RU2546245C1 - Enzymatic agent based on immobilised butyrylcholin esterase and method for its preparation - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: invention proposes a preparation method of an enzymatic agent based on immobilised butyrylcholin esterase and an enzymatic agent for determination of carbamates and organic phosphorus compounds. Starch or gelatine gel is prepared in a phosphate buffer pH 7.9-8.0. The gel is cooled down to 31-35°C. Then, it is mixed with the buffer solution of butyrylcholin esterase and a buffer solution of an indicator of thiolic groups of 5,5'-dithio-bis(2-nitrobenzoic acid) with a concentration of 0.56 M. The obtained mixture is dosed to a hydrophobic substrate and dried at the temperature of 8°C. The inventions allow simplifying an enzyme immobilisation procedure, increasing storage time of an enzymatic agent without any loss of its activity during more than one year.

EFFECT: agent has high sensitivity to action of organic phosphorus compounds and carbamates.

2 cl, 7 dwg, 7 ex

Description

The invention relates to biotechnology, biochemistry and engineering enzymology, in particular to methods for producing immobilized enzymes, and can be used in chemistry, biochemistry, medicine, microbiology, ecology and agriculture for the analytical determination of the toxicity of individual substances and the integral toxicity of mixtures of various substances, including including those with anticholinesterase action.

This invention is intended for the production of highly active preparations of immobilized cholinesterase, which can be used as a biological module in devices of the "biosensor" type to control the degree of environmental pollution by cholinesterase inhibitors, including pesticides used in agriculture, which are derivatives of carbamic acid, as well as organophosphorus compounds.

Cholinesterase is highly sensitive to carbamates and organophosphorus compounds: organophosphate insecticides (chlorophos, dichlorvos) used in households, pesticides used in agriculture to protect plants, and drugs. Organophosphates phosphorylate, and carbamates acetylate the serine residue in the active center of the enzyme; as a result of such irreversible inhibition of the enzyme, cholinesterase activity remains low for a long time.

Currently, for the analysis of cholinesterase inhibitors, enzyme preparations are used, which are cholinesterase immobilized on various carriers by physical and chemical methods.

A known method of obtaining immobilized cholinesterase [copyright certificate SU 1409656, IPC C12N 11/14, publ. 07/15/1988], which consists in covalently linking the enzyme to the acid chloride derivative of silochrome C-80, which in turn is obtained from the carboxyl derivative of silochrome C-80 by heating it with thionyl chloride in a mixture with dimethyl foramide and absolute chloroform. Immobilization is carried out in phosphate buffer for 1 hour, by mixing the cholinesterase preparation with the carrier while cooling, then the preparation is washed with a cooled NaCl solution and water. The obtained immobilized preparation remains active for 80 days at room temperature.

The disadvantages of the known drug and the method of its preparation are the complexity of the procedure for obtaining a carrier, the use of toxic chloroform for this purpose, as well as the relatively short time of maintaining the activity of the enzyme.

