• Domestic science and medicine in the 19th - early 20th centuries Chemistry of the 19th century in medicine
• What are peptides? Types and types of peptides. All about peptides and anti-aging cosmetics with peptides Additional peptides are not formed
• Toxic effect on humans of hazardous chemicals
• Radioautography Method of autoradiography in cytology
• Identification of bacteria by antigenic structure
• Organic matter in wastewater What are organic compounds in water
• Hydrogen index (pH factor)
• What is the pH of water and why is it important to know it?
• Redox potential Redox systems
• Reversible redox system Reversible redox systems

• Specialty HAC RF03.00.23
• Number of pages 106

II. LITERATURE REVIEW.

Chapter 1. STRUCTURE, MECHANISM OF ACTION AND NEW APPLICATIONS OF NATIVE AND RECOMBINANT PEROXIDASES.

1.1. Introduction.

1.2. Crystal structures of peroxidases.

1.3. Acid-base catalysis of peroxidases.

1.4. Metal-binding sites.

1.5. Architecture of the HRP active center.

1.5.1 The structure of the distal region of HRP and the system of hydrogen bonds.

1.5.2. The structure of the proximal region of HRP and the system of hydrogen bonds.

1.5.3. Binding site for aromatic substrates.

1.6. The role of the protein environment in peroxidase catalysis.

Chapter 2. BIOTECHNOLOGICAL APPLICATIONS OF PEROXIDASES.

2.1 Use of peroxidases in dushunoenzymatic analysis.

2.2. Biochemical analysis of physiologically active substances.

2.3. Use of peroxidases in biosensors.

2.4. Application of peroxidases in biotransformations.

2.5. The use of peroxidases for biobleaching in the pulp and paper industry.

Chapter 3

OF DISULPHIDE BONDS OF EUKARYOTIC PROTEINS EXPRESSED IN E. coli CELLS IN INCLUTION BODIES.

III. EXPERIMENTAL PART.

Chapter 4. MATERIALS AND METHODS.

4.1. Reagents.

4.2. Research methods.

4.2.1 Cloning of recombinant forms of HRP.

4.2.2 Obtaining a recombinant conjugate of HRP with BPFA.

4.2.3 Expression of recombinant HRP in E. coli cells.

4.2.4 Protocols for refolding and purification of recombinant HRP.

4.2.5 Physico-chemical properties of recombinant forms of HRP.

4.2.6 Adsorption and electrochemical measurements.

4.2.7 Calculation of structural changes in mutant forms of horseradish peroxidase.

IV. RESULTS AND ITS DISCUSSION.

Chapter 5. EXPRESSION OF RECOMBINANT HORSERADISE PEROXIDASE

IN E.coli CELLS: OPTIMIZATION OF THE SYSTEM OF EXPRESSION, REFOLDING, REACTIVATION AND PURIFICATION.

5.1. Expression of HRP in the periplasmic region of E. coli BL21(DE3) cells.

5.2. Optimization of the HRP refolding system from inclusion bodies when expressed in the cytoplasmic region of E. coli BL21(DE3)pLysS cells.

5.3. Properties of recombinant PCS 6xHis at the C-terminus.

5.4. Obtaining and properties of mutant forms of HRP.

Chapter 6. AMPEROMETRIC SENSORS BASED ON

RECOMBINANT HRP FORMS.

6.1 Amperometric determination of H2O2.

6.2 Biosensor signal stability.

6.3 Development of bienzymatic biosensors.

Chapter 7. PREPARATION AND PROPERTIES OF THE RECOMBINANT CONJUGATE

HORSERADISE PEROXIDASE WITH BPFA.

7.1 Cloning and expression of the recombinant HRP-PAFA conjugate in E. coli cells.

7.2 Physicochemical and immunological properties of the recombinant HRP-BPFA conjugate.

V. CONCLUSIONS

Recommended list of dissertations

• Enzyme-linked immunosorbent assay of a protein that binds fatty acids based on recombinant reagents 2004, candidate of chemical sciences Andreeva, Irina Petrovna

• Recombinant tobacco peroxidase: preparation and catalytic properties 2007, candidate of chemical sciences Khushpulyan, Dmitry Mikhailovich

• Creation and study of the properties of recombinant human proteins with a potential antitumor effect 2007, candidate of chemical sciences Savvateeva, Lyudmila Vladimirovna

• Conformational Differences between Native and Recombinant Forms of Isoenzyme C of Horseradish Peroxidase Detected by Radiochemical and Kinetic Methods

• Study of the organization of the phosphate-binding region of bacterial uridine phosphorylases 2000, candidate of biological sciences Chebotaev, Dmitry Vladimirovich

Introduction to the thesis (part of the abstract) on the topic "Obtaining, properties and application of recombinant horseradish peroxidase"

Isoenzyme C of horseradish peroxidase (EC 1.11.1.7) is a glycoprotein, molecular weight 44 kDa, belongs to the superfamily of plant peroxidases, contains the protoporphyrin-IX prosthetic group, two Ca2+ ions. HRP catalyzes the one-electron oxidation of a large number of organic and inorganic substrates with hydrogen peroxide. HRP has found wide application in analytical biochemistry and biotechnology as a marker of antibodies, DNA, and low molecular weight compounds (analytes).

In recent years, the progress made in cloning and heterologous expression of HRP genes opens up new opportunities for studying structural and functional relationships using protein engineering methods.

To obtain recombinant HRP, the expression system in E. coli cells has found wide distribution. Other modes of expression exist, such as insect cell expression, which allows the enzyme to be obtained in an active, soluble, and glycosylated form, and yeast expression. However, these expression systems are quite expensive and laborious, and besides, the insect cell expression system cannot be scaled up, so they have not found such a wide distribution as the expression in E. coli cells.

Recombinant HRP is expressed in E. coli cells in an insoluble, inactive form in inclusion bodies. The process of refolding and reactivation of apo-peroxidase by the prosthetic group is multistage and rather laborious.

For applications such as, for example, use in biosensors, a more efficient and reliable method for obtaining recombinant HRP is needed. Thus, one of the main objectives of this work was to find ways to optimize the previously described protocols for refolding, reactivation, and purification of recombinant HRP, as well as to study the possibility of periplasmic expression to obtain a soluble, active enzyme.

