SU1472505A1 - Method of immobilizing enzymes - Google Patents

Abstract

This invention relates to biotechnology and may find application in the medical, chemical and microbiological industry. The aim of the invention is to increase the hydrodynamic stability of grafted enzymes, simplifying and accelerating the process. According to the method, a titanium sponge, washed with water and dried at 100 ° C, is activated by treating with hydrogen peroxide with a concentration of 30-35% at a ratio of titanium sponge: hydrogen peroxide 1: / 3-5 / for 45 minutes at 55-67 ° C, after which an enzyme is immobilized on the carrier thus activated in a known manner. 1 hp ff, 1 tab.

Description

one

The invention relates to biotechnology, in particular, to the technology of biochemical transformation of organic substances of plant or animal origin, and may find application in the medical, chemical and microbiological industries.

The purpose of the invention is to increase the hydrodynamic stability of grafted enzymes, simplifying and accelerating the process of immobilization.

The method is carried out as follows.

Hydrogen peroxide is poured in to distilled water and dried titanium sponge with a concentration of 30-35% at a ratio of titanium sponge. Hydrogen peroxide is 1: (3-5)

respectively. The mixture was thermostatic at 55-67 ° C with shaking for 45 minutes, then rinsed with distilled water until neutral and dried at 150 ° C for 1 hour. The titanium sponge so activated was cooled to 0-4 ° C, after which the enzyme is immobilized on it.

The titanium sponge is a by-product when a pure guitar is produced by the method of magnesium thermal reduction of tetrachloride.

titanium.

The titanium sponge has a high mechanical strength, a large specific surface area of 0.06-0.11, and a low saturated mass in the range of 0.7-1.1 g / cm.

Example 1. Getting activated media

1.5 ml of hydrogen peroxide with a concentration of 30-35% is added to 5 g of titanium sponge washed in distilled water and dried at 100 ° C. The mixture is thermostated at 55 ° C with shaking for 45 minutes, a boiled preparation with an enzyme activity of 132 u / g, which is 66% of the activity of the titanative enzyme.

Example 5 Immunization of E. coli Cell Suspensions,

As an enzymatically active material, a suspension is used.

however, they are washed with distilled water Q cells of E. coli with aspartase active20

25

thirty

until neutral and dried for 1 hour. The activated, taKHM titanium sponge is cooled to and used to immobilize enzymes, 15

Example 2. Immobilization of pepsin.

To 2 g of titanium sponge, activated according to Example 1, 10 ml of a 0.5% solution of pepsin in O, 1 M acetate buffer at pH 4.0 are poured. The mixture is agitated on a mechanical shaker for 1 hour at 4 ° C and held in for 10 hours. The titanium sponge C is washed with immobilized enzymes from an unbound enzyme with 490 ml of distilled water. An immobilized preparation is obtained with an activity of 1800 units / g, t, e, 62% of the activity of the native enzyme.

Example 3 Immobilization of Aspergillus niger pectinase,

To 2 g of titanium sponge, activated in Example 1, 5 ml of 3% pectinase solution in 0.1 M acetate buffer at pH 4.0 are poured. The mixture is thoroughly mixed and agitated on a mechanical shaker for 2 hours at 0 ° C and leave for 10 hours. Sponge wash s: wipe with 100 ml of distilled water until neutral. An immobilized preparation with an enzyme activity of 83.2 U / g is obtained, which accounts for 90% of the activity of the native enzyme. Example 4, the mobilization of protease from Aspergillus oryzae,

To 5 g of titanium sponge, activated according to Example 1, 10 ml of a 0.5% solution of protorizin in 0.1 M universal buffer solution at pH 4 (0.1 M boric acid, 0.1 M phosphoric acid, 0 , 1 M acetic acid in 1 M alkali) См The mixture is thoroughly mixed and shaken on a mechanical rocker for 2 hours at and left at this temperature for 10 hours. Titanium sponge is washed with 100 ml of distilled water until neutral. Immobilized

40

45

50

2 С

55

ness. E. coli cells are separated from the culture medium and washed with distilled water, followed by centrifugation for 10 mi of 2 g of titanium sponge, activated according to example 1, 10 ml of E. coli cells are poured in. The mixture is cooled to 6-8 ° C, 9 ml of 0.1 M phosphate buffer at pH 8.0 are poured. The mixture is thoroughly mixed and stirred on a mechanical shaker for 2 hours at 4 ° C and left for 10 hours. A new sponge with an immobilized cell suspension is washed onto the filter. from unbound cells with a 0.1 M aqueous solution of sodium chloride until the light disappears at 280 nm in prom water and O, 1 K acetate buffer at pH 4.5, to the absence of chlorine ions. A preparation is obtained with a total aspartase activity of 69520 units, which is 90% of the initial total aspartase activity of the introduced free cells.

 Example 6, Preparation of Activated Enzyme Carrier.

To 5 g of water washed in distilled water, and then a titanium sponge dried at 100 ° C, 25 ml of concentrated hydroperoxide are added. The mixture was thermostatted at 67 ° C with indentation for 45 minutes, then washed with distilled water until neutral and dried at 150 ° C for 1 hour. The titanium sponge activated in the same way is cooled. Prepared activated titanium sponge is used to bilize enzymes on it.