The closest technical solution, selected as a prototype, is a drug that includes cholinesterase from horse serum, dye FDC Blue No. 1 and one of the specialized additives (glycerin, carboxymethyl cellulose, Carbowax-400 (polyethylene glycol 400), Kraystay K (CAS No. 8038-11-7), sodium chloride, sorbitol, sucrose, Triton X-100, dimethyl sulfoxide), immobilized in starch gel, and method for its preparation [Batjman EK, Goodson LH, Thomson JR Stabilization of serum cholinesterase in dried starch gel. // Analytical Biochemistry, 1967. V.19, N 3. P.587-492.]. A specialized additive is added to the preparation to allow the starch to dry and to keep the starch films attached to the substrate. As the substrate, polyurethane foam is used. The immobilization method consists in carrying out the following sequence of procedures. Sheets of polyurethane foam are cut into pieces measuring 4 × 6 inches (10.6 × 15.24 cm). A mixture is prepared containing warm 8% starch gel (47 ° C), cholinesterase and any of the additives. Pour 10 ml of the mixture into the syringe (without using a needle) and place the contents of the syringe in the center of the prepared polyurethane substrate, which is then placed on a warm glass plate. Then, using a household rolling pin, the starch mixture is very quickly distributed in a thin layer over the entire surface of the polyurethane substrate so that the blue color of the mixture is uniform, and as little as possible remains on the rolling pin and the glass plate of the starch mixture. The prepared glass plates were placed in a refrigerator to set the gel, and then dried in a vacuum evaporator under reduced pressure overnight. The obtained dried enzyme layer has a circular shape, it is cut into smaller pieces with a diameter of 0.95 cm, folded into a bottle with a screw cap containing a layer of magnesium perchlorate as a desiccant. To determine cholinesterase inhibitors in air or water, the activity of cholinesterase immobilized in this way is evaluated by the Ellman method. For this, the initially obtained enzyme preparation is placed in 5 ml of 0.1 M Tris pH 7.4 buffer containing 10 units of α-amylase activity and incubated for 30-60 minutes at room temperature. Then an aliquot of the obtained supernatant is added to a cuvette containing 2.5 ml of the following mixture: 2 ml of a solution of 5.5 ′ dithiobis (2-nitrobenzoic acid) concentration of 396 mg / 100 ml; 0.4 ml of a freshly prepared solution of butyrylcholine iodide (21.6 mg / ml) and 47.5 ml of 0.1 M Tris buffer pH 7.4. The rate of change in optical density at a wavelength of 412 nm, measured using a spectrophotometer, determines the activity of cholinesterase.

The disadvantages of the drug and its preparation method are the complexity and multi-stage procedure for its preparation, a long analysis time, since it is necessary to pre-incubate the drug for 30-60 minutes, add an additional α-amylase reagent, and the need to include an additional component, the FDC Blue dye, in the preparation No.1, with the help of which the authors try to provide a more uniform distribution of starch mixture on the surface of the substrate. However, the use of such a technology for the distribution of a viscous solution leads to an increase in the differences between batches of enzyme preparations, since the degree of uniformity is determined “by eye”.

The technical result of the invention is to simplify the procedure for immobilizing the enzyme, increasing the storage time of the enzyme preparation without losing activity for more than one year, increasing the accuracy of measuring cholinesterase activity, in particular during lengthy analyzes, as well as significantly simplifying the analysis, reducing its time and reducing its cost.

The technical result is achieved in that in an enzyme preparation based on immobilized butyrylcholinesterase containing butyrylcholinesterase immobilized in a gel, the novelty is that it contains an indicator of thiol groups 5.5 ′ dithiobis (2-nitrobenzoic acid).

The technical result is also achieved by the method of preparation of an enzyme preparation based on immobilized butyrylcholinesterase, including preparing the gel in a phosphate buffer pH 7.9-8.0, cooling the gel to 31-35 ° C, mixing a buffer solution of butyrylcholinesterase and a solution of the indicator of thiol groups 5.5 Dithio-bis (2-nitrobenzoic acid) with gel, dosing on a hydrophobic substrate, drying at 8 ° C.

Comparison of the claimed technical solutions with the prototype and other technical solutions from this technical field made it possible to establish their compliance with the criteria of "novelty" and "inventive step".

The invention is illustrated by drawings. Figure 1 presents a comparison of the activities of the preparations of soluble cholinesterase and immobilized in starch gel, as well as cholinesterase immobilized in starch gel together with an indicator of thiol groups, determined by the rate of hydrolysis of butyrylthiocholine iodide cholinesterase. Figure 2 shows the dependence of the rate of hydrolysis of butyrylthiocholine iodide on the amount of butyrylcholinesterase in an immobilized starch gel-based preparation. Figure 3 presents a comparison of the activities of the preparations of soluble cholinesterase and cholinesterase, immobilized together with the indicator in a gelatin gel, determined by the rate of hydrolysis of butyrylthiocholine iodide cholinesterase. Figure 4 shows the dependence of the rate of hydrolysis of butyrylthiocholine iodide on the amount of butyrylcholinesterase in an immobilized gelatin gel preparation. Figure 5 presents the activity of butyrylcholinesterase, immobilized together with an indicator of thiol groups in starch gel, depending on the storage time of the drug. Figure 6 shows the activity of butyrylcholinesterase immobilized together with an indicator of thiol groups in a gelatin gel, depending on the storage time of the preparation. Figure 7 shows the dependence of the enzymatic hydrolysis rate of S-BuCh-I with a starch gel-based preparation on the concentration of pyrimiphosmethyl.

