The method of producing lysozyme from egg white

 

(57) Abstract:

The invention relates to a process for the production of lysozyme from egg white. The invention consists in the obtaining of lysozyme undiluted egg protein by column chromatographic process on the cation exchanger, providing processing for 1 cycle of large quantities of protein by a small number of the cation, the receipt of the eluate of high purity with a high concentration of lysozyme. For that egg protein is passed through a column of strongly acidic, macroporous cation exchange resin, then the cation exchanger is washed with water or sodium chloride solution with a concentration of not higher than 0.2 M, then elute lysozyme, skipping alkaline solutions of inorganic salts with a pH of 8.5-13,2. The composition of the eluent may include soda Na2CO3or potash (K2CO3, chlorides, sulfates or phosphates of sodium or potassium in a concentration of 0.05-2 M, and ethyl alcohol at a concentration of 10-40%. 3 C.p. f-crystals, 1 table.

The invention relates to techniques for the production of egg white lysozyme, used as a preservative additive in veterinary medicine, medicine, food industry.

A method of obtaining lysozyme, in which egg protein is treated with solutions of the om-known method is the low activity of the obtained lysozyme, the complexity and duration of the process. Obtained in this method the saline waste egg whites and the products are of very limited use.

A method of obtaining lysozyme from egg white by processing undiluted egg protein ion-exchange sorbents with subsequent desorption of lysozyme aqueous solutions of salts. As the ion exchange sorbent use macroporous weak acid cation exchangers, for example, "Duolith C-464" [2] According to this method, native, undiluted with water or buffer solutions of egg protein is brought into contact with a weak acid cation exchange resin in the reactor, then the cation exchanger is washed from the ballast proteins salt solution with a salt concentration of no higher than 0.2 M or water and then elute lysozyme aqueous salt solution, for example, 0.1 M nutrifaster buffer (pH 7.5 to 8.3) containing 0.15 M sodium chloride. Later in the collected eluate add inorganic salt crystallization or vysalivaniya lysozyme, or the eluate provide for subsequent drying.

The use of weak acid cation exchangers type "Duolith" for the first time allowed to obtain the lysozyme of raw materials were extracted only the target product, and the consumer properties of egg white, the confectionery industry, in the formulation of powder products, which resulted in a wide spread of this method. The method, however, is not without disadvantages.

The most important disadvantages of this method include the following:

all weak acid cation exchangers, including "Duolith", strong (3-5 times) change its volume when changing the pH or ionic strength balancing their solutions, which makes it almost impossible to carry out a chromatographic process in the column. The implementation of such a process on an industrial scale is possible only in the reactor, which is technically difficult and is associated also with a strong deterioration of the cation;

only a relatively small amount of egg protein can be processed within 1 cycle a certain amount of weak acid cation exchanger (3-4 volume protein 1 by volume of cation exchange resin);

after processing of the cation salt solutions with the purpose of desorption of lysozyme latter is recovered in the form of a highly diluted solution (1-3 mg/ml), which may contain large (up to 50%) of the amount of ballast proteins.

In General, these shortcomings lead to the increasing complexity of technology and the rise of lysozyme.

The present invention assessory process to process large quantities of raw materials at low cost sorbents and to obtain a sufficiently pure eluate with a high concentration of lysozyme.

The invention consists in the following.

Egg protein is passed through a column of strongly acidic (pKa 1-2) macroporous cation exchange resin, in particular KU-2S, and desorption of lysozyme are eluent with a pH of 8.5-13,2. The composition of the eluent may include soda or potash at a concentration of 0.05-2 M Eluent may contain additional or other inorganic salts chlorides or sulfates or phosphates of sodium or potassium in concentrations less than or equal to the concentration of soda. The composition of the eluent can also include ethyl alcohol at a concentration of 10-40%

Use nanabhai macroporous strongly acidic cation exchange resin provides the possibility of lysozyme in a column process, which allows to increase the performance of the method and reduce the cost of obtaining lysozyme.

The substance used soda, potash, neutral salts and ethanol are equivalent from the point of view of their debilitating effect on the electrostatic interaction of polyelectrolytes in water. The specified range of pH values is due to the fact that at pH below 8.5 does not occur desorption of Lisa what is fact, when the concentration is below 0.05 M not going desorption of lysozyme, and at concentrations above 2 M there is a "salting out" of lysozyme on the cation exchanger, which reduces the degree of desorption. These concentrations of ethanol are determined by the same circumstances.

Specific values of the concentrations of salts within the specified range is chosen based on the nature of the subsequent procedures. So, if after desorption conduct desalting of the eluate with subsequent drying, it is advisable to use an eluent with a low salt concentrations (e.g., 0.1 M). If after desorption conduct salting out of lysozyme from the eluate, it is advisable to use an eluent with high concentrations of salts (for example, 0.4 M). Acceptable from the point of view of Economics of the process are the concentration (total) salts of 0.4 M, ethanol 20% In General, the higher the concentration of salts and ethanol eluent, the more concentrated (but not less than 10 mg/mg) is the target solution of lysozyme (eluate).

The method is as follows.

The original egg white pass with the speed of 15-45 ml/h/cm2through a column of cation exchange resin KU-2P 7-15 volume of the cation exchanger. After that, the sorption stop eluent until while the lysozyme is not desorbed, with speeds up to 45 ml/h/cm2. Thereafter, the eluate can be obessolivanie and dry or to carry out the crystallization of lysozyme by known methods.

Examples of implementation of the method.

Example 1. Through the column with a volume of 8 ml (1.8 to 2.1 cm) with a cation exchange resin KU-2P missed a 200 ml egg whites with a speed of 6 ml/hour at a temperature of 20oC, then distilled water 30 ml, then 0,11 NaOH (pH 13,2) in an amount of 100 ml of the cation exchanger was servirovaci 70% of the original amount (activity) of lysozyme, desorbitados 40% of the adsorbed lysozyme.

Examples 2 to 15, are shown in table illustrate the efficiency of eluting solutions with different salt composition, different pH and different ethanol content.

Example 16. Through the column with a volume of 0.8 l (1010,2 cm) with a cation exchange resin KU-2P missed 8 l egg whites with a speed of 420 ml/HR, then 3 liters of 0.2 M NaCl, and then 2 l of eluent composition 0.2 M NaCl, 0.2 M Na2CO3and 30% ethanol at a speed of 250 ml/hour. As a result, the cation exchanger was servirovaci 83% of the original amount of lysozyme was desorbitados 80% of the adsorbed amount of lysozyme. Next, the eluate was absoluely known manner and subjected to lifelinebeta.

1. The method of producing lysozyme from egg white by processing undiluted egg protein ion-exchange sorbents with subsequent desorption of lysozyme eluted salts, characterized in that the ion exchange sorbent using a strongly acidic cation exchangers and desorption exercise eluted salts with a pH of 8.5 to 13.2.

2. The method according to p. 1, characterized in that the composition of the eluting solution is injected soda or potash at a concentration of 0.05 to 2 M

3. The method according to p. 1 or 2, characterized in that the composition of the eluting solution is further added chlorides, or sulfates, or phosphates of sodium or potassium in a concentration of 0.05 to 2M.

4. The method according to p. 1, or 2, or 3, characterized in that the composition of the eluting solution is further added ethyl alcohol at a concentration of 10 - 40%

 

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