CS269267B1 - Process for preparing cellulosic materials with immobilized cholinesterase - Google Patents

Abstract

The solution belongs to the field of environmental control, agrochemistry and pharmaceutical analysis and concerns the preparation of cellulose materials with immobilized cholinesterase, usable for the detection and determination of acetylcholinesterase inhibitors. The essence of the solution is a method for preparing cellulose materials with immobilized cholinesterase, which consists in capturing the enzyme on raw or bleached cellulose fibers or fabric, cellulose filter paper or microcrystalline and pearl cellulose by sorption from a buffered aqueous solution of the enzyme, containing a non-ionic surfactant to increase the wettability of the materials and carbohydrates such as dextran or sucrose as a stabilizing additive of the sorption-bound enzyme on the cellulose carrier. The materials prepared according to the invention can be used in the construction of continuous detectors, analyzers and reactors and for the detection and determination of small amounts and very low concentrations of acetylcholinesterase inhibitors from aqueous solutions and extracts, after capture from the air for needs, environmental monitoring and in agriculture.

Description

The invention relates to a method for preparing a cellulose-based material with immobilized cholinesterase. These filter absorbent cellulose materials with immobilized cholinesterase solve a number of problems associated mainly with the capture and determination of cholinesterase inhibitors, compounds used in agriculture, important in the field of industrial chemistry, in the control of water, feed, food and the environment.

The biochemical cholinesterase reaction, as a very sensitive and relatively selective method, is widely used in a number of modifications and applications for the detection and determination of a large number of cholinesterase inhibitors /AMMON, R. - VOSS, G., Pflugers Arch. ges. Physiol., 235 (1935), p. 393; MICHEL, 0., H., J. Lab. clin. Med., 34 (1949), p. 1564/. Various types of technical means for the detection and determination of cholinesterase inhibitors are based on the cholinesterase reaction, starting with complicated automatic air and water analyzers, through simpler designs such as tube detectors to the simplest detection identification papers /FRANKE, S.: Lehrbuch der Militarchemie, Militarverlag DDR, vol. II., Berlin 1977, p. 481/. The fundamental problem of using filter or sorption materials including paper is the fixation and stabilization of enzymes. A partial solution to this problem is the immobilization of cholinesterase on a carrier by chemical bonding. However, this method of immobilization is disadvantageous, as it is complex, requires the use of a special derivatized carrier, expensive highly purified enzyme and is usually accompanied by a significant loss of enzyme activity, while the extension of the expiration of the bound enzyme is not great /BODOGA, P., BADILESCU, F., CIOBANI, O.: RO No. 82028/. The closest known solution to the method of preparing enzyme indicator paper is immobilization on special chromatographic paper with ion exchange function in a mixture with dextran gel and indication by chromogenic substrate /DAHLGREN, E., BERGEK, S., MARTENSSON, K.: WO 85-04423/, when the required sensitivity is achieved by extending the shelf life of indicator papers, and the above procedure additionally requires expensive and difficult to obtain special materials and chemicals. The mentioned shortcomings of the individual methods of preparing materials, i.e. insufficient fixation and stabilization of the enzyme on the carrier, short expiration, low sensitivity, high price due to the unavailability of special materials and purified enzymes and especially the impossibility of repeated and long-term measurement, or even processing of large volumes of aqueous samples due to insufficient mechanical resistance of the material, are eliminated by the method of preparing cellulose materials with immobilized cholinesterase according to the invention.

The essence of the invention is a method for preparing cellulose materials with immobilized cholinesterase, which consists in capturing the enzyme by sorption on cellulose carrier fibers, such as raw or bleached cotton fibers or fabric or cellulose filter paper or microcrystalline and pearl cellulose, by immersing the cellulose material in a buffered enzyme solution containing a nonionic surfactant and carbohydrates or by applying the solution to a thin layer of cellulose material.

The method of preparing cellulose materials with immobilized cholinesterase achieves a new effect by allowing the prepared filtration and sorption materials to be used repeatedly and for a long time until the loss of hydrolytic activity without the need to use reactive materials developed for enzyme immobilization or special chromatographic materials. A higher effect is achieved by using other cellulose materials in addition to paper, pearl and microcrystalline cellulose, as well as cotton fabric for cholinesterase immobilization as a filtration and sorption material, allowing highly selective capture of even very low concentrations of cholinesterase inhibitors from water, aqueous extracts, colored or turbid solutions and, after wetting the cellulose material, from the air, thus fully utilizing the high sensitivity of individual modifications of the cholinesterase reaction, including devices, reactors and detectors based on this reaction. A higher effect is also achieved due to significantly increased thermal stability, long-term at temperatures up to 70°C, which is confirmed by an expiration period of more than 5 years. The higher effect is also significantly manifested by the use of available domestically available, very cheap raw materials, e.g. the use of raw homogenate

CS 269 267 Bl bovine brain tissue as a substitute for highly purified and extremely expensive enzymes.