The high catalytic activity of HRP in the hydrogen peroxide reduction reaction makes it possible to use peroxidase as a highly efficient bioelectrocatalyst involved in the direct electron transfer between the electrode, the active site of the enzyme, and the substrate. In the presence of a substrate, at a certain electrode potential, proportionality is observed between the measured reduction current and the H2O2 concentration. In this case, the efficiency of direct (mediator-free electron transfer) is one of the most important conditions for the development of amperometric biosensor systems based on HRP. From the foregoing, the following goal of this work organically follows - modification of the surface of recombinant HRP by site-directed mutagenesis with functional groups to ensure efficient oriented immobilization of the HRP molecule on the electrode surface. The paper proposed a strategy for introducing short oligohistidine sequences into the surface regions of HRP, which, on the one hand, should ensure efficient purification of the recombinant enzyme using metal chelate chromatography (Ni-NTA agarose), and at the same time promote efficient adsorption of the corresponding mutants on the surface. gold electrodes (it is known that histidines are irreversibly sorbed on gold).

Horseradish peroxidase has found wide application as a marker enzyme for enzyme immunoassay. Chemical conjugation of proteins and haptens leads to partial inactivation of the enzyme and heterogeneity of conjugates, which in turn affects the specificity and sensitivity of the assay.

With the development of genetic engineering, it became possible to create recombinant conjugates of the marker enzyme with antibodies and protein antigens. Such conjugates have a number of advantages over chemically prepared conjugates, in particular, they are homogeneous in composition, have a 1:1 stoichiometry, and are reproducible. In addition, recombinant conjugates retain 100% of both enzymatic and immunological activity.

Recent advances in heterologous expression of HRP in E. coli cells and reactivation of recombinant horseradish peroxidase make it possible to obtain recombinant conjugates with peroxidase as a marker enzyme and use them in enzyme immunoassay. The aim of this stage of work was to obtain a recombinant conjugate of horseradish peroxidase with human heart-type fatty acid binding protein (FABP). BPFA is a new highly specific marker of myocardial infarction. Obtaining a recombinant conjugate of HRP with BPFA will make it possible to create new express systems for diagnosing myocardial infarction.

II. LITERATURE REVIEW

Similar theses in the specialty "Biotechnology", 03.00.23 VAK code

• Conformational Differences between Native and Recombinant Forms of Isoenzyme C of Horseradish Peroxidase Detected by Radiochemical and Kinetic Methods 2002, candidate of chemical sciences Chubar, Tatyana Anatolyevna

• Recombinant analogues of glutaryl-7-aminocephalosporanic acid acylase from the bacterium Brevundimonas diminuta 2009, candidate of biological sciences Zakirova, Svetlana Anatolyevna

• Obtaining recombinant proteins of West Siberian isolates of Borrelia burgdorferi sensu lato and studying their antigenic properties 2009, Candidate of Biological Sciences Ryabchenko, Alexander Vladimirovich

• Increasing the antitumor activity of the TRAIL cytokine by changing the amino acid composition of the protein 2010, candidate of biological sciences Yagolovich, Anna Valerievna

• Use of the beta-helical domain and peptidyl-prolyl isomerase to obtain fibrillar adhesin of bacteriophage T4 2012, candidate of biological sciences Chuprov-Netochin, Roman Nikolaevich

Dissertation conclusion on the topic "Biotechnology", Grigorenko, Vitaly Georgievich

1. When expressing recombinant horseradish peroxidase in the replasma of E. coli strain BL21(DE3) cells with the pETpelHRPhis expression vector, a soluble, functionally active enzyme is formed with a total yield of about 0.5 mg of protein per 1 liter of cell culture.

2. The cytoplasmic system for the expression of recombinant HRP in E. coli strain BL21(DE3)pLysS cells with the pETHRPhis expression vector provides a high level of HRP expression in the form of inclusion bodies (the target product is about 30% of the total cellular protein of the cell). The introduction of a hexahistidine sequence (6xHis) into the C-terminal region of HRP allowed efficient metal chelate chromatography to isolate and purify the recombinant enzyme preparation.

3. Based on the data on the effect of various reagents (urea, imidazole, glutathione,.) on the properties of HRP, a new scheme was developed for refolding and reactivation of recombinant HRP from inclusion bodies, which makes it possible to obtain an active holo-enzyme, with a yield of 8-10 mg from 1 liters of E.coli culture.

4. It has been shown that the introduction of hexahistidine sequences into the N- and C-terminal regions of the enzyme, as well as the replacement of amino acid residues of two internal surface regions (57-61) and (211-216) by site-directed mutagenesis with histidine ones, respectively, affect the on the catalytic properties of recombinant HRP.

5. Gold electrodes modified with recombinant forms of HRP exhibit a high and stable amperometric signal of H2O2 bioelectrocatalytic reduction due to efficient direct electron transfer between the gold surface and the active site of the enzyme, which serves as the basis for creating a universal sensor for H2O2, as well as for creating bienzymatic sensors based on on the co-immobilization of HRP and the corresponding H2O2-forming oxidases (lysine oxidase, glucose oxidase,.).

6. Recombinant conjugate of horseradish peroxidase with human heart fatty acid transfer protein (HFFA) has both catalytic properties of HRP and immunogenic properties of FAFA, which allows its use in enzyme immunoassay. The yield of the target product was 12 mg per 1 liter of E. coli culture.

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The method involves crushing and homogenization of sprouted horseradish roots, extraction of horseradish root homogenizate with 0.15±0.01 M sodium chloride solution. Ballast proteins are separated and peroxidase is precipitated with ammonium sulfate salts of 45-48% saturation and 85-90% saturation, respectively. Gel filtration of peroxidase solution is carried out on Sephadex G-100 with elution with 0.15-0.2 M sodium chloride solution. Chromatographic purification is carried out on carboxymethyl cellulose. Freeze drying of the target peroxidase is carried out. Dialysis is carried out against sodium acetate buffer with pH 4.4-5.0 and dialysis against potassium phosphate buffer with pH 8.0±0.1. Carry out concentration with further purification on DEAE-cellulose with elution with the same buffer, followed by dialysis against deionized water. The method allows to obtain peroxidase with high specific activity and high yield. The output of peroxidase is 2.52-3.50 g/kg of horseradish roots, the specific activity is 640-700 EA/mg of protein. 5 z.p. f-ly, 2 ill., 1 tab.