An enzyme preparation based on immobilized butyrylcholinesterase is an enzymatically active substance consisting of butyrylcholinesterase, an indicator of the thiol groups 5,5 ′ dithiobis (2-nitrobenzoic acid) and a buffer co-immobilized into a gel.

The resulting preparation is highly sensitive to the action of organophosphorus compounds. For example, the concentration of pyrimiphosmethyl, at which a 50% decrease in the rate of enzymatic hydrolysis is observed, is 50 μM.

In the process of preparation, automatic dosing of the starch mixture is carried out, which allows to obtain preparations that do not differ in enzyme activity from batch to batch. In addition, dosing is carried out on a substrate having hydrophobic properties. As a result, after drying, the enzyme preparation is separated from the substrate, and thus, preparations are obtained that are free of the substrate and intended for one measurement. Drying the immobilized preparation is carried out without the use of a vacuum evaporator.

Example 1

The immobilization of butyrylcholinesterase in starch gel is carried out as follows: a solution of butyrylcholinesterase (BuChE) is prepared on 0.05 M potassium phosphate buffer pH 7.9-8.0. A solution of sulfur-containing butyrylthiocholine iodide (S-BuC-I) is prepared in distilled water. A solution of 5.5′-dithio-bis (2-nitrobenzoic acid) is prepared in 0.05 M potassium phosphate buffer, pH 7.9-8.0. A sample of 0.312 g of starch is placed in 12 ml of 0.05 M potassium phosphate buffer, pH 7.9-8.0, mixed vigorously and boiled for 2 minutes. The resulting mixture was cooled to 31-35 ° C, then 100 μl of a BuChE solution containing 12.25 units was added. enzyme activity, and 300 μl of a 0.56 M solution of 5.5′-dithiobis (2-nitrobenzoic acid). Dose the resulting mixture of 25 μl onto a hydrophobic substrate using an automatic station epMotion 5075 (Eppendorf, Germany), dried at a temperature of 8 ° C for 24 hours. The resulting preparation is activated by adding 100 μl of 0.12 mm solution of S-BuCh-I. The change in optical density is recorded at 412 nm for 5 minutes on a UVIKON 943 A spectrophotometer (Contron Instruments, Italy). The drug is preincubated for 5 minutes in phosphate buffer.

The concentration of 5.5′-dithiobis (2-nitrobenzoic acid) in one preparation is 0.16 M, BuChE - 0.03 U.

The activity of the drug is directly proportional to the rate of enzymatic hydrolysis of S-BuCh-I. The enzymatic hydrolysis rate of S-BuCh-I is determined by the Ellman method.

The introduction of the preparation of 5,5′-dithiobis (2-nitrobenzoic acid) does not change the activity of immobilized BuChE (figure 1). The activity of a preparation containing butyrylcholinesterase and 5.5′-dithiobis (2-nitrobenzoic acid) co-immobilized in a starch gel corresponds to the activity of a soluble enzyme. Thus, in the process of immobilization with an indicator in starch gel, the enzyme retains 100% activity.

Example 2

The immobilization of BuChE and the indicator on the thiol group in the starch gel is carried out in a manner analogous to example 1. To 10 ml of gel cooled to 31-35 ° C, solutions with different contents of BuChE and 0.56 M solution of 5.5′-dithiobis are added (2 nitrobenzoic acid). Dose the resulting mixture of 25 μl onto a hydrophobic surface using an automatic station epMotion 5075, dried at a temperature of 8 ° C for 24 hours.

Preparations containing 0.003 U, 0.006 U, 0.015 U, 0.03 U, 0.05 U, 0.06 U of BuChE activity are obtained, and preparations containing, in addition to BuChE, also an indicator on the thiol group. The concentration of 5,5'-dithiobis (2-nitrobenzoic acid) in one preparation is 0.16 M. The activity of the obtained preparations is determined by a method analogous to example 1.

