RU2388819C2 - Method for production of horseradish peroxidase - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: method for production of horseradish peroxidase includes homogenising of horseradish roots, extraction of ferment, concentration by ultrafiltration and depositing proteins with ammonium sulfate. Deposit of proteins is dialysed against water and 0.01-0.03 M of solution of TEA-HCl buffer, with further cleaning of peroxidase from ballast proteins in column with DEAE-cellulose in the same buffer. Then cleaning is continued in 0.01-0.03 M of MOPS-NaOH buffer solution in column with KM-cellulose with values of pH and pK of buffers equal to 7.1 -7.4 and 7.5-7.6 accordingly. Cleaned target product is dialysed against water and 0.01-0.03 M of NaCl, and then freeze-dried.

EFFECT: simplified production of highly pure peroxidase of horseradish and its increased yield.

3 cl, 4 ex

Description

The invention relates to biochemistry and biotechnology, and in particular to the production of highly purified horseradish peroxidase by adsorption chromatography.

Highly purified horseradish peroxidase (purification criterion A ₄₀₃ / A ₂₇₅ ≥3.1 - RZ from Reinheitszahl - purity indicator) is one of the most popular enzymes for biochemistry and biotechnology. In particular, it is widely used in enzyme-linked immunosorbent assay methods, for example, to determine HIV infection, hepatitis and other socially significant human diseases.

A known method of producing peroxidase, including the homogenization of horseradish roots, extraction of the enzyme with water or saline, fractionation of the extract with ammonium sulfate, purification by gel filtration, alcohol precipitation, re-precipitation with ammonium chloride, filtration through Sephadex G-50, ion-exchange chromatography and dialysis [1].

The disadvantages of this method are the complexity and duration of the preparation, the relatively low purity of the obtained preparation (RZ ~ 2.7), the difficulty of obtaining a large amount of the enzyme.

There is also a method of producing peroxidase, including the homogenization of horseradish roots, enzyme extraction, fractionation of the extract with ammonium sulfate, gel filtration [2].

The disadvantage of this method is the low yield of the enzyme, its low purity.

The objective of the invention is to increase the yield of highly purified enzyme while simplifying the production method.

The problem is solved by the fact that the proposed method of producing horseradish peroxidase, including the homogenization of horseradish roots, enzyme extraction, concentration by ultrafiltration, protein precipitation with ammonium sulfate, dialysis of protein precipitate against a 0.01-0.03 M solution of TEA (triethanolamine) -HCl buffer, sequential purification of peroxidase from ballast proteins on a column with DEAE cellulose in the same buffer, and then in a 0.01-0.03 M solution of MOPS (N-morpholinopropanesulfonic acid) -NaOH buffer on a column with KM cellulose, at pH and pK buffers close to isoto ke of the target product, and the concentration that ensures the movement of peroxidase down the column, according to the principle of adsorption-desorption, at a rate lower than that of some ballast proteins having the same charge with a charge on the column, while the other part remains at the start due to ion exchange ; the solution of the purified target product is dialyzed against water and 0.01-0.03 M NaCl, followed by lyophilization, and the enzyme is extracted with water after root homogenization, ammonium sulfate is added at a concentration of 70-75% of saturation, and the pH and pK of the buffers are 7, 1-7.4 and 7.5-7.6, respectively.

The difference between this method is the simultaneous use of the same carrier for adsorption chromatography of the target product and for purification from ballast proteins at pH values close to the yield of the target product, as well as the use of low concentration buffers with pK values close to the yield of the enzyme, and the components buffers have bulky electron-donor or electron-withdrawing groups competing for adsorbent with peroxidase macromolecule groups. Horseradish peroxidase is obtained from the juice of horseradish roots, collected at the very beginning of flowering, at this time the content of the enzyme in the roots is 3-4 times higher compared to the prototype.

The technical result of solving the problem is to obtain peroxidase with RZ of 3.35 and an activity of 1000 u / mg of the enzyme (substrate 4-aminoantipyrine) in one stage. The yield of the enzyme is 3.2 g from 100 kg of roots, which is 6 times higher compared to the prototype. The proposed method allows to reduce the number of stages and thereby reduce the time to obtain the target product and increase its yield by 1.5-2 times.

