CS211176B1 - A method of preparing high-binding capacity biologically active carrier carriers - Google Patents

Abstract

The invention relates to a method for preparing carriers of biologically active substances with high binding capacity. The essence of the invention lies in the fact that the xerogel. of silicic acid, formed by polymerization of acidified aqueous solutions of alkali metal silicates, is treated in the first stage with inorganic acids, in the second stage the obtained xerogel is washed with water and successively with solvents of decreasing polarity, in the third stage it is freed from organic solvents, dried and calcined, after which in the fourth stage the dried xerogel is allowed to react with aminoalkylsilane and the obtained aminoalkylsilane is activated for reaction with biologically active substances. Biologically active substances immobilized on carriers with high binding activity are used for the preparation of immobilized enzymes, for immobilization of affinity sorbents, cells, lectins and immunochemically active substances, suitable for use in analytics, medicine and catalysis of enzyme processes.

Description

The invention relates to a process for the preparation of carriers of biologically active substances having a high binding capacity. It is an object of the invention to provide xerogel. of silicic acid formed by polymerization of acidified aqueous solutions of alkali metal silicates is treated in the first step with inorganic acids, in the second step the obtained xerogel is washed with water and successive solvents of decreasing polarity, freed from organic solvents in step 3 the dried xerogel is reacted with an aminoalkylsilane, and the obtained aminoalkylsilane is activated to react with biologically active substances. Biologically active substances immobilized on carriers with high binding activity have applications for the preparation of immobilized enzymes, for the immobilization of affinity sorbents, cells, lectins and immunochemically active substances suitable for use in analytics, medicine and catalysis of enzyme processes.

The present invention relates to the preparation of enzymatic carriers, affinity carriers and other biologically active substances with high binding activity.

The preparation of carriers of biologically active agents for the immobilization of enzymes and the covalent binding of affinants is described in detail, e.g. in Methods in Enzymology Vol. XLIV, Immobilized Enzymes, Ed. Klaus Morbach, Academic Press, New York, San Francisco, London, 1976, or Jaroslava Turkova: Affinity Chromatography, Elsevier Scientific Publishing Company, Amsterdam, Oxford, New York, 1978. cellulose, polyamides, polyacrylamide and the like. they have certain disadvantages as carriers because they do not have sufficient mechanical stability, are compressible and cannot be used at higher pressures, are sensitive to changes in solvents, to changes in pH and ionic strength, and are biodegradable. For this reason, inorganic substances with biologically active substances bound by a covalent bond by means of specific functional groups are often preferred.

Of the inorganic materials used as carriers, the oxides of aluminum, nickel, titanium, iron, zirconium, hydroxyapatite, silicates and porous glass are described. Methods in Enzymology, Vol. XLIV, listed from above. The disadvantages of said materials are their relatively low specific surface area (up to 100 m ² / g), in the case of metal oxides, the surface inertness and finally the relatively high production costs which make glass-based carriers of controlled size compare difficult to access for wider technological applications. Since carriers of inorganic origin do not have functional groups capable of immediate chemical reaction with the enzyme, the affinity, and other biologically active substances, their treatment, e.g. with aminoalkylsilanes to generate organic functional groups on the surface (e.g. alkylamine /. The degree of substitution of inorganic carrier supports by organic functional groups depends on the number of reactable inorganic functional groups. The number of reactable inorganic functional groups is proportional to the specific surface area of the enzyme support. So eg. The degree of alkylaminosilane (CPG 10-2000), the glass having a controlled pore size of 12.5 m ² and a mean pore size of 120 nm is 80 mol / kg. It is important, however, that in the case of carriers with a high specific surface, the mechanical properties do not deteriorate sufficiently so that they cannot be applied in the process.

Much of the SiOH group is useless for the binding of enzymes for spatial reasons because it is found in micropores. The volume of comparison of silica gel or porous glass carriers rarely exceeds 1 cm @ 2 / g. Of these reasons, the binding capacity of inorganic enzyme carriers is usually relatively low. The preparation of inorganic carriers of biologically active substances of high binding capacity which overcomes the above-mentioned drawbacks of inorganic flood carriers is included in the present invention.

