DE2248843A1 - METHOD FOR MANUFACTURING LYSOZYME - Google Patents

Description

Patent attorneys Dipl -Ing. F. Ψβιοκμλγτν,

Dipl.-Ing. H.Weickmann, Dipl.-Phys.

Dipl.-Ing. R A.Weickmann, Dipl.-Chem. B. Huber

hk

F.2623 / P. - (SPA 052)

8 MUNICH 86, DEN

PO Box 860 820

MDHLSTRASSE 22, CALL NUMBER 98 3921/22

SPA - Societa Prodotti Antibiotic! 3.pA 8 Via Biella
Kailand / Italy

Process for the production of lysozyme

The invention relates to a method for extraction and Purification of lysozyme from protein. . . , ·

The isolation of lysozyme by Fleming led because of the strong non-specific immunological effect of this compound for their extensive use as pharmaceuticals for the treatment of viral and bacterial infections

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People. Lysozyme has also been used, among other things, as an anti-inflammatory agent and as an agent for the formation of leukocytes. For parenteral administration, therefore, constantly increasing amounts of lysozyme with high activity and high purity were required.

In G3-PS 1 110 466 is a method for the production of Lysozyme from protein described, according to which native protein with a weakly acidic ion exchanger at pH 6 to 7 and temperatures Treated below room temperature and the ion exchanger separated will. Subsequently, protein impurities are removed from the ion exchanger with a maximum of 0.2 molar salt solution eluted with a pH of 7 or less. Then, the lysozyme is eluted with an aqueous saline solution and from the obtained one Eluate precipitated by increasing the salt concentration. One of the advantages of this process is that once the Lyaozyme remaining egg is neither diluted nor contaminated and can be used for animal feed.

Since lysozyme is contained in protein in amounts of 0.3 to 0.5 percent by weight and is considerable for certain therapeutic treatments Amounts, in some cases up to 1 g / day or more, of very pure lysozyme are required, it can be seen that for Very large amounts of protein have to be processed to meet the needs. This inevitably leads to numerous problems, e.g. in storage, freezing and transport, and of course also making the end product more expensive.

The object of the present invention was therefore to provide a. simple and to provide an economical process for the production of high quality lysozyme that

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does not have the disadvantages mentioned. This task is carried out by the Invention solved.

The invention thus relates to a process for the production of lysozyme from protein by treatment with a weakly acidic ion exchanger at pH 6 to 7 and temperatures below room temperature, separating the ion exchanger, eluting protein impurities with a maximum 0.2 molar salt solution with a pH of at most 7 ₅ subsequent elution of the lysozyme with an aqueous salt solution and precipitation of the lysozyme from the resulting eluate by increasing the. Salt concentration, which is characterized in that the protein is concentrated by ultrafiltration before treatment with the weakly acidic ion exchanger. ^;

The present process thus represents an improvement of the process known from GB-PS 1 110 4-66, the process known from US Pat aforementioned patent known extraction of lysozyme is applied to protein, which previously by ultrafiltration 2- to Was concentrated 3 times.

In the process according to the invention, the aqueous lysozyme solution obtained during the elution of the ion exchanger can also be concentrated v / obei the concentration can also be done by ultrafiltration. In this way a considerable part of the the salt required to precipitate the lysozyme "from the eluate, e.g. ammonium sulfate, is saved.

It is also pointed out that in the known methods the lysozyme almost always in the last stage from aqueous solutions is precipitated by adding salts, whereby "a certain amount of these salts, e.g. sodium chloride, is converted into the end product

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remain. Ira method according to the invention, however, succeeds Produce lysozyme with a very low salt content if the lysozyme solution is subjected to ultrafiltration before drying will.

Ultrafiltration is already being used on a large scale, e.g. in sugar refining has been used for concentrating solutions and in wastewater purification.

Egg white contains proteins with low molecular weights, such as lysozyme, and proteins with much higher molecular weights, such as Ovalbumin, conalbumin, ovomucoid and avidin. These proteins are dissolved in an aqueous medium that also contains compounds with much lower molecular weights, v / ie glucose, and contains inorganic ions.

Depending on the membrane used, the protein can the applied pressure and the number of circulations can be concentrated up to 3 times or more, the lysozyme content of protein increases to 0.9 percent or more.

Some of the advantages achieved by reducing the original protein volume have already been described above. Another, even more important advantage is that in the ultrafiltration according to the invention, a large part of the glucose, the causing the dried egg white to turn brown, is removed. The remaining concentrated protein that is not with contaminated with toxic or inedible compounds, can be dried with considerable heat saving or after other procedures can be processed, with one for purposes of use usable by-product is obtained.

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In the process according to the invention, the semipermeable membranes are in particular made of organic polymers, e.g. made of vinyl and Acrylic polymers, existing membranes preferred. It can however, numerous other types of membrane can also be used, ■ provided that they have such a pore size, "at the Proteins, including lysozyme, are retained while water, glucose and inorganic ions pass through the membrane.

The examples illustrate the invention.

example 1

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 0.9 to 1.5 percent by weight. The concentrated solution obtained in this way is treated with 15 liters of a weakly acidic ion exchanger ("Amberlite" IRG-50) adjusted to pH 6.5. The temperature of the mixture is maintained at 0 to 5 ° C until the lysozyme concentration in the protein concentrate has dropped to 100 to 150 meg / ml. The ion exchanger is separated off and washed with water / water and then with a 0.2 M sodium phosphate buffer with a pH of 6.5 until no more protein-containing s- material is eluted. The lysozyme is then eluted with an 8 percent strength by weight ammonium sulfate solution. The resulting solution is then concentrated by ultrafiltration. The concentrated solution is mixed with ammonium sulfate up to a concentration of about 4-0 percent by weight, the lysozyme completely precipitating out.

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Example 2

For the production of lysozyme hydrochloride, the procedure described in Example 1 described procedure repeated. The precipitated lysozyme is dissolved in water by adding hydrochloric acid up to pH 3> 3. the Solution is filtered to separate the insoluble material, and the lysozyme hydrochloride is then added by adding a aqueous sodium chloride solution precipitated.

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ORIGINAL INSPECTED

Claims (2)

Claims 1. Process for the production of lysozyme from protein by treatment with a weakly acidic ion exchanger at pH 6 to ' ¹ / · and temperatures below room temperature, separating the ion exchanger, eluting protein impurities with a maximum 0.2 molar salt solution with a pH of at most 7i subsequent elution of the lysozyme with an aqueous salt solution and precipitation of the lysozyme from the eluate obtained by increasing the salt concentration, characterized in that the protein is concentrated by ultrafiltration before treatment with the weakly acidic ion exchanger. 2. The method according to claim 1, characterized in that the eluate containing the lysozyme is added before the lysozyme is precipitated concentrated by a second ultrafiltration. 309815/0892 fiCj + 'itf-ii