It is known that protein adsorption on a sorbent is associated with various non-covalent interactions of macromolecular groups with the surface of this sorbent. However, their total energy is small compared, for example, with protein-inhibitor binding in affinity chromatography or ionic interactions on an ion exchanger. Thus, the adsorption interaction is masked by other, stronger interactions between the surface of the protein and the surface of the carrier. Therefore, if it is supposed to purify this protein by adsorption chromatography, it is necessary to create such conditions that the ballast proteins either bind on the column or move along it faster than the target protein, i.e. so that only this protein can slowly move on the principle of adsorption-desorption. Such conditions are created if cellulose is used as a carrier for adsorption chromatography, and DEAE and KM groups are used for purification from ballast proteins. In order to achieve simultaneous movement of the target enzyme according to the principle of adsorption chromatography, and part of the ballast proteins due to the repulsion of the charged protein from the same charged groups, it is necessary to combine these groups and the cellulose carrier, i.e. use DEAE and KM cellulose. If chromatography is carried out at pH values close to the target enzyme, some of the ballast proteins are delayed at the start due to ion exchange, and some quickly move down due to interaction with the same charges of the ion exchanger. An uncharged or weakly charged enzyme will slowly move, as compared to ballast proteins, down both on DEAE and KM celluloses in the case of competition between the enzyme groups and the components of the buffer for the sorbent.

The buffer has:

1) a small concentration, since with its increase the competition of the components of the buffer can be so strong that the target product will move at a speed comparable to the speed of movement of ballast proteins, and purification will not occur;

2) since the buffer should provide a stable pH value at a low concentration, it should have a pK close to the yield of the target product;

3) the components of the buffer must have bulky groups for effective competition with the surface groups of the target product. Ballast proteins will be charged at this pH value and, depending on the charge, either remain at the start ("ion exchange"), or move much faster than the target product (interaction of the same charges). An uncharged or weakly charged target product will be separated from both the proteins remaining at the start and fast-moving proteins. Only proteins that have iso-points close to those of peroxidase could bind adsorptively and thereby reduce the degree of purification, but, as experience shows, such proteins are absent or in a minor amount in horseradish root juice.

Purification of the target product:

1) proteins from horseradish root juice after their homogenization are extracted with water, ultrafiltered to the initial volume of juice, precipitated with ammonium sulfate, dialyzed against water, and then against 0.01-0.03 M TEA-HCl buffer, pH 7.1-7-7 , 4 and sequentially applied to columns with DEAE cellulose in the Cl ^- form and KM cellulose in the Na ⁺ form, balanced respectively 0.01-0.03 M TEA-HCl and 0.01-0.03 M MOPS- NaOH buffers, pH 7.1-7.4 (pK buffers 7.5 and 7.6; iso peroxidase 7.2);

2) the slow movement of the brown ring of the enzyme is monitored visually;

3) after the enzyme leaves the KM cellulose column, it is dialysed sequentially against water and 0.01-0.03 M NaCl, diluted to 2-5 mg / ml with a solution of 0.01-0.03 M NaCl and lyophilized;

4) measure the RZ value of the product and its activity on 4-aminoantipyrine.

To measure peroxidase activity, 1.5 ml of a phenol-aminoantipyrine solution is added to the cuvette (810 mg of phenol is dissolved in 40 ml of water, 25 mg of 4-aminoantipyrine is added and diluted with water to 50 ml), 1.4 ml of hydrogen peroxide solution (1 ml 30 % H ₂ O _{2 is} diluted with water to 100 ml; then 1 ml of this solution is diluted with 0.2 M potassium phosphate buffer, pH 7.0 to 50 ml), 0.1 ml of the enzyme solution. The measurement of optical density is carried out at 510 nm and a temperature of 25 ° C. The speed is determined from the linear part of the curve:

u / mg = ( _Δ A ₅₁₀ / min) / 6.58 × mg of enzyme / ml.

The concentration of the enzyme is equal to: mg of the enzyme / ml = A ₄₀₃ × 0.44.

The following examples illustrate these points.

Example 1. 100 kg of horseradish roots of two years old, collected at the beginning of flowering, washed with water, homogenized, extracted with water, concentrated by ultrafiltration to the initial volume of juice, protein precipitated with ammonium sulfate (70% saturation). The precipitate is dissolved in a minimal amount of water and dialyzed against it, and then against 0.01 M TEA-HCl buffer, pH 7.1. The protein concentration was adjusted to 80 mg / ml by concentration by ultrafiltration (determination of the protein by Bradford or Lowry) and applied to a column (10 × 20 cm) with DEAE cellulose balanced with 0.01 M TEA-HCl buffer, pH 7.1 and pK 7 ,5. After gravity passing the brown ring, it is applied to a column of the same volume with KM cellulose, balanced with 0.01 M MOPS-NaOH buffer, pH 7.1 and pK 7.5. After the enzyme leaves the KM cellulose column, it is dialysed sequentially against water and 0.01 M NaCl, diluted to 2-5 mg / ml with a solution of 0.01 M NaCl and lyophilized.

