SU578893A3 - Method of preparing immobilized enzymes - Google Patents

Description

(54) METHOD FOR OBTAINING LESHOBILIZED ENZYMES

Examples of carriers include brick, silica, alumina, clay, sand, agarose, starch, polydextra, cellulose, polymers such as polybutadiene, polystyrene, mesh or non-mesh, copolymers of methacrylic acid, copolymers of maleic anhydride and ethylene.

The carrier is modified or activated in such a way as to provide it with one or more functional groups that react with the enzyme. Modification is carried out by known methods depending on the nature of the carrier taken and the enzyme to be fixed. The activation is carried out by diazotization or by halogenation or by sulfonation. You can call the reaction of the carrier, for example, with the halides of sulphuryl, cyanuryl, dicyan, thionyl, graft copolymerization of the carrier, namely polymers with polyfunctional gadro;

Such carriers are used in the form of grains, the size of which in most cases is larger than 100 microns, can vary greatly. They must be free from all products that inhibit or denature the enzyme, and from all traces of water.

As an organic medium that does not dissolve the enzyme, aliphatic, cycloaliphatic, aromatic hydrocarbons are used, for example, hexane, benzene, toluene, xylene, chloroform, carbon tetrachloride, dichloroethane, trichloroethane, trichloroethylene, perchlorethylene, dioxane, tetrahydrofurate, and tetrachloride. Hydrocarbons are used alone or in a mixture.

The process is carried out as follows.

The active carrier and one or more enzymes are dispersed simultaneously or sequentially in a hydrocarbon, then the reaction mixture is heated to the reaction temperature, preferably to the boiling point, for the time required for fixing the enzyme. The enzyme is taken in excess with respect to the functional groups of the carrier. The amount of hydrocarbon used varies depending on the nature of the carrier and the enzyme. The process is conducted at 30-120 ° C, the temperature depends on the nature of the hydrocarbon and the enzyme. To accelerate the reaction, it is possible to operate at elevated pressure. In contrast to the known method, the pH of the medium during the fixation process does not affect the properties of the fixed enzyme. The reaction time ranges from a few minutes to 2 hours. After fixation, the hydrocarbon is easily separated and the carrier – enzyme complex obtained is washed with a buffer solution taken at such a pH and composition that it does not denature the complex. As a result, the fergent is adsorbed on the nostopic. Then the enzyme can be used again in peaKiQiH fixation.

The resulting carrier-enzyme complexes consist of one or more enzymes fixed on the carrier. In the case when the complex

contains several enzymes, the latter can be fixed either simultaneously or sequentially. Good complexes are obtained by mixing two complexes.

The amount of enzyme fixed by covalent bonds depends on the nature of the enzyme, as well as on the nature and structure of the carrier.

The proposed complexes carrier-enzyme

can exist at higher temperatures, as well as known complexes obtained in an aqueous medium. In addition, it is advantageous to use them continuously for quite a long time, since the enzyme does not lose its activity.

Example. Pectinase fixation on brick.

Pounded and sifted homogeneous. brick with a grain size of 0.5-0.8 mm is distilled, then with an aqueous solution of 1N. hydrochloric acid and distilled again

water After calcination for 15 hours in an atmosphere of oxygen at 700 ° C, an activated brick is obtained which does not contain impurities. 2 g of bricks are added to 40 ml of a 10% by volume solution of sulfuryl chloride in anhydrous benzene. Then

the suspension is stirred, brought to a boiling point and held at this temperature for 20 hours. The active active brick is separated.

0.5 g of commercial pectinase and then 2 g of active brick is dispersed using sound in

50 ml of anhydrous benzene. The resulting dispersion is heated at boiling point for 1 hour. After cooling, the solid is drained, washed with 20 ml of 1 M sodium chloride solution, leaving the mixture to stand for 15 hours at 4 ° C, centrifuged and

dried The removal is carried out 7 times, which allows desorption of the enzyme adsorbed on the carrier, which can be used again. After each washing, a solid substance consisting of a carrier-enzyme complex, in which

the enzyme is fixed on the carrier by covalent bond, enzyme activity is determined. This is done as follows.

