Method of obtaining avidin and lysozyme

FIELD: chemistry; biochemistry.

SUBSTANCE: present invention pertains to biochemistry. To obtain lysozyme enzyme and avidin protein from albumen, chromatography of albumen homogenisate is carried out on silochrome C-80 at neutral pH (7.0-7.5). Ballast proteins are removed in a 0.2 M glycine - NaOH buffer in the presence of 0.3 M NaCl at pH 9.3-9.5. Target proteins are eluted together with carbonate buffer with 0.5-0.8 M NaCl, with pH 10.8-11.0, and subsequent separation of proteins through gel filtration on a column with sephadex G-75.

EFFECT: simplification of the method of obtaining electrophoretically pure proteins and increasing the efficiency of the method.

1 dwg, 4 ex

 

The invention relates to biotechnology, in particular to methods for enzyme lysozyme and protein avidin. Lysozyme is used as a medicinal drug, preservative in the food industry, biotechnology. Avidin is a biologically active compound which are used in medicine, chemical, microbiological and food industry.

A method of obtaining lysozyme from egg white by direct crystallization by adding 5% by weight NaCl and bringing the pH to 9.8 with an alkaline agent, followed by purification of lysozyme by double recrystallization, and stages of crystallization and recrystallization add the seed crystals, as the alkaline agent used NaHCO3and to egg protein, released from lysozyme, add HCl, which allows its use in the food industry, for example, to obtain egg powder [1].

A method of obtaining avidin homogenization egg whites with the subsequent deposition of homogenizate ammonium sulfate in the range of 40-100%, saturation by dissolving the precipitate in water, repeated precipitation with a polar organic solvent (ethanol) at the final concentration of the latter 40-75%, extraction of sediment avidin acidic buffer solution at pH 1-6, chromatography of the extract on the column KM - cellulose with the following the elution, dialysis and allocation avidin [2].

The lack of these cues is multi-stage and duration, thus produce only one product, i.e. when receiving avidin lost lysozyme and Vice versa.

The objective of the invention is to simplify the method of producing lysozyme and avidin and increase its effectiveness.

The problem is solved in that a method for obtaining lysozyme and avidin of egg proteins, including homogenization egg protein chromatography of homogenizate with subsequent elution and selection of target products, and homogenized passed through a column of highly porous silochrome C-80 when neutral (7,0-7,5) pH values, then remove the ballast proteins in 0.2 glycine-NaOH buffer at a pH of 9.3 to 9.5 in the presence of 0.3 M NaCl, and the target proteins elute together carbonate buffer with 0.5-0.8 M NaCl, at a pH of about 10.8-11,0 with subsequent separation of proteins by gel-filtration on a column of Sephadex G-75.

The difference of the proposed method is the use of highly porous (52 nm) silochrome C-80. Sorption of proteins occurs at neutral pH values, and the elution of the target products with sorbent is carried out jointly carbonate buffer with NaCl at pH near or above the value of isotocin target products, further separated by gel-filtration on Sephadex G-75. The pH value in column silochrome quickly (2-3 minutes, to generate is to kilogram was under alkaline conditions minimum time) is lowered to neutral values, rinsing with water.

Silagra known as silica sorbent with a very weak ion exchange properties at neutral pH values. So far such sorbents are widely used only for cleaning viruses [3], but not proteins. As his iStock equal to 2.0 to 2.5, and RK˜9,0 (the pH at which it is charged half of the groups), at neutral pH values, and determining role in the sorption of proteins are not ionic, and hydrogen bonds and hydrophobic interactions between the protein and the sorbent. Only when approaching the pH to 9,0, i.e. to the value of RK, the role of ionic interactions increases dramatically. Therefore, alkaline proteins (i.e. having high stocki) suiryudan later proteins with lower stockli. Thereby obtaining the purified proteins.

The invention is illustrated by the following examples.

Example 1. Protein derived from 100 eggs, diluted 6-fold with 0.02 M phosphate buffer, pH 7.0 and passed at room temperature through a column with silochrome 80 (8×15), equilibrated with the same buffer until the optical density in the wash water at 280 nm of 0.01. After this column balance 0.05 M glycine - NaOH buffer pH to 9.3 with 0.3 M NaCl and washed them as well, until the optical density at 280 nm of 0.01. Column offset of 0.5 M carbonate buffer pH to 10.8 with 0.5 M NaCl and elute avidin with lysozyme at 80 ml/hour, a protein Solution is neutralized with 5 M of the criminal code is usnei acid, brought to a pH of 6.0 alkali (NaOH), concentrate on the membrane, cut-off molecular weight of >10 kDa to 4-5 ml and placed on a column (2×50), balanced 0.05 M acetate buffer, pH of 6.0. This was followed by gel filtration with a speed of 10-12 ml/H. the First peak (smaller) - avidin. The second peak - lysozyme. Both get protein electrophoretic net (see drawing). The activity of lysozyme and its output is compared with the total activity in primary homogenizate check hydrolysis of dried cells of Micrococcus lysodeicticus [4]. Activity avidin and its output is checked for binding with d-Biotin [5].