Drawings to the RF patent 2353652

The invention relates to the field of biotechnology, namely the production of enzymes from plant materials, and can be used for laboratory and industrial production of the peroxidase enzyme from horseradish roots for immunology and immunochemistry as the main component of conjugates for enzyme immunoassays.

Various methods are known for obtaining peroxidase from horseradish roots.

A known method for producing peroxidase from horseradish roots, including homogenization of horseradish roots, extraction of the enzyme with saline, precipitation of the enzyme with ammonium sulfate salts, gel filtration, alcohol precipitation, electrophoresis, reprecipitation with ammonium chloride, filtration through Sephadex G-50 and DEAE-cellulose and dialysis.

The main disadvantages of this method are the low activity and purity of the resulting product, as well as the complexity of its production and a large number of process steps.

There is also known a method for producing peroxidase from horseradish roots, in which horseradish roots are homogenized, then the enzyme is extracted with water and the peroxidase is precipitated with ammonium sulfate salts, followed by gel filtration [US Pat. Hungary 172872, C07G 7/022].

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

A known method [A.S. Bulgaria 46675, C12N 9/08, 15.02.90], according to which horseradish roots are germinated for 2-3 days, then homogenized and the enzyme is extracted with water for a day. The aqueous extract is centrifuged, followed by fractionation of proteins with ammonium sulfate salts, then the ammonium sulfate precipitate of the enzyme is dissolved in distilled water and subjected to ultrafiltration on Milipor PTGC 000 05 filters. 0.5 M phosphate buffer (pH 8) is added to the resulting filtrate in a ratio of 100 parts of buffer per 1 part of the filtrate and passed through a column with an ion exchanger DEAE-Sephadex A-50, then successively ultrafiltered on Milipor PTGC 000 05, RTNK 000 05, PTGC 000 05 and subjected to freeze drying.

The disadvantage of this method is the insufficiently high yield of peroxidase, high labor intensity and duration of the process.

The closest is the way [US Pat. RF 2130070, C12N 9/08, 10.05.1999], in which the water-washed horseradish roots are cleaned by 1/3 of the mass in the presence of a 0.25% solution of food ascorbic acid used as an extracting solution. Peroxidase from the purifications is extracted for 1 hour with a 0.25% solution of ascorbic acid, then the extract is filtered and centrifuged. To the supernatant add 5% sodium sulfite and incubated for 24 h at room temperature for the "maturation" of the enzyme. The "matured" enzyme solution is concentrated on ultrafiltration fiber devices with filters having a pore diameter of less than 40 kDa. Ammonium sulfate is added to the solution concentrated 10 times to a final saturation of 85-90%, centrifuged, the precipitate is dissolved in a tenfold volume of bidistilled water and applied to a column filled with Sephadex G-25, elution is carried out with bidistilled water. Collect fractions containing peroxidase with R Z >0.1. Ammonium sulfate is added to the collected fractions until saturation of 85-90%, centrifuged, the precipitate is dissolved in a 3-fold amount of bidistilled water and applied to a gel filtration column filled with Sephadex G-50, elution is carried out with bidistilled water. Collect fractions containing peroxidase, with the value of R Z >0.5. The fractions are mixed, dotitrated to pH 4.4 and subjected to purification on carboxymethyl cellulose, the enzyme is eluted in a concentration gradient from 5 mm to 0.15 M acetate buffer (pH 4.4) (V=S-500 ml, R-500 ml). Collect fractions with an R Z value >2.7 and with an enzyme concentration of at least 10 mg/ml. Fractions are combined, titrated to pH 5.0 and freeze-dried.

The disadvantages of this method are insufficiently high purity and activity and low yields of peroxidase.

The invention solves the problem of creating an industrial method for producing peroxidase from horseradish roots, which makes it possible to obtain peroxidase with high purity, high specific activity and high yield.

The problem is solved by a method for obtaining the peroxidase enzyme from horseradish roots, which includes grinding and homogenization of sprouted horseradish roots, extraction of horseradish root homogenizate, separation of ballast proteins and precipitation of peroxidase with ammonium sulfate salts, gel filtration of a peroxidase solution on Sephadex, chromatographic purification on carboxymethyl cellulose, freeze-drying of the target peroxidase , while crushed horseradish roots are extracted with 0.15±0.01 M sodium chloride solution with pH=4.4±0.2; gel filtration of peroxidase solution is carried out on Sephadex G-100 with elution with 0.15-0.2 M sodium chloride solution with pH 4.4-5.0, dialysis is performed against sodium acetate buffer with pH 4.4-5.0 and dialysis against potassium phosphate buffer with pH 8.0 ± 0.1 and concentration with further purification on DEAE-cellulose with elution with the same buffer, followed by dialysis against deionized water.

Protein precipitation with ammonium sulfate salts is used twice: 45-48% saturation is used to separate ballast proteins, and 85-90% saturation is used to precipitate peroxidase,

Chromatographic purification of peroxidase on carboxymethyl cellulose is preceded by dialysis against sodium acetate buffer pH 4.4-5.0.

Freeze drying is preceded by dialysis of the peroxidase solution against deionized water.

Figure 1 shows a schematic diagram of the isolation of peroxidase from horseradish roots.

Horseradish roots are washed under running water and germinated for 140-160 hours at a temperature of +25±1°C. Sprouted roots are crushed and extracted with 0.15 M sodium chloride solution for 12±2 hours with constant stirring, then centrifuged.