The activity of a multicomponent immobilized enzyme preparation, obtained on the basis of starch gel, depends on the amount of enzyme in the preparation (figure 2). The activity of the obtained drugs are in the range from 0.012 to 0.085 srvc. and make up at least 80% of the activity of butyrylcholinesterase immobilized without an indicator.

Example 3

An immobilized enzyme preparation, based on a gelatin gel, is prepared as follows: a solution of butyrylcholinesterase (BuChE) is prepared on 0.05 M phosphate buffer pH 7.9-8.0. A solution of sulfur-containing butyrylthiocholine iodide (S-BuC-I) is prepared in distilled water. A solution of 5.5′-dithio-bis (2-nitrobenzoic acid) is prepared on 0.05 M phosphate buffer pH 7.9-8.0. A sample of 0.187 g of gelatin is placed in 10 ml of 0.05 M phosphate buffer pH 7.9-8.0, mixed vigorously and left for 30 minutes. Then the resulting mixture is heated to 80 ° C. The resulting mixture was cooled to 31-35 ° C, then 100 μl of a BuChE solution containing 12.25 units was added. enzyme activity, and 300 μl of a 0.56 M solution of 5.5′-dithiobis (2-nitrobenzoic acid). Dose the resulting mixture of 25 μl onto a hydrophobic substrate using an automatic station epMotion 5075, dried at a temperature of 8 ° C for 24 hours. The enzyme preparation is activated by adding 100 μl of a 0.12 mm solution of S-BuCh-I. The change in optical density is recorded at 412 for 5 minutes on a UVIKON 943 A spectrophotometer (Contron Instruments, Italy). The drug is preincubated for 5 minutes in phosphate buffer.

The concentration of 5,5′-dithiobis (2-nitrobenzoic acid) in one preparation is 0.16 M, BuChE - 0.03 U. The activity of the enzyme preparation is directly proportional to the rate of S-BuCh-I enzymatic hydrolysis. The enzymatic hydrolysis rate of S-BuCh-I is determined by the Ellman method.

The introduction of the composition of the drug 5,5'-dithiobis (2-nitrobenzoic acid) does not lead to a significant change in the activity of BuChE (figure 3). The activity of the resulting preparation is at least 80% of the activity of soluble butyrylcholinesterase.

Example 4

The immobilization of BuChE and the indicator of thiol groups in a gelatin gel is carried out in a manner analogous to example 3. To 10 ml of gelatin gel cooled to 31-35 ° C, solutions with different contents of BuChE and a 0.56 M solution of 5.5′-dithiobis are added (2 nitrobenzoic acid). Dose the resulting mixture of 25 μl onto a hydrophobic surface using an automatic station epMotion 5075, dried at a temperature of 8 ° C for 24 hours.

Preparations are obtained containing an indicator of thiol groups and 0.003 U, 0.006 U, 0.015 U, 0.03 U, 0.05 U, 0.06 U. The concentration of 5.5'-dithio-bis (2-nitrobenzoic acid) in one preparation is 0.16 M. The activity of the obtained preparations is determined in a manner analogous to example 1.

Based on a gelatin gel, active enzyme preparations are prepared (FIG. 4). The activity of immobilized preparations based on gelatin gel depends on the amount of enzyme included in their composition.

Example 5

The immobilization of BuChE and the indicator of thiol groups in the starch gel is carried out in a manner analogous to example 1. BuChE solution containing 44 U and a 0.56 M solution of 5.5′-dithiobis is added to 10 ml of gel cooled to 31-35 ° C nitrobenzoic acid). Dose the resulting mixture of 25 μl onto a hydrophobic surface, dry at a temperature of 8 ° C for 24 hours.

The concentration of 5.5′-dithiobis (2-nitrobenzoic acid) in one preparation is 0.16 M, BuChE - 0.11 U.

The enzyme preparation is activated by adding 100 μl of 120 · 10 ^-3 M S-BuCh-I solution. The change in optical density was recorded at 412 nm for 5 minutes on a UVIKON 943 A spectrophotometer (Contron Instruments, Italy) at a temperature of 25 ° C. The drug is preincubated for 5 minutes in phosphate buffer.