Example 7, Sin Immobilization, V.

- To 2 g of titanium sponge, activated in Example 6, 10.5% aqueous solution of pepsin in 0.1 acetate buffer at pH 4.0 is poured. The mixture is stirred up on a mechanical rocker for 1 "at 4 ° C and holding for 10 h. Titanium sponge with

The enzyme activity is 132 U / g, which is 66% of the activity of the titanative enzyme.

Example 5 Immobilizing E. coli Cell Suspensions

As an enzymatically active material, a suspension is used.

E. coli cells with aspartaznoy active0

five

0

five

35

40

45

50

55

ness. E. coli cells are separated from the culture medium and washed with distilled water, followed by centrifugation for 10 minutes. To 2 g of titanium sponge, activated according to example 1, 10 ml of E. coli cell suspension is poured. The mixture is cooled to 6-8 ° C, 9 ml of 0.1 M phosphate buffer at pH 8.0 is poured. The mixture is thoroughly mixed and shaken on a mechanical shaker for 2 hours at 4 ° C and left for 10 hours. A titanium sponge with immobilized cell suspension is washed on the filter of unbound cells with a 0.1 M aqueous solution of sodium chloride before IC. The efficiency of light absorption at 280 nm in prom water and O, 1 K acetate buffer at pH 4.5 to the absence of chlorine ions. A preparation with a total aspartase activity of 69520 units is obtained, which is 90% of the initial total aspartase activity of the introduced free cells.

 Example 6 Preparation of Activated Enzyme Carrier

25 ml of concentrated hydroperoxide is added to 5 g of the titanium sponge dried in distilled water and then the titanium sponge dried at 100 ° C. The mixture was thermostatic at 67 ° C while stirring for 45 minutes, then washed with distilled water until neutral and dried at 150 ° C for 1 hour. The titanium sponge thus activated was cooled to. Prepared activated titanium sponge is used to immobilize enzymes on it.

Example 7 Immobilization of Pepsin, V.

- To 2 g of titanium sponge, activated according to example 6, 10 ml of a 0.5% aqueous solution of pepsin in 0.1 K acetate buffer at pH 4.0 are poured. The mixture is agitated on a mechanical rocking chair for 1 "at 4 ° C and maintained for 10 hours. The titanium sponge with immobilized enzymes is washed with 490 ml of distilled water from an unbound enzyme. An immobilized preparation with an activity of 1870 units / g, i.e. 64.4% of the activity of the native drug.

Example 8. Immobilization of the thease from Aspergillus oryzae.

To 5 g of titanium sponge, activated according to Example 6, 10 ml of a 0.5% aqueous solution of protorizin in O, 1 M universal buffer solution at a pH of 4 are poured. The mixture is thoroughly mixed and stirred on a mechanical rocker for 2 hours at 2 ° C and left at this temperature for 10 hours. A titanium sponge is washed with 100 ml of distilled water to a neutral medium. An immobilized preparation with an enzyme activity of 140 U / g is obtained, which accounts for 70% of the activity of the native enzyme.

Example 9. Getting activated carrier enzyme

To 5 g washed in distilled water, and then dried with a titanium sponge, is added 20 ml of concentrated hydroperoxide. The mixture was thermostatted at 60 ° C with shaking for 45 minutes, then washed with distilled water until a neutral reaction and dried at 150 ° C for 1 hour. The titanium sponge activated in such images was cooled to 0 ° C. The prepared activated titanium sponge is used to immobilize enzymes on it.

Example 10 Immobilization of Pepsin

To 2 g of titanium sponge, activated according to example 9, 10 ml of a 0.5% aqueous solution of pepsin in 0.1 M acetate buffer at pH 4, O, are poured. The mixture is stirred in a mechanical rocking chair

for 1 h at 4 ° C and incubated for 10 h. The titanium sponge with immobilized enzymes is extracted from unbound enzyme with 490 ml of distilled water. Immobilized drug is obtained from 1780 units / g, that is, „61.5% of the active enzyme.

activity activity on

Examples 11-18. The method is carried out in a similar manner, but with a different ratio of titanium sponge and hydroperoxide when the carrier is activated.

The results are shown in the table.

As can be seen from the table, when the titanium sponge: peroxide ratio conditions are in place, the activity of the immobilized enzyme decreases.

Claims (2)

Invention Formula 1, A method for immobilizing enzymes, involving the activation of a titanium-based carrier and the subsequent binding of enzymes to an activated carrier, is also different from the fact that, in order to increase the hydrodynamic stability of the grafted enzymes, to simplify and speed up the process, titanium is used as a carrier the sponge and activation were obtained by treating with 30-35% hydrogen peroxide at a titanium sponge: hydrogen peroxide ratio of 1: (3-5), respectively, and a temperature of 55-67 s. 2. The method according to claim. 1, about tl and h. y u u and so that the treatment with hydrogen peroxide 45 45 min. lead for 1D72505 eight Table continuation