16.7 ± 0.05 kg (45-48% saturation) of ammonium sulfate are added to supernatant-1 (Figure 1) with continuous stirring, the precipitate is separated by centrifugation, and another 11 ,8±0.05 kg (85% saturation) of ammonium sulfate, the precipitate (Figure 1) is separated by centrifugation and dissolved with distilled water to a final volume of 200±10 ml. After centrifugation, the supernatant containing peroxidase is applied to a column filled with Sephadex G-100 and eluted with a 0.15 M sodium chloride solution at a rate of 100 ml/h; fractions of 20 ml are taken during the elution. The collected fractions measure R Z =D 408 /D 275 . Fractions in which R Z is not less than 0.8 are pooled and dialyzed against sodium acetate buffer (pH 4.4 ± 0.2) (buffer-1), then the desalted peroxidase solution is layered on a column packed with carboxymethyl cellulose and equilibrated with buffer-1 , and elute with a linear gradient of 5 mm - 0.1 M acetate buffer (pH 4.4±0.2) (V=S-0.5 l, R-0.5 l). In protein fractions measure the value of R Z . Fractions in which the value of R Z is not less than 2.5 are combined and dialyzed against potassium phosphate buffer (pH 8.0 ± 0.1) (buffer-2), the dialyzed enzyme solution is layered on a column filled with DEAE-cellulose, and eluted buffer-2. Fractions in which the value of R Z is not less than 3.0 are combined and dialyzed against deionized water, then transferred into a sterile heat-resistant flask, frozen with liquid nitrogen and freeze-dried until the drug is completely dry.

The essential distinguishing features of the proposed method for obtaining biologically active substances are:

The use of gel filtration on Sephadex G-100 for the most complete purification of peroxidase from low molecular weight impurities;

Dialysis against sodium acetate buffer (pH 4.4-5.0), which makes it possible to desalt the peroxidase solution and prepare it for chromatography on carboxymethyl cellulose;

Dialysis against potassium phosphate buffer (pH 8.0 ± 0.1), which makes it possible to transfer the peroxidase solution to the optimal buffer with the optimal pH for application to DEAE-cellulose;

Ion-exchange chromatography of peroxidase on DEAE-cellulose as a method providing not only additional purification, but also concentration of the enzyme solution.

Thus, we propose a new approach that allows the processing of horseradish roots with the production of a peroxidase enzyme of high purity and specific activity.

The difference of the proposed method from the closest analogue and prototype is as follows.

In the claimed method, crushed horseradish roots are extracted with a 0.15±0.01 M sodium chloride solution with pH=4.4±0.2, thereby achieving the most complete transition of the enzyme into the solution, while the enzyme does not lose its activity.

In the analogue of the invention [US Pat. RF 2130070, C12N 9/08, 10.05.1999] peroxidase from purifications (without homogenization!) is extracted for 1 h with a 0.25% solution of food ascorbic acid, which may affect the yield of peroxidase, since purifications are not homogenized, and therefore, the enzyme goes into solution far from completely, while the extraction proceeds under acidic conditions, which can lead to partial inactivation of the enzyme. In the prototype of the invention [A.S. Bulgaria 46675, C12N 9/08, 15.02.90] the enzyme from the homogenizate of horseradish roots is extracted with water, which also leads to a low yield of peroxidase from the homogenizate into solution.

In contrast to the analogue of the invention, where peroxidase is precipitated twice with 85-90% saturation with ammonium sulfate, and the prototype of the invention, where proteins are precipitated four times with ammonium sulfate salts, in the claimed method, ballast proteins are separated with 45-48% saturation, and then peroxidase 85 is precipitated. % saturation.

In the analog and prototype of the invention, the concentration is carried out by ultrafiltration, while in the analog of the invention, ultrafiltration precedes ammonium sulfate precipitation of peroxidase, which leads to a high probability of co-precipitation of impurity proteins, and, accordingly, to a lower purity of the final product. In the claimed method, peroxidase is subjected to concentration on the DEAE-cellulose ion-exchange sorbent at the final stage of the technological process, which also leads to additional purification of the enzyme.

In the claimed method, gel filtration is carried out on Sephadex G-100 with elution with a 0.15 ± 0.01 M sodium chloride solution (pH 4.4-5.0), which allows you to completely get rid of the greenish tint of the peroxidase solution due to the presence of chlorophyll and related compounds , and substances with a smaller molecular size than that of peroxidase. In an analogue of the invention, gel filtration is carried out on Sephadex G-25, which, in terms of its ability to retain impurity particles, given the size of the peroxidase molecule (about 40 kDa), is inferior to Sephadex G-100.

The essence of the invention is illustrated by the following examples.

Example 1 Preparation of a coarse extract

50 kg of horseradish roots are washed under running water and germinated for 140-160 hours at a temperature of +25±1°C. Sprouted roots are crushed on a bladed homogenizer and pour 50 l of 0.15-0.2 M sodium chloride solution (pH 4.4±0.2). The suspension is extracted for 12±2 h with constant stirring, then centrifuged.

Ammonium sulfate is added to supernatant-1 with a volume of 60 l to separate ballast proteins with continuous stirring to 45-48% saturation. (By 100% saturation is meant the amount of salt, upon addition of which the solution becomes saturated, and upon further addition of salt, the solution becomes supersaturated, and the salt precipitates. For solutions with ammonium sulfate, 100% saturation is the addition and complete dissolution of 70, 7 g of ammonium sulfate in 100 ml of distilled water.) Precipitate-1 is separated by centrifugation. Next, the resulting supernatant-2 with a volume of 55 l to precipitate peroxidase is saturated with ammonium sulfate to 85%, sediment-2 is separated by centrifugation. The resulting precipitate-2 is dissolved with deionized water to a final volume of 200±10 ml, the undissolved precipitate-3 is separated by centrifugation.

Gel chromatography on Sephadex G-100.

The peroxidase solution is applied to a 1 L column filled with Sephadex G-100. The elution is carried out with a 0.15-0.2 M sodium chloride solution (pH 4.4-5.0) at a rate of 100 ml/h, during the elution fractions of 20 ml are taken. The collected fractions measure R Z =D 408 /D 275 . Fractions in which R Z is not less than 0.8 are pooled.

5 l of 5±0.1 mm sodium acetate buffer (pH 4.4-5.0) (buffer-1) and a dialysis bag with combined peroxidase fractions are placed in the collection. Dialysis is carried out with constant stirring for 24 hours, changing the buffer in the collection three times.

The desalted enzyme solution is layered onto a 1 liter column filled with carboxymethyl cellulose and equilibrated with buffer-1. The elution is carried out at a rate of 50 ml/h in a linear gradient of 5 mm-0.1 M acetate buffer (pH 4.4±0.2) (V=S-0.5 l, R-0.5 l). In protein fractions measure the value of R Z . Fractions in which the R Z value is not less than 2.5 are pooled.