Example 1.

Preparation of acetylcholinesterase solution

According to another envisaged use, bovine caudate nucleus brain tissue is osmogenized in a ratio of 1:20 or 1:100 in a solution of 0.2 mol/l phosphate buffer pH 7.4 containing 5% dextran for infusion, 15% sucrose and 0.5% Slovasol SF-10.

Example 2 · .

Immobilization of acetylcholinesterase on cotton fabric.

The fabric is immersed in the solution prepared according to Example 1 for 30 minutes, after removal from the solution the fabric is dried at room temperature.

Example 3

Immobilization of cholinesterase on pearl cellulose

The dry pearl cellulose is added to a solution prepared by dissolving lyophilized plasma cholinesterase in a pH 8.0 solution containing 0.5% Slovasol SF-10, 5% dextran for infusion and the mixture is stirred for 30 minutes. After filtering the solution, the pearl cellulose with immobilized cholinesterase is dried at room temperature.

Example 4

Use of cotton fabric with immobilized acetylcholinesterase for detection of cholinesterase inhibitors in water

The fabric treated according to Example 2 is placed in the analyzed aqueous solution for 30 minutes. Then the fabric is removed and after 10 minutes a strip of dry filter paper saturated with an ethanol solution containing 0.15% 5,5'-dithio-bis-2-dinitrobenzoic acid and 0.5% acetylthiocholine iodide is placed on the white fabric for 1 minute. After removing the paper, the fabric turns yellow within 1 minute, in the presence of inhibitors in the analyzed water it remains white.

Example 5

Limit of detection mg.l when using cotton fabric with immobilized acetylcholinesterase according to example 4 for the detection of insecticidal preparations (without determining the content of the active substance) in water and % inhibition of acetylcholinesterase when analyzing the same solution by the spectrophotometric method according to Ellman /ELLMAN, G.L., COURTNEY, K.D., ANDRES, V., FEATHERSTONE, R.M.: Biochem. Pharmacol. 7, 88-95 (1961)/.

,-1 mg.l % inhibition Sevin 1.0 69.7 Pirimacarb 5.0 76.7 Methathion 10.0 46.5 Nuvan 6.0 79.8 Baygon 1.0 38.2 Dimefox 30.0 40.6

Example 6

Use of pearl cellulose with immobilized cholinesterase for detection of inhibitors in air

Pearl cellulose prepared according to Example 3, volume 1 ml in a glass column is moistened with 1 ml of water and air is filtered through the column. After the filtration is completed, 1.5 ml of a solution containing 0.5 mg of butyrylthiocholine iodide and 0.005 mg of 5,5'-dithio-bis-2-dinitrobenzoic acid is introduced into the closed column. The solution in the column turns yellow within 2 minutes, and remains colorless in the presence of inhibitors in the analyzed air.

CS 269 267 Bl

The invention can be used for the preparation of a number of types of filtration and sorption materials based on cellulose with immobilized cholinesterase exhibiting long-term hydrolytic activity towards substrates of the acetylcholine type and high affinity to acetylcholinesterase inhibitors. These can be used in a number of sectors of the national economy, pharmaceutical and chemical industries, agriculture, in environmental monitoring, in the construction of continuous detectors, analyzers and as a filling for reactors in biotechnical production.

The Seventy-Seventh Invention

Claims (3)

1. A method for preparing cellulose materials with immobilized cholinesterase, characterized in that cholinesterase is immobilized on the cellulose material by sorption from a buffered solution. 2. The method according to item 1, characterized in that for immobilization, an aqueous homogenate of brain tissue buffered to pH 6 to 8 or a solution of lyophilized plasma cholinesterase or erythrocyte acetylcholinesterase containing 1 to 10% nonionic surfactant and 5 to 30% carbohydrates, preferably dextran or sucrose, is used. 3. The method according to item 1, characterized in that raw or bleached cotton fibers or fabric or microcrystalline cellulose or pearl cellulose or filter paper is used for immobilization.