The introduction of the indicator of thiol groups does not affect the activity of the enzyme and ensures the preservation of activity throughout the year (figure 5).

Example 6

The immobilization of BuChE and the indicator of thiol groups in a gelatin gel is carried out in a manner analogous to example 3. BuChE solution containing 44 U and a 0.56 M solution of 5.5′-dithiobis is added to 10 ml of gel cooled to 31-35 ° C nitrobenzoic acid). Dose the resulting mixture of 25 μl onto a hydrophobic surface, dry at a temperature of 8 ° C for 24 hours.

The concentration of 5.5′-dithio-bis (2-nitrobenzoic acid) in one preparation is 0.16 M, BuChE - 0.11 U.

The enzyme preparation is activated by adding 100 μl of a 0.12 mm solution of S-BuCh-I. The change in optical density was recorded at 412 nm for 5 minutes on a UVIKON 943 A spectrophotometer (Contron Instruments, Italy) at a temperature of 25 ° C. The drug is preincubated for 5 minutes in phosphate buffer.

The introduction of the indicator of thiol groups does not affect the activity of the enzyme and ensures the preservation of activity throughout the year (Fig.6).

Example 7

The immobilization of BuChE and the indicator of thiol groups in the starch gel is carried out in a manner analogous to example 1. BuChE solution containing 44 U and a 0.56 M solution of 5.5′-dithiobis is added to 10 ml of gel cooled to 31-35 ° C nitrobenzoic acid). Dose the resulting mixture of 25 μl onto a hydrophobic surface, dry at a temperature of 8 ° C for 24 hours.

The concentration of 5.5′-dithio-bis (2-nitrobenzoic acid) in one preparation is 0.16 M, BuChE - 0.11 U.

The sensitivity of the drug to the action of pyrimifosmethyl is determined. To measure the rate of change in optical density, one disk of the preparation, 1.8 ml of distilled water and 100 μl of pyrimifosmethyl solution are added to the UVIKON 943 A spectrophotometer cuvette (Contron Instruments, Italy) and incubated for 5 minutes. The enzyme preparation is activated by adding 100 μl of 120 · 10 ^-3 M S-BuCh-I solution. The change in optical density is recorded at 412 nm for 10 minutes at a temperature of 25 ° C.

The dependence of the hydrolysis rate of S-BuCh-I preparation on the concentration of pyrimiphosmethyl is shown in Fig.7.

The claimed invention is a dosed preparation intended for one analysis, and can be used in laboratory and field conditions. The drug is intended for the analysis of the toxicity of various environments, including determining the quality of natural, waste water and aqueous solutions; determination of toxicity of industrial and household waste, water extracts from the soil and bottom sediments; air toxicity assessments; determination of the presence of harmful substances in food products and agriculture.

The additional introduction of the indicator of thiol groups 5,5′-dithiobis (2-nitrobenzoic acid) into the preparation makes it possible to improve the characteristics of the preparation by increasing the accuracy of measuring cholinesterase activity, significantly simplifying the analysis and shortening its time.

Thus, the use of the claimed invention allows to simplify the procedure of immobilization of the enzyme, to increase the storage time of the enzyme preparation without losing activity for more than one year, to increase the accuracy of measuring the activity of cholinesterase, in particular during long-term analyzes, and also to significantly simplify the analysis, reduce its time and reduce it cost.

Claims (2)

1. A method of preparing an enzyme preparation based on immobilized butyrylcholinesterase, including the preparation of starch or gelatin gel in phosphate buffer pH 7.9-8.0, cooling the gel to 31-35ºC, mixing a buffer solution of butyrylcholinesterase and a buffer solution of 5.5 'thiol group indicator -dithio-bis (2-nitrobenzoic acid) with a concentration of 0.56 Μ with a gel, dosing on a hydrophobic substrate, drying at a temperature of 8ºC. 2. An enzyme preparation for the determination of carbamates and organophosphorus compounds obtained by the method according to claim 1.

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