5 l of 20 ± 0.1 mM potassium phosphate buffer (pH 8.0 ± 0.1) (buffer-2) and a dialysis bag with pooled enzyme fractions are placed in the collection. Dialysis is carried out in the same way.

Concentration chromatography on DEAE-cellulose.

An enzyme solution with a pH of 8.0 ± 0.1 is layered on a 300 ml column filled with DEAE-cellulose. The elution is carried out with buffer-2 at a rate of 50 ml/h. Fractions in which the R Z value is not less than 3.0 are pooled.

5 l of deionized water and a dialysis bag with peroxidase solution are placed in the collector. Dialysis is carried out in the same way.

Freeze drying.

The peroxidase solution is transferred into a sterile heat-resistant flask, frozen with liquid nitrogen and freeze-dried until the drug is completely dry.

Example 2 (comparative)

In this example, the conditions for obtaining peroxidase according to the prototype are maintained, but to improve the main indicators of peroxidase, two stages existing in the prototype are changed, namely: for ultrafiltration concentration of the peroxidase solution, two types of columns with hollow fibers are used, in contrast to the prototype, where one type of fibers is used, and fractional fractionation of proteins with ammonium sulfate salts (as in the claimed method).

Obtaining a rough extract.

50 kg of horseradish roots are washed under running water and cleaned by 1/3 of the mass in the presence of 33.5 l of a 0.25% solution of food grade ascorbic acid, in which the obtained cleanings are kept for 1 hour to extract the peroxidase enzyme. Next, the extract is centrifuged in a flow centrifuge and incubated for 24 h for the "maturation" of the enzyme.

Primary purification and concentration.

1.7 kg (5% by weight) of sodium sulfite is added to the obtained extract, and the extract is subjected to concentration and primary purification by ultrafiltration in a UPV-6 hollow polymer fiber unit equipped with columns with filters having pore sizes of 60 kDa and 5 kDa. A schematic diagram of the installation is shown in Fig.2, which shows a centrifugal pump 1; prefilter 2; column with fibers having a pore size of 60 kDa 3; a column with fibers having a pore size of 5 kDa 4; peroxidase filtrate 5; concentrated solution of peroxidase 6; filtrate containing low molecular weight proteins 7.

Fractionation of proteins with ammonium sulfate salts.

Ammonium sulfate is added to the concentrate with a volume of 6 l to separate ballast proteins with continuous stirring to 45-48% saturation. The precipitate is separated by centrifugation. Next, the resulting supernatant with a volume of 5.7 l to precipitate peroxidase is saturated with ammonium sulfate to 85%, the precipitate is separated by centrifugation, which is dissolved with bidistilled water to a final volume of 200±10 ml, the undissolved precipitate is separated by centrifugation.

Gel chromatography on Sephadex G-25 and G-50.

Further purification of the peroxidase is carried out on a gel filtration column packed with Sephadex G-25 and equilibrated with bidistilled water. The peroxidase solution is applied to the column, the elution is carried out with bidistilled water. Collect fractions in which R Z is not less than 0.2. Ammonium sulfate is added to the collected fractions to 90% saturation, stirred until the salt is completely dissolved and centrifuged. The precipitate is dissolved in 3 times the volume of bidistilled water and applied to a gel filtration column filled with Sephadex G-50 and equilibrated with bidistilled water. Elution is carried out with bidistilled water. Collect fractions in which R Z is not less than 0.6. With 50% acetic acid, the pH of the peroxidase fractions is adjusted to 4.4.

Chromatography on carboxymethyl cellulose.

The peroxidase fractions are layered onto a 1 liter column filled with carboxymethyl cellulose pre-equilibrated with 5 mM acetate buffer pH 4.4. Elution is carried out with a linear gradient of 5 mm-0.15 M acetate buffer (pH 4.4±0.2) (V=S-0.5 l, R-0.5 l) for 1 hour. In protein fractions measure the value of R Z . Fractions in which the value of R Z is not less than 2.7 are combined, adjusted to pH 5.0 by adding ammonia and freeze-dried.

Comparative data for example 1 and 2 are shown in the table.

Table Comparative data on the main parameters of the quality of peroxidase from horseradish roots using two production methods.
The name of the technological parameter, units of measurement.	Example 1 (claimed method)	Example 2 (known way)
Mass yield of peroxidase, g/kg of horseradish roots.	2,52-3,50	0,21-0,85
Specific activity of the drug, EA/mg of protein*	640-700	560-610
Spectrophotometer purity R Z =D 408 /D 275	3,00-3,20	2,70-3,00
* - Specific activity was calculated using the data given in STP 103.34-83. Rules for calculating and processing the results of quantitative analysis. NICKTI BAV, 1983.

Thus, as can be seen from the examples and the table, the claimed method for obtaining peroxidase from horseradish roots (example 1) makes it possible to obtain peroxidase with high yields, with a purity and activity sufficient to use this enzyme as an integral part of conjugates for enzyme immunoassays. And under the conditions of the prototype (example 2), even with the improvement of two stages that improve performance, the yield, specific activity and purity of the target peroxidase are lower than those of the proposed method.

This method can be used in the industrial production of peroxidase from horseradish roots.

CLAIM

1. A method for obtaining peroxidase enzyme from horseradish roots, including grinding and homogenization of germinated horseradish roots, extraction of horseradish root homogenate, separation of ballast proteins and precipitation of peroxidase with ammonium sulfate salts, gel filtration of a peroxidase solution on Sephadex, chromatographic purification on carboxymethyl cellulose, freeze-drying of the target peroxidase, differing the fact that the crushed horseradish roots are extracted with 0.15±0.01 M sodium chloride solution; gel filtration of the peroxidase solution is carried out on Sephadex G-100, dialysis is carried out against sodium acetate buffer with pH 4.4-5.0 and dialysis against potassium phosphate buffer with pH 8.0±0.1, and concentration with additional purification on DEAE -cellulose eluting with the same buffer followed by dialysis against deionized water.

2. The method according to claim 1, characterized in that the crushed horseradish roots are extracted with a 0.15 ± 0.01 M sodium chloride solution with a pH of 4.4 ± 0.2.