1 g of the complex is dispersed in 10 mlO, 4% by weight solution of polygalacturonic acid in

acetate 0.01 M buffer solution with a pH of 4. The dispersion is heated at 35 ° C for 30 minutes. After cooling and decanting, 5 ml of the solution is taken out, then OD of 9 ml of a 9% by weight solution of Zinc sulfate in water and 0.3 ml of 1 N is added. A suitable soda solution is allowed to stand. After centrifugation, the amount of the recovered compounds in the remaining part is determined using the dinitrosalicylate method. For comparison, each experiment was repeated by fixing in an aqueous

environment. In this case, take a solution of 0.5 mg of pectinase in 50 ml of water, 2 g of active brick and hold at 4 ° C for 7 hours.

The results are shown in Table. 1 where they are expressed as a percentage of activity

complex in relation to the activity of the enzyme before fixation.

As can be seen from the table. 1, enzyme fixation in benzene is faster than in an aqueous medium. In addition, pectinase, fixed in an organic solvent, is more stable than pectinase, fixed in an aqueous medium. On the other hand, for pectinase fixed in an organic medium, a slight decrease in activity is first observed, then the activity remains constant after the 3rd line; after the first phase, while the activity of pectinase fixed in an aqueous medium is continuously reduced. Thus, the amount of enzyme covalently fixed in an organic medium is definitely higher than the amount fixed in an aqueous medium.

Example 2. Fixing papain on a brick.

2 mg of papain and 2 g of active brick, similar to npifMepy 1, are suspended in 20 ml of hexane. The resulting suspension is heated under reflux for 1 hour. After cooling, the solid is separated, washed with water, 20 ml of 1 M sodium chloride solution and again with water. Then, the enzymatic activity of the carrier – enzyme complex obtained was measured; the suspension was subsequently suspended in 1 ml of water; 9 ml of a 0.3% by weight casein solution in phosphate-citrate 0.025 M buffer solution with a pH of 7 was added. The suspension was heated at 37 ° C 10 min. After cooling and decanting, 4 ml of the solution are taken out and 4 ml of a 10% w / w aqueous solution of trichloroacetic acid are added. The casein is precipitated, it is filtered and the peptide content in the filtrate is determined by the Lowry method. The carrier-enzyme complex is again rinsed with water, 1 M sodium chloride and water, and the enzymatic activity is measured again. These operations are repeated several times. These comparisons fix papain on the same nosttel in an aqueous medium at 40 ° C for 7 hours. After washing, the enzymatic activity is measured by the indicated method. The results (in micrograms of free peptides per ml / min) are presented in table. 2

As can be seen from the table. 2 papain hexases are faster in hexane, and the amount of fixed enzyme is 10 times greater than in water.

Example 3. Trypsin fixation on brick.

The experience of example 2 is repeated, replacing papaya with trypsin. The results are shown in Table. 3

As can be seen from the table. 3, trypsin fixation in hexane is faster than in water, the amount of fixed enzyme is high, and the enzyme is more stable.

The complex and the enzyme obtained in hexane are relatively stable, despite successive washes that are harmful to the complex. This stability is even greater when the complex is continuously consumed.

Thus, 50 g of the active brick-trypsin complex, prepared as above, is placed in a column where the temperature is maintained at 37 ° C. 37 (kt solution (by weight) casein in phosphate-citrate 0.025 M buffer solution with pH 7 for 15 hours at a rate of 60 ml / h are continuously passed through the column. Activity is measured in this manner for 24 hours. After 15 hours, the activity decreases by 5%. This well demonstrates the stability of the proposed complexes.

Example 4 Fixed ribonuclease on polybutadiene.

Polybutadiene is activated by reaction with acetyl chloride.