Lysozyme activity is 12,000 IU/mg.

Yield 84%.

Activity avidin 12 u/mg

Yield 88%.

Example 2. Protein derived from 100 eggs, diluted 7-fold with 0.02 M phosphate buffer, pH 7.5 and passed at room temperature through a column with silochrome 80 (8×15), equilibrated with the same buffer until the optical density in the wash water at 280 nm of 0.01. After this column balance 0.05 M glycine - NaOH buffer pH to 9.5 with 0.3 M NaCl and washed them as well, until the optical density at 280 nm of 0.01. Column offset of 0.5 M carbonate buffer pH of 11.0 with 0.8 M NaCl and elute avidin with lysozyme at 80 ml/hour, a Solution of proteins neutralize 5 M acetic acid, brought to a pH of 6.0 alkali (NaOH), concentrate on the membrane, cut-off molecular m is ssy > 10 kDa to 4-5 ml and placed on a column (2×50), balanced 0.05 M acetate buffer, pH of 6.0. This was followed by gel filtration with a speed of 10-12 ml/H. the First peak (smaller) - avidin. The second peak - lysozyme. Both get protein electrophoretic net (see drawing). The activity of lysozyme and its output is compared with the total activity in primary homogenizate check hydrolysis of dried cells of Micrococcus lysodeicticus [4]. Activity avidin and its output is checked for binding with d-Biotin [5].

Lysozyme activity is 12,000 IU/mg.

Yield 85%.

Activity avidin 12 IU/mg.

Output 90%.

Example 3. Protein obtained from 20 chicken eggs, diluted as in example 1, the same phosphate buffer and placed in a plastic Cup with a cylindrical wall and a flat bottom. Cooled to 4-5°contribute silagra C-80 of 125 ml and gently stirred for at least 10 hours at a temperature of 4-5°With mechanical stirrer, 50-60 rpm). Stop the mixer, silagra transferred to column (4×10 cm) and at room temperature, washed as in example 1. Next, as in example 1, using glycine - NaOH and carbonate buffer. A solution of proteins, buervenich this buffer is acidified, as in the previously mentioned example, 5 M acetic acid, made alkaline to pH 6,0, also concentrate on the membrane to a volume of 3-4 ml and placed on a column DL the gel filtration (2× 50), balanced 0.05 M acetate buffer, pH of 6.0. Elute in the same way as in example 1. The release of lysozyme and avidin 80% and 85%, respectively.

Example 4. Protein obtained from 20 chicken eggs, diluted as in example 2, the same phosphate buffer and placed in a plastic Cup with a cylindrical wall and a flat bottom. Cooled to 4-5°contribute silagra C-80 of 125 ml and gently stirred for at least 10 hours at a temperature of 4-5°With mechanical stirrer, 50-60 rpm). Stop the mixer, silagra transferred to column (4×10 cm) and at room temperature, washed as in example 2. Further, as in example 2, using glycine - NaOH and carbonate buffer. A solution of proteins, buervenich this buffer is acidified, as in the previously mentioned example, 5 M acetic acid, made alkaline to pH 6,0, also concentrate on the membrane to a volume of 3-4 ml and placed on a column for gel filtration (2×50), balanced 0.05 M acetate buffer, pH of 6.0. Elute as in example 2. The release of lysozyme and avidin 85% and 90%, respectively.

Thus, the proposed method, in contrast to the known [1, 2], allows to obtain lysozyme and avidin with the release of 80-85% and 85-90%, respectively, thereby increasing the efficiency of the method, and also to reduce the number of stages of selection. Silorane column, the proposed method can tolerate, at the very the least, 10 cycles without loss of activity.

Information taken into consideration

1. RU patent No. 94025573 A1, Kachkaev B. I., etc. a Method of producing lysozyme from egg white (1996).

2. SU 1643554 A1, Ikelite and other Way of getting avidin (1991), Bulletin No. 15.

3. Maslan, Geesepeace, Shebester and others (1971), Virology, No. 4, s-482.

4. D.Shugar (1952), Biochim. Biophys. Acta, v.8, p.302-306.

5. N.M.Green (1963), Biochem J., v.89, p.599-603.

The method of producing lysozyme and avidin of egg proteins, including homogenization egg protein chromatography of homogenizate with subsequent elution and selection of target products, characterized in that homogenized passed through a column of highly porous silochrome C-80 when neutral (7,0-7,5) pH values, then remove the ballast proteins in glycine-NaOH buffer at a pH of 9.3 to 9.5 with 0.3 M NaCl, and the target proteins elute together carbonate buffer at pH 10,8-11,0 with 0.5 M NaCl, followed by separation by gel-filtration on a column of Sephadex G-75.



 

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