3. The method according to claim 1, characterized in that precipitation of proteins with ammonium sulfate salts is used twice: 45-48% saturation is used to separate ballast proteins, and 85-90% saturation is used to precipitate peroxidase.

4. The method according to claim 1, characterized in that gel filtration is carried out on Sephadex G-100 with elution with a 0.15-0.2 M sodium chloride solution with a pH of 4.4-5.0.

5. The method according to claim 1, characterized in that the chromatographic purification of peroxidase on carboxymethyl cellulose is preceded by dialysis against sodium acetate buffer with a pH of 4.4-5.0.

6. The method according to claim 1, characterized in that freeze-drying is preceded by dialysis of the peroxidase solution against deionized water.

"hacked" dissertation

I didn't have to look for a topic. All his childhood was connected with horseradish. Village grandmothers did not have pills. Health was corrected by the gifts of nature. And as soon as the snow melted, they went to the garden to dig a healing root. Wrote and "hacked" his thesis himself.

I thought that I licked the sour cream without revealing the essence. For agricultural technology, I missed something important, - the scientist explains his extraordinary act.

In pursuit of the truth, he reasoned something like this. In the homeland of horseradish, in Russia, for 60 years it was practically written off. Of the 500 varieties that existed, only 2-3 remained. And in Europe, where it appeared 200 years ago, today there are 2.5 thousand varieties. In America, where it was brought in 1900 at the initiative of the Ministry of Agriculture, there are already 3.5 thousand. And work on its breeding continues. What for?

The first semi-hidden information that a horseradish drug can cure cancer leaked out of Israel. Later, America and Europe "lit up". I turned to scientists from the FMD Institute and the Institute of Microbiology. They deciphered the formula of the drug - peroxidase. From a ton of pureed horseradish, only half a gram was obtained. He began to pester, as if to "feel" him and see, - says Emelin.

It was just right to involve scouts in the case. And so it happened. With their help, we found a French company that traded in our oil. Clicked. They promised to build a plant for the production of peroxidase. But the technology for its production remained under lock and key.

Right from the field, so that the most valuable thing did not have time to disappear, state farm horseradish was sent for analysis to a laboratory in France. The analysis is delayed. Sent a second time.

And it turned out that in our horseradish the content of peroxidase is 50 times higher than in imported. That is, from one ton we can get not 0.5 grams, but 25 grams. “You will breed at least 30 thousand varieties of horseradish, but you will never get the same as we have in Russia. This is our property,” Emelin triumphed then.

It turned out that in our horseradish the content of peroxidase is 50 times higher than in imported.

And the defense of his dissertation on horseradish still took place. Marina Vladimirovna Alekseeva, a student of Ivan Vladimirovich Michurin, a professor at the Agricultural Institute in Balashikha, was the scientific adviser. The opponents are the luminaries of vegetable growing, who hardly believed that some collective farmer from the Vladimir hinterland swung at such a topic. The result of the defense was a manual on the industrial cultivation of horseradish. Meanwhile, scientists from two institutes in Vladimir, FMD and Virology, to which Emelin supplied raw materials from his farm, created a technology for producing domestic peroxidase.

We were on the verge of industrial production of a substance whose price on the world market reached 7,000 dollars per gram. If 100 years ago Suzdal people traded very profitably with Germany, sending there 40 wagons of horseradish roots a year, then this technology could become a gold mine for the country, - Yuri Anatolyevich is convinced.

But perestroika began, and the country had no time for hell. Scientific research was curtailed, and the scale of its industrial cultivation was reduced to one field at the Stavrovsky state farm, whose director by that time was Emelin.

Garden Serpent Gorynych

Or maybe it’s good that they limited themselves to one field? - I think aloud, remembering how difficult it is to fight him. It is necessary to get horseradish in the garden, you will never bring it out. The story of the famous artist, and now the governor Mikhail Evdokimov, comes to mind about how he tried to get rid of horseradish, but even tol did not help.

What then to do?

You don't have to fight him. It can only be tamed. Horseradish gets along well with onions, potatoes and other garden crops and protects them from pests. This has long been noted by our ancestors. Even 500 years ago they said: a garden without horseradish is like a flock without a shepherd. And when gardens were started, first of all they determined the place where horseradish would grow.

It is no coincidence that there were horseradish and onion plantations on Suzdal land. First they removed the onion, and then the horseradish. The same with potatoes. In September, they dug it up, and in October horseradish came up. We sowed wheat and rye. And they received 50-60 centners per hectare, - says Emelin.

Studying horseradish, Emelin came to "archaeological" excavations. In the area where he grew up, he sifted the ground and counted the roots. And he concluded that there are two types of horseradish. Rooted to a depth of 15 meters, he called the mother. Formed in the upper layer of soil from small roots left after harvesting - riding.

True horseradish - maternal. He takes out from the subsoil layers, the so-called Adam's earth, those nutrients and elements that are no longer here. But horseradish-weed can also become maternal. Give it time, because in a year it goes into the ground by 60-70 cm.

Means, "infection" a horse-radish is quite probable? - I express concern to Emelin. And in response, he presents a new argument:

Horseradish is a self-regulating culture. Just as you cannot pour two barrels of water into one empty barrel, so it will not grow more than it should be on one piece of land.

And how much is due?

Its yield is a maximum of 4-5 tons of roots per hectare. Such a sharp limitation in terms of productivity distinguishes horseradish from other crops. I say this with full responsibility as a person who has devoted more than 30 years to studying it. But if he settled, then for centuries. On the land where horseradish is grown, it will be born in 100 and even 300 years.

So, Mikhail Evdokimov is right and only a nuclear bomb will save him? - I continue the argument on behalf of all gardeners.

You will not, after picking apples, cut an apple tree? She will give a harvest next year. Same with hell. It used to be dug in the month with the letter "r" in its name: September, October, November (the rest of the time it does not have that sharpness and mustard smell) ... Cut off the top as much as you can, and by the next season on this place will grow even more.

When in Rus' they talked about the Serpent Gorynych, in which three heads grow in place of one cut head, they probably meant horseradish, ”explains Emelin.

Look at the root

SPK "Stavrovsky" is one of the rare places in Russia where you can see a field of flowering horseradish. And they say that bees fly over it for days. True, the director did not have to eat shitty honey. But he gave flowers. They, Emelin believes, are of extraordinary beauty.

