PS 11 10 466 a process for the production of lysozyme from protein is described, after which native protein with a weakly acidic ion exchanger at pH 6 to 7 and temperatures Treated below room temperature and the ion exchanger is separated. Then be Protein contamination from the ion exchanger with a maximum of 0.2 molar salt solution with a pH 7 or less eluted. The lysozyme is then eluted with an aqueous salt solution and removed from the obtained eluate precipitated by increasing the salt concentration. One of the advantages of this procedure is that the protein that remains after the lysozyme has been separated is neither diluted nor contaminated and closed Can be used for feeding purposes.

Because lysozyme is contained in protein in amounts from 0.3 to 0.5 percent by weight and for certain therapeutic treatments in considerable amounts, in some cases up to 1 g / day or more, of very high pure lysozyme are required, it can be seen that very large amounts of protein are required to cover the requirement need to be worked up. This inevitably leads to numerous problems, e.g. B. in storage, the Freezing and transportation, and of course also making the end product more expensive.

The object of the present invention was therefore to provide a simple and economical process for production of high quality lysozyme available that has the aforementioned disadvantages does not have. This object is achieved by the invention

solved

The subject matter of the invention is thus the subject matter defined in the claims.

In the method according to the invention, the Elution of the ion exchanger obtained aqueous lysozyme solution can be concentrated, the concentrating takes place by ultrafiltration. In this way, a considerable part of the precipitate of the Lysozyme from the eluate required salt, e.g. B.

Ammonium sulfate, saved

It should also be noted that in the known method, the lysozyme in the last stage is precipitated from aqueous solutions by adding salts, whereby a certain amount of these

Salts, e.g. B. sodium chloride, Im remains in the end product In contrast, the method according to the invention succeeds in producing lysozyme with a very low salt content, when the lysozyme solution is subjected to ultrafiltration before drying.

The ultrafiltration is already on a large scale z. B.

Used in sugar refining to concentrate solutions and in wastewater treatment been.

Egg white contains proteins with low molecular weights, like lysozyme, and with much higher molecular weights like ovalbumin, conalbumin, ovomucoid and avidin. These proteins are dissolved in an aqueous medium that also has compounds with contains a great deal of low molecular weights, such as glucose, and inorganic ions.

Depending on the membrane used, the pressure applied and the number of circulations, the protein can be concentrated up to 3 times or more, with the lysozyme content of the protein increasing to 0.4 percent or more.

Above, some of the levels achieved by reducing the original protein volume have already been achieved Advantages described. Another, even more important advantage is that the invention-Ben used ultrafiltration a large part of the glucose, which is the browning of the dried Egg white is removed. The remaining concentrated protein can under considerable Heat saving dried or processed by other methods, with one for Feed usable by-product is obtained.

In the method according to the invention, as semipermeable membranes, in particular made of organic polymers, eg. B. made of vinyl and acrylic polymers, existing membranes are preferred. However, numerous other types of membranes can be used provided they have a pore size such that proteins, including lysozyme, are retained while water, glucose and inorganic ions pass through the membrane.
The examples illustrate the invention.

example

52 kg of native protein are adjusted to pH 6 to 7 with dilute hydrochloric acid or acetic acid and then concentrated by ultrafiltration to a lysozyme content of about 0.9 to 1.5 percent by weight The concentrated solution obtained is mixed with 15 liters of a weakly acidic ion exchanger adjusted to pH 6.5 treated. The temperature of the mixture is kept at 0 to 5 ° C until the lysozyme concentration in the protein concentrate dropped to 100 to 150 mcg / ml

When the ion exchanger is separated and washed with ammonium sulfate solution eluted The solution obtained

Water and then with a 0.2 M sodium phosphate is then concentrated by ultrafiltration

Washed phate buffer with a pH of 6.5 until concentrated solution is made up to with ammonium sulfate

no more proteinaceous material is eluted then at a concentration of about 40 percent by weight

the lysozyme is mixed with an 8 weight percent s, whereby the lysozyme completely precipitates

Claims (2)

Patent claims: 1. A method for obtaining lysozyme from protein, characterized in that one the lysozyme is concentrated in the egg white by ultrafiltration of the egg white so that the obtained Concentrate with a weakly acidic ion exchanger at pH 6 to 7 and temperatures below Treated room temperature that after separating the ion exchanger from this Protein contamination with a salt solution of 0.2 molar or less with a pH of 7 or less Eluieit that you then the lysozyme from the ion exchanger with an aqueous salt solution eluted and that the lysozyme from the eluate obtained by increasing the salt concentration fails 2. The method according to claim 1, characterized in that the eluate containing the lysozyme concentrated before the precipitation of the lysozyme by a second ultrafiltration.