Yield: 3.2 g of peroxidase with RZ 3.35 and activity of 1000 u / mg of the enzyme in 4-aminoantipyrine.

Example 2. 100 kg of horseradish roots of two years old, collected at the beginning of flowering, washed with water, homogenized, extracted with water, concentrated by ultrafiltration to the initial volume of juice, protein precipitated with ammonium sulfate (75% saturation). The precipitate is dissolved in a minimal amount of water and dialyzed against it, and then against 0.03 M TEA-HCl buffer, pH 7.4. The protein concentration was adjusted to 80 mg / ml by concentration by ultrafiltration and applied to a column (10 × 20 cm) with DEAE cellulose balanced with 0.03 M TEA-HCl buffer, pH 7.4, pK 7.6. Next, the target product is chromatographed, as in Example 1, on KM cellulose, but in 0.03 M MOPS-NaOH buffer, pH 7.4, pK 7.6. After the enzyme leaves the KM cellulose column, it is dialysed sequentially against water and 0.03 M NaCl, diluted to 2-5 mg / ml with a 0.03 M NaCl solution and lyophilized.

Yield: 3.2 g of peroxidase with RZ 3.35 and activity of 1000 u / mg of the enzyme in 4-aminoantipyrine.

Example 3. 15 g of lyophilized peroxidase powder with RZ 0.3 are suspended in 0.01 M TEA-HCl buffer, pH 7.1 is centrifuged (20 min, 3000 g) and 300 ml of the supernatant is applied to a column (10 × 20 cm) s DEAE cellulose balanced with the same buffer. After gravity passing the brown ring, it is applied to a column of the same volume with KM cellulose, balanced with 0.01 M MOPS-NaOH buffer, pH 7.1, pK 7.5. After the enzyme leaves the KM cellulose column, it is dialysed sequentially against water and 0.01 M NaCl, diluted to 2-5 mg / ml with a solution of 0.01 M NaCl and lyophilized.

The yield is 1.5 g of the target product with RZ 3.35 and activity of 1000 u / mg of the enzyme for 4-aminoantipyrine.

Example 4. 15 g of lyophilized peroxidase powder with RZ 0.3 are suspended in 0.03 M TEA-HCl buffer, pH 7.4, centrifuged (20 min, 3000 g) and 300 ml of the supernatant are applied to a column (10 × 20 cm) with DEAE cellulose balanced with the same buffer. After gravity passing the brown ring, it is applied to a column of the same volume with KM cellulose, equilibrated with 0.03 M MOPS-NaOH buffer, pH 7.4, pK 7.6. After the enzyme leaves the KM cellulose column, it is dialysed sequentially against water and 0.03 M NaCl, diluted to 2-5 mg / ml with a 0.03 M NaCl solution and lyophilized.

The yield is 1.5 g of the target product with RZ 3.35 and activity of 1000 u / mg of the enzyme for 4-aminoantipyrine.

Thus, the proposed method in comparison with the prototype allows to obtain the target product with an RZ of 3.35 and an activity of 1000 u / mg of the enzyme for 4-aminoantipyrine. The yield of the enzyme is 3.2 g per 100 kg of roots, which exceeds the yield of the prototype ~ 6 times. In addition, the resulting target product has a higher purity compared to the prototype (RZ, respectively, 3.35 and 2.7). The simplification of the method is expressed in the reduction of biochemical stages from four to one. This method also allows the isolation of highly purified peroxidase from the lyophilized, low-purified enzyme.

Sources of literature

1. Paul K.G. The Enzymes. New York, Acad. Press, 1963.

2. RF patent №2130070.

Claims (3)

1. A method of producing horseradish peroxidase, including homogenization of horseradish roots, enzyme extraction, concentration by ultrafiltration, precipitation of proteins with ammonium sulfate, dialysis of the precipitate, characterized in that the protein precipitate is dialyzed against water and 0.01-0.03 M TEA-HCl buffer with subsequent purification of peroxidase from ballast proteins on a column with DEAE cellulose in the same buffer, and then in a 0.01-0.03 M solution of MOPS-NaOH buffer on a column with KM cellulose, at pH and pK buffers close to target product 7.1-7.4 and 7.5-7.6, respectively, purified the target product is dialyzed against water and 0.01-0.03 M NaCl, and then lyophilized. 2. The method according to claim 1, characterized in that the enzyme is extracted with water after homogenization of the roots. 3. The method according to claim 1, characterized in that the ammonium sulfate contribute at a concentration of 70-75% saturation.