And horseradish seeds are formed. But if you sow them, nothing will come up. Horseradish reproduces only with the help of roots - vegetatively. Why? Yemelin did not find an answer. But on the other hand, this is one of the moments that allows the scientist to think about its ancient origin. Having survived the strongest cataclysms, the plant apparently adapted and survived in this way.

There is still no exact answer: is it a vegetable, a medicinal plant or a spice? The absence of clear species boundaries is also, according to Emelin, a confirmation of the hypothesis that horseradish is the messenger of Atlantis.

The origin of many cultures has long been known. Where did the horseradish come from, there is a lot of confusion in science. In many sources, I read that under Tutankhamun, the Egyptians somewhere took the root worth its weight in gold from the barbarians. But I don’t have a firm conviction that this is horseradish, ”Emelin does not hide.

But, deifying the subject of his research, he remains a practitioner. Having traveled about 250 vegetable gardens in the Vladimir and Yaroslavl regions in search of breeding material, he developed a new variety of horseradish - "Tolpukhovsky" (after the name of the central estate of his farm) and planted its plantation on the steep bank of the Koloksha River. Says: until the next civilization.

Emelin himself uses horseradish as a medicine. But unlike us, the uneducated, it takes into account that the beneficial properties in mashed horseradish last only 7 days.

In horseradish, which for months "dangles" on store shelves, and in our pantries, there is not a damn thing! - says the scientist.

What to do?

They dug 5-6 kilograms out of the ground in the fall, put them in the cellar, sprinkled them with earth, and let them lie. Get out of there a little, so that it is enough for a 250-gram jar. It can be taken twice a year: in winter and early spring. In winter - from the cellar. And in the spring, when the earth has thawed a little, you can safely dig up until the leaves grow up to 5 centimeters. So did our ancestors, who ate a pig with fresh horseradish on Easter.

Emelin can be trusted. Recently, he released three books in succession: "Fuck in your garden", "Fuck your doctor" and "Fuck on your table." Now he is preparing for publication "Hrenolechebnik". And he continues to refute the stereotype that sucks means bad.

Or maybe, really, when we say that life is completely worthless, they mean only the fate of Russian horseradish?

The invention relates to the field of biochemistry and can be used for laboratory and industrial production of high quality peroxidase from horseradish roots for diagnostic purposes. The crushed biomass of horseradish roots is kept in a 0.1 M buffer solution of sodium phosphate pH 7.0, pre-purged with nitrogen, in the presence of 5 μm solution of hemin and 5 mm calcium chloride. The extract is separated by decantation, followed by filtration and concentration by ultrafiltration through ultrafilters with a pore size of less than 30 kDa. The enzyme extract is saturated with ammonium sulfate up to 35% of saturation and applied to a column with phenylsepharose, after intensive washing of the buffer with sulfate, the active fractions are removed with an ammonium sulfate gradient (35%-0%) and an increase in pH to 8.0. The enzyme is purified by gel filtration on Toyopearl HW55F, dialyzed and lyophilized. The use of hemin-containing buffers at the stage of extraction and gel filtration makes it possible to obtain a highly active enzyme with a high yield due to 100% saturation of the enzyme with hemin. This makes it possible to accelerate the production of the enzyme and significantly improve its catalytic characteristics and stability. 6 w.p. f-ly, 1 tab.

The invention relates to the field of biochemistry and can be used for laboratory and industrial production of high quality peroxidase from horseradish roots, intended for diagnostic purposes.

Known (Paul K.G. The Enzymes. New Jork, Acad. Press, 1963) method for producing peroxidase, including homogenization of horseradish roots, extraction of the enzyme with water or saline and fractionation of the extract, the fractionation being carried out by sequential treatment of the extract with ammonium sulfate, gel filtration, alcohol precipitation , electrophoresis, reprecipitation with ammonium chloride, filtration through Sephadex G 50 and DEAE-cellulose and dialysis.

The main disadvantages of this method are the low purity and activity of the resulting drug, as well as the complexity and duration of the process of obtaining it. In addition, this method does not imply waste-free production.

Also known (HU, patent No. 172872) a method for producing peroxidase, including homogenization of horseradish roots, extraction of the enzyme with water, fractionation of the extract with ammonium sulfate and its gel filtration.

The disadvantage of this method is the insufficiently high yield of the enzyme.

Known (BG, patent 46675) is a method for producing an enzyme from production waste, including homogenization, extraction of the enzyme with water, precipitation of the extract with ammonium sulfate, purification and concentration of the enzyme by ultrafiltration and gel filtration, and subsequent lyophilization.

Also known (RU, patent No. 2130070) is a method for producing peroxidase, including homogenization of horseradish plant tissue, enzyme extraction, separation of the extract with precipitation of the enzyme with ammonium sulfate, purification of the enzyme by concentration by ultrafiltration and gel filtration, subsequent lyophilization of the target product, and horseradish plant tissue is used Waste from stripping roots in the production of food products, before ultrafiltration, sodium sulfite is added to the extract in an effective amount, the enzyme is precipitated with ammonium sulfate from the ultrafiltrate, and after gel filtration, the enzyme is further purified by ion-exchange chromatography.

The known method is chosen as the closest analogue of the developed invention.

The disadvantages of these methods is the duration of the purification process with loss of activity, which leads to a deterioration in the quality, namely the specific activity, of the final product.

The technical problem to be solved by the developed technical solution is to develop a method for producing peroxidase, which ensures the maximum yield of the active drug and its high specific activity.

The technical result obtained by implementing the developed method consists in reducing the consumption of horseradish roots and increasing the yield of peroxidase with a high degree of purity and activity, as well as speeding up the method.

To achieve this technical result, it is proposed to use a method for producing peroxidase, including homogenization of plant tissue of horseradish root, extraction of the enzyme, separation of the extract, purification of the enzyme by hydrophobic chromatography, concentration by ultrafiltration and gel filtration, subsequent lyophilization of the target product, and hemin and calcium chloride are added to the extract, the extract is saturated with nitrogen by purging and the enzyme is purified by hydrophobic chromatography.