SUMMARY OF THE INVENTION The moist silicic acid xerogel resulting from the polymerization of acidified aqueous alkali metal silicate solutions is treated in the first step with inorganic acids, preferably sulfuric acid or hydrochloric acid or nitric acid or phosphoric acid or organic acids, such as e.g. trichloroacetic acid and in the second step the xerogel obtained is first washed with water and successive solvents of decreasing polarity, preferably ethanol and acetone, after which the product in the third step is freed from organic solvents, dried and calcined at temperatures of 200 to 900 ° C, preferably at 700 to 750 ° C, whereupon the dried xerogel in the fourth step is reacted with an aminoalkylsilane (preferably gamma-aminopropyltriethoxysilane) at a temperature of 100 to 110 ° C for 2 to 10 hours and the aminoalkylsilane obtained is activated by known methods for reacting with biologically active substances.

An advantage of the process for preparing carriers of biologically active substances according to the invention is:

- high porosity and concentration - SiOH groups resulting in

- high degree of substitution of silica gel with organic functional groups (up to 1.76 mol / kg)

- satisfactory mechanical properties for both hatching and column use, including high pressure columns

low production costs and correspondingly low cost of the prepared carrier.

Examples of design

Example 1

200 ml of an aqueous solution of sodium silicate (water glass) containing 9.20 JS by weight. 130 ml of 3 wt. nitric acid and homogenize. The resulting solidified mass of silica hydrogel is mechanically disrupted and mixed with 250 ml of 65 / S nitric acid or 160 ml of conc. sulfuric acid or 300 ml of 55% by weight, phosphoric acid or 200 g of trichloroacetic acid and allowed to mix occasionally into the second dithe, when the mixture is diluted with distilled water, filtered and washed thoroughly with distilled water until neutral. finally acetone. The material is freed from the organic solvent residue by drying and stabilized at temperatures of 200 to 900 ° C, preferably at 700 to 750 ° C.

The obtained silica gel (10 g) with a high specific surface area (500 µg / g) and a pore volume of 2 to ml g -1, and having an average pore size of 20 nm, was tempered for 10 hours at 550 ° C. g) was used for silanization, which was carried out in a 10% solution of gamma-aminopropyltriethoxysilane in toluene (100 ml), at 110 ° C, for 10 hours. After silanization, the product was washed with an excess of teluene (600 ml) and in an additional excess of toluene (400 ml) the product obtained was heated for 4 hours at 110 ° C to completely extract unreacted gamma-aminopropyltriethoxysilane. The washed silica gel derivative was dried overnight at 100 ° C and characterized for primary amino content using a 2,4,6-trinitrobenzenesulfonic acid / H method. Wand, M. Rudel, H. Dantzemberg, Z. Chemie, 18, 224 (1978). The primary amino content was 1.42 mol / kg. The primary amino-containing silica gel (10 g) was further reacted with thiophosgene. A 10% solution of thiophosgene in 200 ml of chloroform was used / at 100 ° C overnight. The derivatized silica gel was washed with an excess of 800 ml of chloroform and dried. Primary amino groups e and quantitatively converted to the isothiocyanate functional groups, corresponding to the binding capacity of the modified gel to the / U-14 _C / L-cysteine / 1.41 mol / kg / a / U- ^ C / L-valine / 1.36 mol / kg /.

The carrier thus prepared can be used to immobilize biologically active substances, e.g. enzymes. Thus peeudocholine esterase / acetylcholine acyl hydroPSea, EC 3.1.1.8/ from horse plasma / 0.5 g; 2 (ukat. Mg.V) was dissolved in 200 ml of 0.02 M borate buffer (pH 8.5) and 10 g of polyisothiocyanate derivative of silica gel was added. X-mobilization was carried out with stirring at room temperature for hours. 2 M NaCl / 500 ml /. the immobilized enzyme retains activity 71 Qika. g ^- ^ in a humidified condition at 4 ° C for 60 days.