20 mg of the enzyme is suspended in 50 ml of benzene, 2 g of active polybutadiene is added, the resulting suspension is heated to boiling and kept at this temperature for 1 hour. The fixation of the enzyme is monitored by IR spectroscopy. The 1720 cm absorption band of the carbonyl group, which is present in the active polybutschgien, disappears during the fixation process, while the absorption band 1640 appears, which is attributed to the imine function. After separation, the resulting complex carriers: the enzyme is washed with 20 ml of carbonate buffer solution with a pH of 10.5 to remove the adsorbed enzyme. The activity of the fixed enzyme with respect to ribonucleic acid (RNA) is then measured. 20 mg of the resulting carrier-enzyme complex are suspended in a mixture of 1 ml of 0.2 M Tris buffer solution c. pH 7.8, containing 0.02 M of methylenediaminetetraacetic acid, 1 ml of water and 1 ml of an aqueous solution of HK in such a concentration that it was 3.3 mg GOK per 1 ml of mixture. The suspension is kept for 2 minutes at 37 ° C, then 1 ml of Fayden's reagent is added and left for 15 minutes at 0 ° C and centrifuged for 5 minutes at a speed of 6000 rpm at 3 ° C to separate the excess RNA. The remaining solution is diluted in a ratio of 1:30 and the absorbance measured at 260 mm, compared with the carrier without enzyme. Enzymatic activity corresponds to 1.2 mg of active enzyme per 1 g of carrier.

When M er 5. Fixation of glucoamylase on silica.

Silica with surface 15 is activated by graft copolymerization with silane having an epoxy function.

1 g of the active carrier is added to a 50 ml chloroform dispersion containing 20 mg of glucoamylase. The resulting suspension is heated under reflux for 2 hours. After cooling, the resulting complex is decanted, dried and washed 3 times with 20 ml of distilled water and then washed for 24 hours with 20 ml of 2 M sodium chloride solution at 4 ° C. For comparison, the same experiment was carried out in an aqueous medium with 50 ml of acetate 0.1 M buffer solution with a pH of 4.5, containing

20 mg of glucoalshla and 1 g of that carrier. The gge solution (x: mei1 at 66 ° C. 66 hours. After decantation and washing, as indicated above, the activity is measured.) Best carrier – enzyme complexes are added to 10 ml of Ennois by weight of starch solution in 0.1 M acetate buffer. the solution with a pH of 4.5 and stirred for 10 minutes at 40 ° C. After separation, the amount of reduced sugar in the liquid phase is determined by a colorimetric method, using 3.5 - - / tinitros / ts lovoy acid. 4 shows the results, expressed in optical th density.

As in previous experiments, the fixation time of glucoamylase in chloroform is less than in water, the resulting complexes are more active and more stable.

Table 1

Fixation medium

Fixation medium

Claims (6)

1. A method of obtaining immobilized enzymes by contacting the enzyme with an activated carrier, characterized in that, in order to improve the stability of the enzyme associated with the carrier, the process is carried out in anhydrous anrotonic solvent at 30-120 ° C. table 2 .Activities with the amount of washing with 1 M NaCl solution, times Activity when the amount of washing with 2 M NaCl solution, times 2. A method according to claim 1, characterized in that an oxidoreductase, a transferase, a hydrolase, a lyase, an isomerase, a ligase are used as an enzyme. 3. A method according to claim 1, characterized in that a mineral, organic or organic-mineral substance is used as a carrier, insoluble in organic matter and having re-acyl-functional functional groups with the help of which Moiyr fix enzymes. 4. A method according to claim 3, characterized in that the carrier is brick, silica, alumina, clay, sand, agarose, starch, polydextran, cellulose, polymers. 5. A method according to claim, characterized in that aliphatic, nicloaliphatic, aromatic hydrocarbons are used as the organic medium. 6. The method according to p. 1, characterized in that the process is conducted during (those time from several minutes to 2 hours Sources of information taken into account in the examination: 1. Zaborsky O. Immobilized Enzymes. CRS. Press, Cleveland, Ohio, 1973.