In some embodiments of the method, the enzyme is extracted in 0.1M sodium or potassium phosphate buffer for 1 hour with continued nitrogen purge.

Preferably, hemin is added to the extract to a concentration of 5 μM and calcium chloride is added to the extract to a concentration of 5 mM.

In some embodiments, hydrophobic chromatography is carried out with the addition of ammonium sulfate in an amount of 35% of saturation.

Predominantly, hydrophobic chromatography is carried out on phenylsepharose, followed by fractionation with RZ>1.5, and gel filtration is carried out on Toyopearl HW55F, followed by fractionation with RZ>2.7.

The developed method includes the following set of features that provide a technical result in all cases for which legal protection is sought:

1) homogenization of plant tissue;

2) both horseradish roots and waste obtained during the stripping of horseradish roots in the manufacture of food products can be used as plant tissue;

3) enzyme extraction;

4) concentration of the extract by ultrafiltration;

5) purification of the enzyme by hydrophobic chromatography;

6) additional purification by gel filtration;

7) lyophilization of the enzyme.

The achievement of the technical result when using the proposed method is explained as follows.

It was experimentally found that the formation of polyphenolic pigments during the destruction of biomass is a consequence of the occurrence of an oxidase reaction, which leads to a partial inactivation of peroxidase. Purge of the extraction buffer with nitrogen for 2 hours reduces the oxygen concentration to 25-30 μM and inhibits the formation of polyphenols by 10-12 times. The inclusion of hemin and calcium chloride in the composition leads to 100% saturation of the active center with hemin and additional stabilization of the enzyme. The authors of the invention proposed for the first time to use these additives for the manufacture of peroxidase, which made it possible to reduce the consumption of horseradish roots and increase the yield of peroxidase per unit mass of roots at a high degree of its purity and activity.

Acceleration of the process of obtaining peroxidase is achieved by introducing a stage of hydrophobic chromatography, which allows you to get rid of expensive and time-consuming precipitation with ammonium sulfate. At the same time, savings of ammonium sulfate are achieved.

Compared with the closest analogue, the distinguishing features of the invention are:

1) the use of additives of hemin, calcium chloride and preliminary purge of the extraction buffer with nitrogen during the extraction of the enzyme;

2) purification and simultaneous concentration of the enzyme by hydrophobic chromatography;

3) additional purification by gel filtration in the presence of hemin and calcium additives.

In the future, the method will be illustrated by an implementation example.

3 kg of water-washed horseradish roots are crushed in a homogenizer and poured into 3 l of 0.1 M sodium phosphate buffer solution, pH 7.0, in the presence of 5 μM hemin solution and 5 mM calcium chloride, the buffer solution being preliminarily purged with nitrogen. Extraction is carried out for an hour. The extract is separated by decantation, kept overnight at 4°C, and filtered through a paper filter and then through ultrafilters with a pore diameter of 0.23 microns to completely remove the remaining biomass particles before the concentration step. Concentration is carried out by ultrafiltration on a flow-through concentration cell with a filter holding 30 kDa for 6 hours. The final volume is 0.5 l. Next, 200 g of ammonium sulfate (35% of saturation) is added to the enzyme solution, kept for 3 hours at 4°C, the precipitate is separated by centrifugation at 9000g, and the supernatant is applied to a column with phenyl sepharose, washed with 1 l of a buffer solution containing ammonium sulfate, and remove the enzyme with a gradient of ammonium sulfate (35-0%) while increasing the pH to 8.0. Fractions are collected with RZ>1.5; the fraction volume is 100 ml.

The enzyme was further purified by gel filtration on a 2 L Toyopearl HW55F column equilibrated with 0.1 M K-phosphate buffer, pH 7.8, in the presence of 5 μM hemin and 5 mM calcium chloride. The fractions of the main peak are collected (tail fractions can be used to obtain preparations of the acid isoform of horseradish peroxidase) with an RZ parameter value>2.7; the volume of fractions is 150 ml. The fractions are dialyzed against 2 l of a 5 mM solution of the same buffer, changing the buffer solution 4 times, and freeze-dried. Dried peroxidase has the appearance of an amorphous mass of bright brown color, easily soluble in aqueous buffer solutions. The yield of the enzyme is 500 mg. The degree of purity of the resulting enzyme is controlled spectrophotometrically by the RZ index (the ratio of absorption values ​​at wavelengths of 403 and 278 nm), the value of which should be at least 2.7. The enzymatic activity of peroxidase is determined by the indicator reaction with ABTS. The drug is considered conditioned if 1 mg contains at least 1000 units of activity. The mass fraction of moisture is determined according to GOST 24061-89.

Thus, the above information indicates that the implementation of the developed method allows to achieve the technical result - reducing the consumption of horseradish roots and increasing the yield of peroxidase of a high degree of purity and activity, as well as speeding up the method.

Thus, the above information indicates that the implementation of the developed method allows to achieve the technical result - reducing the consumption of horseradish roots and increasing the yield of peroxidase of a high degree of purity and activity, as well as speeding up the method.

1. A method for obtaining peroxidase, including homogenization of plant tissue of horseradish roots, extraction of the enzyme, separation of the extract, followed by concentration by ultrafiltration and gel filtration and lyophilization of the target product, characterized in that between the stages of ultrafiltration and gel filtration, the enzyme is purified by hydrophobic chromatography, and the extracting buffer is preliminarily saturated with nitrogen by purge and make hemin and calcium chloride.

2. The method according to claim 1, characterized in that the extraction of the enzyme is carried out in a 0.1 M buffer solution of sodium or potassium phosphate for 1 hour while continuing to purge with nitrogen.

3. The method according to claim 1, characterized in that hemin is added to the extract to a concentration of 5 μM.

4. The method according to claim 1, characterized in that calcium chloride is added to the extract to a concentration of 5 mm.

5. The method according to claim 1, characterized in that hydrophobic chromatography is carried out with the addition of ammonium sulfate 35% of saturation.

6. The method according to claim 1, characterized in that hydrophobic chromatography is carried out on phenylsepharose, followed by the selection of fractions with a parameter RZ>1.5.

7. The method according to claim 1, characterized in that the gel filtration is carried out on a Toyopearl HW55F, followed by the selection of fractions with a parameter RZ>2.7.

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