CS204368B1 - Process for preparing immobilised enzymes with high specific activity - Google Patents

Description

The invention relates to a process for preparing immobilized enzymes of high specific activity.

Immobilization of enzymes on water-insoluble carriers, organic or inorganic substances by functional groups capable of reacting with amino acids and proteins of the enzyme to form a covalent bond is well described (e.g., Methods in Enzymology Vol. XLIV, Immobilized Enzymes, Ed. Klaus Morbach, Academic Press, New York, San Francisco, London, 1976). Organic substances e.g. cellulose, polyamides, polyacrylamide and the like. they have certain disadvantages as carriers because they do not have sufficient mechanical stability, are sensitive to solvents, pH and ionic strength changes and are often attacked by microorganisms. For this reason, inorganic substances with enzymes bound by a covalent bond by means of specific functional groups are often preferred. Since the carrier of the inorganic basin generally does not have these functional groups, it is necessary to pretreat it, e.g. with aminoalkylsilanes, whereby the surface acquires organic functional groups, e.g. alkylamines which form a covalent bond with the inorganic substance. Further treatment of the material thus obtained can be carried out e.g. using thiophosgene, phosgene, dicarboxylic acid anhydride, or in other ways.

Among the inorganic materials, suitable enzymatic supports are aluminum, iron, nickel, titanium, zirconium, hydroxylapatite, silicate, and porous glass oxides. The pore size of the porous glass allows for the accessibility of the enzymes in the immobilization process as well as the substrates during the reaction with the immobilized enzyme into the interior of the support. However, the efficiency of the enzyme immobilization reaction, the specific activity of the immobilized enzyme and its storage stability on the surface of the support, however, depend on the length and substituents of the compound (spacer) containing a functional group suitable for anchoring the enzyme. The preparation of immobilized enzymes with high specific activity, high reaction efficiency with enzyme protein and long storage stability of immobilized enzyme is included in the present invention.

SUMMARY OF THE INVENTION The glass surface is reacted with χ-aminopropyltriethoxysilane at 100 to 120 ° C for 4 to 10 hours, wherein the obtained aminoalkyl-containing product is reacted in a second step with thiophosgene at 70 to 90 ° C after for 2 to 8 hours and the obtained polyisothiocyanate is reacted in a third step with 1,6 diamino-2,5-dihydroxyhexane for 2 to 8 hours at 80 to 100 ° C and the obtained derivative containing free primary amino groups is modified in the reaction with suitable agents, e.g. thiophosgene in the fourth step at 70 to 90 ° C for 2 to 6 hours, or succinic anhydride, and in the last step, at. the carrier thus treated immobilizes the enzyme with stirring for 1 to 20 hours at 0 to 40 ° C, at a pH of 4 to 6.

The advantage of said method of immobilization is that by using a bifunctional hexagonal substance containing hydroxyl groups in the system to reactive groups (e.g. - NH ₂ ), the effective surface is increased, allowing up to 96% of enzyme applied to the support in active form (DL Latigue, Immobilized Enzymes for Industrial Reactors, London 1975, p. 127), even under the best conditions, only about 80% of the enzyme remains in active form after immobilization on the support. The decrease in the activity of immobilized enzyme anchored to the glass modified by the above method during storage is 10 to 30% lower than that of the carriers without the modification or with the introduction of a bifunctional substance longer or shorter than 6 carbon. The accessibility of the active center of the immobilized enzyme for reaction with the substrate or inhibitor is better than that of carriers not modified in this manner.