Example 2

Silica gel (10 g) containing primary amino groups, prepared as described in Example 1, was homogeneously quenched with 2 ml of water and 10 ml of 2-chloromethylaxirane was added. The reaction was run at room temperature for 3 hours and then at 40 ° C for 30 minutes. The reaction product was washed with ethanol, dried and characterized by an oxirane functionality of 1.2 mol / kg. The carrier thus prepared (10 g) was used to immobilize pseudocholinesterase from horse plasma (0.5 g);

(ukat. mg-1) (dissolved in 200 ml of 0.02 M borate buffer (pH 8.5)). Immobilization was performed with shaking on a mechanical shaker for 4 hours at room temperature. The unreacted soluble enzyme was eluted with 2 M NaCl (500 ml). .

The immobilized enzyme retained 67.5 ukat activity. g '' in a humidified state at 4 ° C for 90 days.

Example 3

So. as in Example 1, except that the silanization reaction of silica gel with gamma-aminopropyltriethoxysilane was carried out at 100 ° C for 2 hours, and the obtained amino propyl silica gel containing 1.76 mol / kg primary amino groups was converted to thiophosgene to give polyisothiocyanate having an isothiocyanate group content of 1.66 mol / kg. Said silica gel polyisothiocyanate (1 g) was used to react with β- (omega-aminomethyl) phenylboronic acid (0.3 g) and the obtained boronic silica derivative was used for the affinity sorption of the serine enzymes in the active center.

Example 4

As shown in Example 1, except that silica gel with a specific surface area of 30 m @ 2 / g was used for the reaction of sianization. The content of primary amino groups in the obtained alkylamino silane was 0.22 mol / kg. The obtained aminoalkyl silicagel (10 g) was treated with succinic anhyuride (3 g) in chloroform (200 ml) at 80 ° C for 2 hours. The reaction product was washed in water (500 ml) and in acetone (300 ml) and dried. A minor modification of silica containing free carboxyl groups (0.19 mol / kg) was carried out in a 2% aqueous solution of 1-cyclohexyl-3- (2-morpholinoethyl) carbodiimide at pH 4.8 with stirring at room temperature for 1 hour. hours. The obtained derivatized silica gel was washed 4 times with water (500 ml) and the pseudochlinesterase was immobilized as described in Example 1, except that the reaction temperature was kept at 0-2 ° C,

The activity of the immobilized pseudocholinesterase was 52 peaks. g * ^.

Example. 5

As described in Example 1, except that glucoamylase / alpha-1,4-glucan glucohydrolase, EC, 3.2.1.3/ from Endomycopsis bispora, was used instead of pseudocholinesterase.

The activity of the immobilized enzyme was 60.2 g / cm @ 2.

Biologically active substances immobilized on said carriers of high binding activity can be used to prepare mobilized enzymes, to mobilize affinity sorbents, cells, lectins and immunochemically active substances suitable for use in analytics, medicine and catalysis of enzyme processes.

Claims (1)

Process for preparing carriers of biologically active substances having a high binding capacity, characterized in that the moist silicic acid xerogel resulting from the polymerization of acidified aqueous alkali metal silicate solutions is treated in the first step with inorganic acids, preferably sulfuric acid or hydrochloric or nitric acid or phosphoric acid by organic acids, preferably trichloroacetic acid, and in the second step, the xerogel obtained is first washed with water and successively decreasingly polarized solvents, preferably ethyl alcohol and acetone, after which the product in the third step is freed from organic solvents, dried and calcined at 200 to 900 ° C, preferably at a temperature of 700 to 750 ° C, after which the dried xerogel in the fourth step is reacted with an aminoalkylsilane, preferably gamma-aminopropyltriethoxysilane, at a temperature of 100 to 110 ° C, for 2 to 10 hours and the aminoalkylsilane obtained is activated by known procedures for reaction with biologically active substances.