Examples of design

Example 1

Glass of controlled pore size (10 g), CPG-10, 1400 with 120/200 grain size, mean pore diameter 1200 A, surface-specific. 12.5 m ² / g (Electronucleonics, USA) was heated in dilute (1: 1) nitric acid at 120 ° C for 5 hours. The glass particles were then washed with an excess (500 ml) of water, distilled water and acetone and dried. The dried glass was reactivated in a stream of oxygen at 400 ° C for 12 hours. Activated glass (10 g) was used for silanization, which was carried out in a 10% solution of χ-aminopropyltriethoxysilane in toluene (160 ml) at 120 ° C for 4 hours. The product of the silinization reaction was washed with excess toluene (400 mL) to remove undiluted. agged χ-aminopropyltriethoxysilane. The black glass was dried at 100 ° C overnight. The obtained primary amino-containing glass was characterized for its content using a 2,4,6-trinitrobenzenesulfonic acid method (H. Wand, M; Rudel and H. Dantzemberg, Z. Chemie, 18, 224 (1978)). with primary amino groups (10 g) was reacted with thiophosgene (10% solution) of thiophosgene in chloroform (200 ml) at 80 ° C overnight The obtained glass containing isothiocyanate functional groups was characterized for their content by reaction with (U). ⁴¹ C) valine (U- ¹⁴ G) with cysteine and mercaptoethanol.

The reaction was carried out by reacting porous glass (10 g) containing isothiocyanate groups with 2.5% 1,6-diamino-2,5-dihydroxyhexane (in 400 ml chloroform) at 80 ° C for 5 hours . Unreacted diaminohexane was eluted with excess (0.6 L) chloroform, (0.5 L) water and (0.5 L) acetone. The reaction of the modified porous glass with thiophosgene was carried out in a 10% solution of thiophosgene in 200 ml of chloroform at 80 ° C for 5 hours.

The carrier thus prepared (10 g) was used to immobilize pseudooholine esterase (acylcholine acyl hydrolase EC 3.1.1.8). Pseudocholinesterase from horse plasma (0.5 g, 120 U / mg) was dissolved in 200 ml of 0.02 M borate buffer (pH 8.5) and 10 g of porous glass with 1.6-diamino-2.5 introduced was added. dihydroxyhexane and isothiocyanate functional groups. The immobilization was carried out with stirring for 2 hours. The unreacted soluble enzyme was eluted with 2 M sodium chloride in water (500 mL). The immobilized enzyme was maintained in a humidified state at 4 ° C and activity decreased by 15% over 1 year. Obtained specific activity of the enzyme 2300 U / g, immobilization efficiency 95%. Example 2

As in Example 1, except that, after the introduction of 1,6-diamino-2,5-dihydroxyhexane, derivatized glass (10 g) with free amino groups is reacted with succinic anhydride (4 g) in 200 ml of chloroform at a temperature of 80 ° C for 2 hours. The reaction product was washed with water (500 mL) and acetone (300 mL) and dried. Further modification of the glass (10 g) containing free carboxyl groups was carried out in a 2% aqueous solution of 1-cyclohexyl-3- (2-morpholinoethyl) carbodiimide pH 4.8 with stirring at room temperature for 1 hour. The obtained derivatized glass was washed 4 times with water (500 mL) prior to enzyme immobilization. The enzyme immobilization was carried out as described in Example 1, except that the temperature in the immobilization reaction was maintained in the range of 0-2 ° C. Specific activity of immobilized enzyme 2050 U / g. Mobilization efficiency 85%. Example 3

As in Example 1, except that glucoamylase (α-1,4-glucan glucohydrolase EC 3.2.1.3) with Endomycopsis bispora is used instead of pseudooholine esterase. Specific activity of immobilized enzyme 1200 U / g, immobilization efficiency 92%.

The immobilized enzymes on the nosia using 1,6 diamino-2,5-dihydroxyhexane are suitable for industrial application and for use in analytical chemistry.

Claims (1)

OBJECT OF THE INVENTION Process for preparing immobilized enzymes of high specific activity, characterized in that the glass surface is reacted with r-aninopropyl-triethoxysilane at a temperature of 100 to 120 ° C for 4 to 10 hours, wherein the product obtained containing aminoalkyl groups is reacted in a second step with thiophosgene at 70 to 90 ° C for 4 to 8 hours and the obtained isothiocyanate alkyl derivative is reacted in the third step, with 1,6-diamino-2,5-dihydroxyhexane for 4 to 8 hours at 80 to 100 ° C, and the obtained derivative containing free primary amino groups is modified in reaction with thiophosgene in the fourth step at a temperature of 70 to 90 ° C for 2 to 8 hours or with succinic anhydride and in the last step in the glass thus treated. is immobilized with stirring for 1 to 20 hours at 0 to 40 ° C and pH 4 to 6.